Phospholipases PLBD1 and PLBD2: Difference between revisions

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=== PLBD2 active site ===
== Introduction ==
 
A surprising number of orphan human enzymes (unknown substrate) still exist ten years after the completion of the human genome project. PLBD1 and PLBD2 are semi-orphans in the sense of being probable phospholipases of B class but with uncertain physiological substrates and thus functionalities. This is especially important in the case of PLBD2 which localizes to the lysosome, as its absence could plausibly lead to a serious yet unrecognized [http://en.wikipedia.org/wiki/Lysosomal_storage_disease lysosomal storage disease].
 
No bioinformatic algorithm or experimental protocol leads with any certainty to determination of function. The gene pair here has [http://www.ncbi.nlm.nih.gov/pubmed/19706171,19237744,17007843,19019078,20093120,16077094,8892229,17105447 eight targeted publications] but cases exist where protein function remains unknown after ten thousand papers (eg PRNP).
 
PLBD1 and PLBD2 constitute a small gene family (sequence homology class) within vertebrates though one that occurs expanded in some early diverging eukaryotes. However, the Pfam clan [http://pfam.janelia.org/clan?acc=CL0052 NTN] (N-terminal nucleophile aminohydrolases) may have, among its ten family members, additional representatives in humans diverged beyond recognizability in primary sequence. These establish the great antiquity of the fold and certain of its features but are not likely to shed additional light on phospholipases specifically.
 
PLBD2 presents a special difficulty in that a sequence of post-translational steps are apparently necessary for its activation. Without these, potential substrates can hardly be assayed. These steps include removal of the signal peptide, mannosylation appropriate to the lysosome targeting receptor, and self-catalytic proteolytic activation (into 28k and 42k fragments which remain associated) to expose the substrate binding site as this becomes appropriate.
 
Because PLBD1 and PLBD2 are full length paralogs, the bioinformatic approach below considers both on an equal footing. PLBD1 has been more amenable to activation whereas PLBD2 has a high-resolution structural determination. Thus comparative genomics allows for annotation transfer, first from PLBD2 to a structural model for PLBD1 (already provided by the SwissModel pipeline), then perhaps transfer of PLBD1 experimental protocols to PLBD2.
 
However the gene duplication event occurred some 650 million years ago and the two genes are quite diverged today. It is not known whether substrates have diverged or merely their cell type of expression. Increased gene dosage per se is seldom an explanation. Yet certain core features remain conserved, including the fold, active site residues, signature motifs, certain glycosylation sites and even the fragmentation pattern, implying these are essential functional features under long-range strong selective pressure for their maintenance.
 
Disulfides are only separately conserved within each paralog but this fortuitously provides a reliable signature for assigning deeply diverged proteins from early eukaryotes to their orthology class. As the respective functions become better known, we can hope to understand how the gene duplication event contributed advantageously to increasing evolutionary complexity, leading to persistence of both enzymes in most species over immense time spans.
 
=== Conservation at critical sites ===
 
The six residues of PLBD2 associated with the active site are completely conserved within vertebrates to within genomic sequencing error. These same six residues are also completely conserved within PLBD1. Indeed 3 of the residues are conserved in the broader NTN hydrolase clan.
 
This is perhaps unsurprising since the active site was established a couple billion years earlier in the bacterial ancestor. However if PLBD2 and PLBD1 have different substrates, this establishes that these six residues are insufficient to distinguish the two active sites. Note H266 and T330 do not contribute their side chain, leaving them and W269 to separate phospholipases from the other NTN hydrolases.
 
The glycosylation sites are surprisingly conserved both within and between PLBD2 and PLBD1. Some of the motifs may be either recently acquired within later vertebrates or spurious glycosylation motifs with N and D both acceptable (or similar small amino acids) in the first slot of the NxS/T motif. Glycosylation is important in correct targeting of lysosomal proteins, more so than in generic endoplasmic reticulum proteins where motifs are often poorly conserved (as in sulfatases).
 
PLBD2 has two established disulfides. Strict sequence conservation of these throughout vertebrates (indeed, throughout metazoan) suggests both play an important role in protein structure and stability.
 
In PLBD1 however, the first disulfide is not a possibility and while an opportunity exists for a disulfide homologous to the the second disulfide of PLBD2, indels cloud the alignment and spacing would have to be different. There is additionally ambiguity given C...CC as to the cysteines involved. Indeed a second distal disulfide may occur utilizing C...CC.............C which has no counterpart in PLBD2. While cysteines can be conserved for many reasons other than disulfide (as in the nucleophile cysteine here), suitably proximity and side chain orientation in the SwissModel of PLBD1 would argue for disulfide. Comparative genomics suggests that C2 and C4 may form an ancient disulfide whereas C1 and C3 might represent a deuterostome innovation.
 
homSap <span style="color: #FF0000;">C</span>NTI<span style="color: #FF0000;">C</span><span style="color: #FF0000;">C</span>REDLNSPNPSPGG<span style="color: #FF0000;">C</span> human PLBD1
braFlo <span style="color: #FF0000;">C</span>SAI<span style="color: #FF0000;">C</span><span style="color: #FF0000;">C</span>RKDLAKVGAKPDG<span style="color: #FF0000;">C</span> Branchiostoma floridae
strPur SKSI<span style="color: #FF0000;">C</span>MRGDLM-TSPMPNG<span style="color: #FF0000;">C</span> Strongylocentrotus purpuratus XM_001192029
nemVec MNAI<span style="color: #FF0000;">C</span>SRGDLIADGPRASG<span style="color: #FF0000;">C</span> Nematostella vectensis XM_001638165
monBre YNAI<span style="color: #FF0000;">C</span>SRGDLESDSPSPGG<span style="color: #FF0000;">C</span> Monosiga brevicollis XM_001745398
SwissModel coordinates for PLBD1 show the 2nd and 4th sulfur atoms separated by 2.03 angstroms:
ATOM  3552  SG  CYS  471      49.680 -13.769 -12.461
ATOM  3579  SG  CYS  475      <font color = blue>49.273 -14.310  -4.881</font>
ATOM  3585  SG  CYS  476      51.067  -9.716  -9.172
ATOM  3678  SG  CYS  490      <font color = blue>50.737 -13.198  -5.750</font>
 
The known human SNPs of PLBD2 are in some cases quite radical substitutions in terms of both physical qualities of the substituted amino acid and the degree of observed phylogenetic conservation at that site. These likely result in unstable and/or inactive enzyme. Both enzymes are autosomal so compensation might occur in the recessive state, or alternately, PLBD2 and PLBD1 could fill for each other to some extent. In either case, lysosomal storage disease might not be clinically observable.
 
Here Q54P may actually be a deleterious mutation in the reference sequence individual (with the SNP representing wildtype) as proline is highly  conserved throughout mammals. The neanderthal genome also has proline at this location (UCSC track: 1C>0A(CCG>CAG)).In A204V, valine is quite a bulky substituent for a site normally restricted to small amino acids; R354C is definitely a serious mutation, no doubt attributable to a CpG hotspot; Q521K appears milder as does R524C.
 
The known human SNPs of PLBD1 can be analyzed similarly. P26Q and V30L may be inconsequential as they occur in the rather unconstrained primary sequence of the N-terminus; V265I occurs at an ILV reduced alphabet; V377A and P534A are much more serious despite the aliphatic nature of alanine and likely give rise to dysfunctional protein.


[[Image:PLBD2activeSiteComp.png|left]]
[[Image:PLBD2activeSiteComp.png|left]]
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  showing conserved side chains (*) and relevant main chains (....)
  showing conserved side chains (*) and relevant main chains (....)
  (adapted from Fig 6 of [http://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19706171 Lakomek et al. BMC Struct Biol.2009;9:56:)]
  (adapted from Fig 6 of [http://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19706171 Lakomek et al. BMC Struct Biol.2009;9:56:)]
                                                *           (*)           *     *
                                            *         (*)       *   *
  PLBD2 phospholipase B-like       gray   3FGR  C249 H266 W269 T330 N432  R463
  PLBD2 phospholipase B-like     gray   3FGR  C244 H261 W264 T325 N427 R458 human numbering
  Cephalosporin acylase           pink   1OQZ  S170 .... H192 .... N413 R443
  PLBD1 phospholipase B-like    ....  pred C228 H245 W248 T303 N402 R433 human numbering [http://swissmodel.expasy.org/repository/?pid=smr03&uid=&token=&query_1_input=Q8VCI0&zid=async SwissModel]
  Conjugated bile acid hydrolase   green   2BJF  C2   ....  D21  .... N175 R228
  PLBD2 phospholipase B-like    ....  .... C225 H242 W246 T302 N401 R432 Dictyostelium numbering
  Penicillin V acylase             yellow 3PVA  C1   ....  D20  .... N175 R228
  Cephalosporin acylase         pink   1OQZ  S170 .... H192 .... N413 R443
  Penicillin G acylase             orange 1K5S  S1    ....  Q23   ....  N241 R263
  Conjugated bile acid hydrolase green 2BJF  C2   .... D21 .... N175 R228
  Penicillin V acylase           yellow 3PVA  C1   .... D20 .... N175 R228
  Penicillin G acylase           orange 1K5S  S1   .... Q23  .... N241 R263
Human SNPs resulting in amino acid substitutions:
PLBD2:                PLBD1:
  Q54P  [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs7965471  rs7965471]   P26Q  [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs1141509  rs1141509]
  A204V [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs12231990  rs12231990]  V30L  [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs12296104  rs12296104]
  R354C  [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs56935204  rs56935204]  V265I  [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs7957558  rs7957558]
  Q521K  [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs17852787  rs17852787]  V377A  [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs2287541  rs2287541]
   R524C  [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs12425042 rs12425042]  P534A [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs1600  rs1600]


[[Image:PLBD2colored.png|left]]
<br clear ="all">
[[Image:PLBD1colored.png|left]]
<br clear ="all">
[[Image:PLDB2consSites.png|left]]
<br clear ="all">
[[Image:PLDB1consSites.png|left]]
<br clear ="all">
<br clear ="all">
  SNPs resulting in amino acid substitions:
 
=== Intron evolution ===
 
PLBD1 and PLBD2, being full length paralogs, clearly indicate an early gene duplication and subsequent divergence to the current low percent identity. Segmental duplications preserve any introns present at the time of the event and these generally persist in both position and phase into living species.
 
However PLBD1 and PLBD2 -- despite having similar numbers of introns -- exhibit very little in common in terms of location as the diagram below shows. One possibility is that a second copy arose as a retroprocessed gene (a mechanism erasing existing introns) and was subsequently intronated at random positions. This is unlikely here given that 10-11 relatively rare events would be needed.
 
The remaining possibility is that the gene duplication took place prior to the main era in early eukaryotes during which the bulk of introns were established. This fits the current state of high divergence despite fairly slow rates of evolution during metazoan times.
 
The last five amino acids of each PLBD1 exon are colored below. Then using an alignment of PLBD1 to PLBD2, the colors are mapped to the homologous five residues within PLBD2. There they fall on the ends of exons only when these correspond to those of PLBD1. The outcome here -- despite uncertainties in alignment gapping --  shows intron positions do not correspond with the exception of the terminal intron (which also is phase 0).
 
While this merely compares human PLBD1 and PLBD2, the collected reference sequences (intronated against their respective genome assemblies) confirm that introns in both genes are deeply conserved.
PLBD1 introns do not correspond well to those of PLBD2:
>PLBD1_homSap Homo sapiens (human) first and last introns are not mappable
0 MTRGGPGGRPGLPQPPPLLLLLLLLPLLLVTAEPPKPA 1
2 GVYYATAYWMPAEKTVQVKNVMDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQTLSNEIIMFVAGFLEG<span style="color: #0066CC;">YLTAP 2</span>
1 HMNDHYTNLYPQLITKPSIMDKV<span style="color: #CC0099;">QDFME 2</span>
1 KQDKWTRKNIKEYKTDSFWRHTGYVMAQIDGLYVGAKKRAI<span style="color: #0066CC;">LEGTK 0</span>
0 PMTLFQIQFLNSVGDLLDLIPSLSPTKNGSLKVFKRWDMGHC<span style="color: #FF0000;">SALIK 0</span>
0 VLPGFENILFAHSSWYTYAAMLRIYKHWDFNVIDKDTSSSRLS<span style="color: #0066CC;">FSSYP 1</span>
2 GFLESLDDFYILSSGLILLQTTNSVFNKTLLKQVIPETLLSWQRVRVANMMADSGKRWADIF<span style="color: #CC0099;">SKYNS 1</span>
2 GTYNNQYMVLDLKKVKLNHSLDKGTLYIVEQIPTYVEYSEQT<span style="color: #0066CC;">DVLRK 1</span>
2 GYWPSYNVPFHEKIYNWSGYPLLVQKLGLDYSYDLAPRAKIFRRDQGKVTDTASMKY<span style="color: #CC0099;">IMRYN 1</span>
2 NYKKDPYSRGDPCNTICCREDLNSPNPSPGG<span style="color: #00CC66;">CYDTK 0</span>
0 VADIYLASQYTSYAISGPTVQGGLPVFRWDRFNKTLHQGMPEVYNFDFITMKPILKLDIK* 0
>PLBD2_homSap Homo sapiens (human)
0 MVGQMYCYPGSHLARALTRALALALVLALLVGPFLSGLAGAIPAPGGRWARDGQVPPASRSRSVLLDVSAGQLLMVDGRHPDAVAWANLTNAIRETG 2
1 WAFLELGTSGQYNDSLQAYAAGVVEA<span style="color: #CC0099;">AVSEE 0</span>
0 LIYMHWMNTVVNYCGPFEYEVGYCERL<span style="color: #0066CC;">KSFLE</span>ANLEWMQEEMESNPDSPYWHQ 0
0 VRLTLLQLKGLEDSYEGRVS<span style="color: #0066CC;">FPAGK</span>FTIKPLGFL 2
1 LLQLSGDLEDLELALNKTKIKPSLGSGSC<span style="color: #FF0000;">SALIK</span>LLPGQSDLLVAHNTWNNYQHMLRVIKKYWLQFREGPW 1
2 GDYPLVPGNKLV<span style="color: #0066CC;">FSSYP</span>GTIFSCDDFYILGSGL 0
  0 VTLETTIGNKNPALWKYVRPRGCVLEWVRNIVANRLASDGATWADIF<span style="color: #CC0099;">KRFNS</span>GT 2
1 YNNQWMIVDYKAFIPGGPSPGSRVLTILEQIP 2
1 GMVVVADKTS<span style="color: #0066CC;">ELYQK</span>TYWASYNIP 2
1 SFETVFNASGLQALVAQYGDWFSYDGSPRAQIFRRNQSLVQDMDSMVR<span style="color: #CC0099;">LMR 2
1 YN</span>DFLHDPLSLCKACNPQPNGENAISARSDLNPANGSYPFQALRQRSHG<span style="color: #00CC66;">GIDVK 0</span>
0 VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD* 0
 
=== Signal peptide compositional anomaly ===
 
The first exon of both PLBD1 and PLBD2 are ill-behaved in alignments. The explanation can be see in their compositional distortion (very high GC content) that specialized masking tools such as seg and gnu recognize. Such dna manifests itself at the protein level by high levels of the amino acids, such as GPL that use those codons in the three reading frames.
 
Such regions are prone to repeated expansions and contractions via replication slippage. Not only do we expect such alleles in human but also that inter-species comparisons will be difficult and alignments problematic (as homology by definition is lost even if the sequences still align).
 
This matters very little to the mature protein since this region is trimmed off during maturation but the question still arises as to how signal peptide variations continue to be recognized efficiently by the signal receptor complex. Indeed a class of mutations could exist in which the signal peptide cannot be processed correctly and the protein never reaches the lysosomal compartment, in effect a knockout mutation.
 
This compositional anomaly may have caused vertebrate-wide sequencing problems. Many assemblies had difficulty sequencing back to the initial methionine and alignment programs also fell short. A set of reliable sequences could only be obtained after careful hand-curation and only then from fewer species than usual in comparative genomics.
 
Even then the set of first exons raises more questions than it answers as it seems to be evolving quite chaotically in fish. Mammals also exhibit a peculiar conserved insertion as placentals diverged from marsupials. And using [http://www.cbs.dtu.dk/services/SignalP/ SignalP 3.0] separately on each sequence, it emerges that the marsupial signal peptide and those of earlier diverging species are much shorter. That isn't a problem per se because signal peptide lengths are [http://www.signalpeptide.de/ quite variable].
 
PLBD1 also exhibits a shift in the location of the signal peptide cleavage site over evolutionary time, crossing the boundary into exon 2 in some clades. (Since the exon break is extremely conserved, this conclusion is independent of alignment gapping.) Here again this would be functionally irrelevant since the co-processing of nascent chain takes place well after mRNA splicing. However this does provide an interesting case of homologous residues not being functionally homologous and so evolving under the different functional constraints.
 
PLBD1:  ATGAcccgcggcggtccgggcgggcgcccggggctgccacagccgccaccgcttctgctgctgctgctgctgctgccgctgttgTTAGTCACCGCGGAGCCGCCGAAACCTGCAG
          MTRxxxxxxxxxxxxxxxxxxxxxxxxxxVTAEPPKPA
          MTRGGPGGRPGLPQPPPLLLLLLLLPLLLVTAEPPKPA
PLBD2: ATGGTGGGCCAGATGTACTGCTACCCCGGCAGCCACCTGGCCCGGGCGCTGACGCGGGCGCTGGCGCTGGCCCTGGTGCTGGCCCTGCTGGTCGGGCCGTTCCTGAGCGGCCTGGCGGGGGCGATCCCAGCGCCGGGGGGCCGCT...
        MVGQMYCYPGSHxxxxxxxxxxxxxxxxxxxGPFLSGLAGAIPAPGGR...
        MVGQMYCYPGSHLARALTRALALALVLALLVGPFLSGLAGAIPAPGGR...
Phylogenetic variation in first exon signal peptide of PLBD2:
              <------ signal peptide ----------------->            <---- start of 3FGW 3FGR 3FGT------------->
<font color = purple>>PLBD2_homSap MVGQMYCYPGSHLARALTRALALALVLALLVGPFLSGLAGA</font><font color = red>IP</font>APGGRWARDGQVPPASRSRSVLLDVSAGQLLMVDGRHPDAVAWANLTNAIRETG
<font color = purple>>PLBD2_panTro MVGQMYCSPGSHLARALTRALALALVLALLVGPFLSGLAGA</font><font color = red>IP</font>APGGRWARDGPVPPASRSRSVLLDVSAGQLLMVDGRHPDAVAWANLTNAIRETG
<font color = purple>>PLBD2_ponAbe MVGQMYGSSGSHLA----RALALALVLALLVGPFLSGLAGA</font><font color = red>IP</font>APGGRWARDGPVTPASRSRSVLLDASAGQLLLVDGRHPDAVAWANLTNAIRETG
<font color = purple>>PLBD2_rheMac MVGQMYCSSGSPLARALTRALALALVLALLVGLFLSGLAGA</font><font color = red>IP</font>APGGRWAHDGPVTPASRSRSVLLHAATGQLLLVDGRQPDAVAWANLTNSIHETG
<font color = purple>>PLBD2_papHam MVGQMYCSSGSPLARALTRALALALVLALLVGLFLSGLAGA</font><font color = red>IP</font>APGGRWAHDGPVTPASRSRSVLLDAATGQLLLVDGRHPDAVAWANLTNAIRETG
<font color = purple>>PLBD2_calJac MVGKMYSSPSSRLAQALTRALALALVLALLAGLFLSGLSGA</font><font color = red>IP</font>APGGRWARDGSVPSGSGSRSVVLDAAAGQLLLVDGRHPDAVAWANLTNAIHETG
<font color = purple>>PLBD2_otoGar MvGPMYGSPGGRLARALTRALALALVLaLLIGLFLSCLAGA</font><font color = red>iP</font>PPGSGRARDGLITPASRSSSVLLDATTDQLRLVDGRHPDAVAWANLSNAIHETG
<font color = purple>>PLBD2_musMus MAAPVDGSSGGWAARALRRALALTSLLASLTGLLLSGPAGA</font><font color = red>LP</font>TLGPGWQRQNPDPPVSRTRSLLLDAASGQLRLEDGFHPDAVAWANLTNAIRETG
<font color = purple>>PLBD2_ratNor MAAPMDRTHGGRAARALRRALA----LASLAGLLLSGLAGA</font><font color = red>LP</font>TLGPGWRRQNPEPPASRTRSLLLDAASGQLRLEYGFHPDAVAWANLTNAIRETG
<font color = purple>>PLBD2_dipOrd MAAPPYGSRGGRPAGSLSRALV----LAVLVGLSPSGPAGA</font><font color = red>VP</font>SPGDRWGRHKPEPPVSRSRSVLVDAASGQLRLVDGLHPGAVAWANLTNAIRETG
<font color = purple>>PLBD2_cavPor MAAPTYVSLDGRPVRARALALA--PALCLLVGLSLGRLAGA</font><font color = red>VP</font>APGPRGARDGPVPAA--CRSVLLDAASGQLRLVDGLQPGAVAWANLTNAIPETG
<font color = purple>>PLBD2_oryCun MVAPRDGCAGGRLARALALALL--------TGLLLGGLAGA</font><font color = red>AP</font>APGGGEQRDPPSPPASCCRSALLDAATGQLRLVDGRHPDAVAWANLTNAIHETG
<font color = purple>>PLBD2_ochPri MAATRDSSAGCRLARVLTRALAL---LALPTGLFLSGPAGA</font><font color = red>IP</font>VRGDGEERGRPAPSGSRCRSVLVDAESGQLRLVDGRHPAAVAWANLTNAIHETG
<font color = purple>>PLBD2_turTru MVDPMYGCPGGRLARALTRALALALVLALLVGLFLSGLTGA</font><font color = red>IP</font>TPRGHRGPGRPVPPASRCRSVLLDPEtGQLRLVDGRHPDAVAWANLTNAIRETG
<font color = purple>>PLBD2_bosTau MVAPMYGSPGGRLARAVTRALALALVLALLVGLFLSGLTGA</font><font color = red>IP</font>TPRGQRGRGMPVPPASRCRSLLLDPETGQLRLVDGRHPDAVAWANLTNAIRETG
<font color = purple>>PLBD2_oviAri MVAPMYGSPGGRLARAVTRALALALVLALLVGLFLSGLTGA</font><font color = red>IP</font>TPRGQRGRGMPVPPASRCRSLLLDPETGQLSLVDGRHPDAVAWANLTNAIRETG
<font color = purple>>PLBD2_susScr MVAPMYGSPGGRLARALTRALALALVLALLVGLFLSGLTSA</font><font color = red>IP</font>TPKGYRGSGRSVPPASRSRSVLLDTETGQLRLVDGRHPDAVAWANLTNAIHENG
<font color = purple>>PLBD2_ursAme MAAPMYGSPGGRLARALTRALALALVLALLVGLFLSGLTGA</font><font color = red>IP</font>ISGRQWGPNGPVPPDSRSRSVLLDAETGQLRLVDGRHPEAVAWANLTNAIRETG
<font color = purple>>PLBD2_musPut      GS-GGRLARALTRALALALVLALLVGLFLSGLTGA</font><font color = red>IP</font>ISGRQWGPKGPVPPDSRSRSVLLDAETGQLRLVDGRHPDAVAWANLTNAIRETG
<font color = purple>>PLBD2_canFam ...................................SGLTGA</font><font color = red>TP</font>VSGRRWGPSGPVPPASRSRSVRLDPQTGQFQLVDGRNPDAVAWANLTNAIRDTG
<font color = purple>>PLBD2_myoLuc MVAPPSRSPGGRLTPALSRAPALAPGLALLAGLFLSGWTGA</font><font color = red>IP</font>TPRDPWGPNGPVPPASRSRSVVLDARTGQLQLVDGRQPDAVAWANLTNAIHETG
<font color = purple>>PLBD2_pteVam MVAPMDRSPGGRLAGALTRTLELTLVLAPLAGLFLSGRTSA</font><font color = red>IQ</font>TPGSRWGSEGPVSPASRSRSVLLDPQTGQLRLVDGRHPDAVAWANLTNAIHETG
<font color = purple>>PLBD2_eriEur MVAPMCGSPGGRPARALTRALALAPALALLVGLFLSSLAGA</font><font color = red>IP</font>PPEDNWGRNGSFPPVSRCRSVLLDSETGQLRLVDGRHPDAVAWANLSNAIHETG
<font color = purple>>PLBD2_loxAfr MVAPVYGSPGGRLARALTQALAVALVLALLVGLFLSGLTGA</font><font color = red>IS</font>LTGHRWGPDGPAPPASRSRSVLLDTATGQLRLVDGRHPDAVAWANLTNAIRETG
<font color = purple>>PLBD2_echTel MVATEYGSPGGRLARALTRAPALALMLALLVGLFLSGLTGA</font><font color = red>IS</font>PAGGRREPNGRVPPASSSRSALLDPATGQLRLADGRHPEAVAWANLTNAIHETG
<font color = green>>PLBD2_macEug mVATMYQ--GGCLALGLALGLGLVLVLS</font><font color = red>LP</font>--------------------QPSLPPPPSRTRSVVMDSATGQLNVVEGWEAGAIAWANLTNAIAETG
<font color = green>>PLBD2_monDom MVATMCQ--GSSLALGLALALGLALG</font><font color = red>LR</font>-------------------PPQPSLPPPAPSRSCSVVLDEASGQLKVVEGAQAGAVAWANLTNAIGETG
<font color = blue>>PLBD2_anoCar MAPAWLLRFFGLALLLARSPA</font><font color = red>RR</font>------------------------PPPFPDPAAVPTRSCSVVLEPGSAALKLVNGWAPGAVAWANLTEGIRQNG
<font color = blue>>PLBD2_galGal MAVVRALLVAAAVAAWVPGVAS</font><font color = red>GP</font>-------------------------------TPPPRSASVLLEPGSGRLRVLPGRQPAAVAWAELTDHIQAVG
<font color = blue>>PLBD2_melGaL MAVVRALLVAAAVAAWVPGVAS</font><font color = red>GP</font>-------------------------------TPPPRSASVLLEPGSGRLRVLPGRQPAAIAWAELTDHIQAVG
<font color = blue>>PLBD2_xenTro MGAQLLLIFMLFSLGAAQQ</font><font color = red>AV</font>---------------------------------------VSVLFDPATGNITTVEEKRVVGAVAWAELKDSILENG
<font color = blue>>PLBD2_xenLae MAPWQLFIFSLFCVGAAQQ</font><font color = red>QA</font>--------------------------------------VVSVLFDPATGNITTVAEKKVAGAAAWAELTDSIQENG
<font color = brown>>PLBD2_oryLat MAFRQNKTVCAKMSTFMKSLLVLGLFWGCGRA</font><font color = red>EI</font>---------------------------RSAVIDKGSGKLTVVEGYHEGFVAWANFTNDIETSG
<font color = brown>>PLBD2_dicLab MASRLNKTSAVGGFSKVLNVLAVLSGLCLLFASVG</font><font color = red>AE</font>-----------------------IRTAVIDKQTGQLSVVDGYREGFVAWANFTDDIKTSG
<font color = brown>>PLBD2_hipHip masrlnktDGVQDKQDVFCGEFSSASVAFYVLCLTCVRA</font><font color = red>EI</font>--------------------KSAVIDGQSGELSVVDGFQKDFVAWANFTDDIQTSG
<font color = brown>>PLBD2_parOli MASRINKMGVEDKQDVSCVEFCVRA</font><font color = red>EI</font>----------------------------------KSAVIDAQSGDLCVRDGFHQDLVAWANFTDDIQTSG
<font color = brown>>PLBD2_gasAcu MASRQNTTVTLRHFKAVLSALFVMCACVQA</font><font color = red>EI</font>-----------------------------RSAVIDKQTGKLSVVEGYREGFVAWSNFTDDINTSG
<font color = brown>>PLBD2_oreNil MACRRNGADRVRSFTEVLGLLKMFLLLFCLFAVRA</font><font color = red>EI</font>-----------------------SRTAVIDKQTGQLSVIEGYQEDFVAWANFTNDIETSG
<font color = brown>>PLBD2_sebCau MASRHNKMFAVGRFKVALSVLSTLCFMCASVGA</font><font color = red>EV</font>--------------------------RTAVVNKQTGQLSVVEGYREDFVAWSNFTDDIKTSG
<font color = brown>>PLBD2_osmMor MAFRLLRLSTTLHLAVFLHVLFLSCSSIKA</font><font color = red>EI</font>-----------------------------STIVLDEKTGQLTILEGYRDDYVAWANFTDDIEHSG
<font color = brown>>PLBD2_onyTsh MADRRTQMSLTTEKMFMFSCVFYLSWTSVRA</font><font color = red>EI</font>----------------------------PSKILDKQTGQLSLEEGFRDDYVAWANFTDDIKNSg
<font color = brown>>PLBD2_salSal madrrtqMSVTTEKMFMFLCVFYLSWTSVGA</font><font color = red>EI</font>----------------------------HSAVLDKQTGQLSLEEGFRDDFVAWANFTDDIKNSG
<font color = brown>>PLBD2_danRer MAHLQLLVSAVCVLLSVCQA</font><font color = red>QI</font>---------------------------------------YSAIYEEETAQLLLIEGARTHSVAEANFTDHINTTG
<font color = #9999FF>>PLBD2_calMil MCVGVRGQGLGLGLPLLLVLAAVGVSPSARG</font><font color = red>HL</font>---------------------------LRSVVLDEHSGRLRVVGGLNPHSIAWANLTDRIRATG
<font color = #9999FF>>PLBD2_braFlo MAACRNIFCGRMLSCLLLFSFVFS</font><font color = red>AV</font>-----------------------------SDGSKLASVRYDEAAKTYQITDKLDPSAAAWANFTDRISSTG
<font color = #9999FF>>PLBD2_acyPis MLSIRCILLSLLFVWALQCSAT</font><font color = red>QK</font>------------------------------NQTLLAVKTDNNRITIQPKHYSVKDKEIIIGKGKFIDRINSTG
<font color = #9999FF>>PLBD2_triAdh MAQCGKFLIYFSIFIITLATLCSC</font><font color = red>QS</font>-------------------------------------GSVIYKDGLYTFSKGINKRAASYGTFTDKIASSG
The two paralogs do not align at all in the signal region and only poorly thereafter:
PLBD2_homSap:    67 DVSAGQLLMVDGRHPDAVAWANLTNAIRETGWAFLEL--GTSGQ-YNDSLQAYAAGVVEAAVSEE 128
                          Q+  V  ++ DA  + N  N+++ TGW  LE+  G  Q  ++ +  + AG +E  ++ 
PLBD1_homSap:   50 AEKTVQVKNVMDKNGDAYGFYN--NSVKTTGWGILEIRAGYGSQTLSNEIIMFVAGFLEGYLTAP 112
   
   
  Q54P   [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs7965471  rs7965471]
  Phylogenetic variation signal peptide location in first two exons of PLBD1:
A204V  [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs12231990 rs12231990]
              <------ signal peptide --------->      <-------------------------- second exon ---------------------------------->
  R354C [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs56935204  rs56935204]
<font color = purple>>PLBD1_homSap MTRGGPGGRPGLPQPPPLLLLLLLLPLLLVTAE<font color = red>PP</font>KPA:GVYYATAYWMPAEKTVQVKN-VMDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQTLSNEIIMFVAGFLEGYLTAP
  Q521K [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs17852787 rs17852787]
>PLBD1_panTro MTRGGPGGRPGLPQPPPLLLLLLLLPLLLVTAE<font color = red>PP</font>KPA:GVYYATAYWMPAEKTVQVKN-VMDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQTLSNEIIMFVAGFLEGYLTAP
  R524C [http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs12425042 rs12425042]
>PLBD1_ponAbe MTRGGPGGRPGLPPPPPLLLLLLLPPLLLVAAE<font color = red>PA</font>NSA:GVYYATAYWMPTEKTVQVKN-VMDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQALSNEIIMFVAGFLEGYLTAP
>PLBD1_rheMac MTRGGPGGCPGLPPPLPLLLRLLLPPLLLVTAE<font color = red>SP</font>NPA:GVYYATAYWMPAEMTVEVKN-IMDKNGDAYGFYNNSVETTGWGILEIRAGYGSQALSNEIIMFVAGFLEGYLTAP
>PLBD1_papHam MTRGGPGGCPGLPPQLPLLLRLLLPPLLLVTAE<font color = red>SP</font>NPA:GVYYATAYWMPAEMTVEVKN-IMDKNGDAYGFYNNSVETTGWGILEIRAGYGSQALSNEIIMFVAGFLEGYLTAP
>PLBD1_calJac MTRGGPGGRLGLPPPPLLLLLLLLLPPLPTTAE<font color = red>PP</font>TPA:GISYATAYWMPAEKTVQVKN-VMDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQALSNEIIMFVAGFLEGYLTAL
>PLBD1_otoGar MANRTLDRRLGLPPPPLLLLLLLPPPPLLVTA<font color = red>AR</font>KNPP:GVYYATAYWKPAEKTVEVKK-VIDKNGDAYGFYNNSMNATGWGILEIRAGYGSQALSNEMTMFVAGVLEGYLTAP
>PLBD1_musMus MCHRSPGRSLRPPSPLLLLLPLLLQPP-WAAA<font color = red>LP</font>ASPT:GVHCATAYWSPESKKVEIKT-VLDKNGDAYGYYNDSIKTTGWGILEIRAGYGSQVLSNEIIMFLAGYLEGYLTAL
>PLBD1_ratNor MCHRSHGRSLRPPSPLLLLLPLLLQSP-WAA<font color = red>AP</font>LRSSA:GVHYATAYWLPDTKAVEIKM-VLDKKGDAYGFYNDSIQTTGWGVLEIKAGYGSQILSNEIIMFLAGYLEGYLTAL
>PLBD1_cavPor MALCGPGCSPGLPPSPLLLLPLLL----LAAA<font color = red>WS</font>PSPP:GIHYATAYWIPDTKTVEVKD-ILDKDGDAYGYYNNSMEATGWGILEIKAGYGSQELTNEIIMFVAGFLEGYLTAL
>PLBD1_speTri MSRRSLGCGRW-PPPPLQLLPLLLLLLPLAAA<font color = red>QP</font>----:EVYYATAYWIPSEKSIKVKH-VMDKSGDAYGYYNDSMETTGWSILEIRAGYGSQALSNEIIMFVAGFLEGYLTAP
>PLBD1_oryCun MALWLPPLLFPLL---------------LAAA<font color = red>EP</font>PSPE:GVSYATAYWMDAEKKVQVRN-VLDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQALSNEIIMFVAGFLEGYLTAP
>PLBD1_turTru MSRRSPDGSLGLLSPPALLLLLL------AAVVPSGLA:<font color = red>GV</font>YYATAYWMPTEKRIQVQN-VLDRNGDAYGFYNNSVKTTGWGILEIRAGYGSRSLSNEIVMFAAGFLEGYLTAP
>PLBD1_bosTau MSRHSQDERLGLPQPPALLPLLLLL----AVAVPLSQA:<font color = red>GV</font>YYATAYWMPTEKTIQVKN-VLDRKGDAYGFYNNSVKTTGWGILEIKAGYGSQSLSNEIIMFAAGFLEGYLTAP
>PLBD1_oviAri MPRHRRDERLGLPPPPARLPLLLLLL---AAAVPLSQA:<font color = red>GV</font>YYATAYWMPTEKRIQVKN-VLDRKGDAYGFYNNSVKTTGWGILEIKAGYGSQSLSNEIIMFAAGFLEGYLTAP
>PLBD1_susScr MSRRSRDGRLGLPAPPAPL-LLLLLL---AAAVPPSLA:<font color = red>GV</font>YYATAYWMPTEKRMLVKN-VLDRNGDAYGFYNDSMKTTGWGILEIRAGYGSQSLSNNIIMFAAGYLEGYLTAP
>PLBD1_equCab MARHRPDGRLGLPAPPAPPLPPLLLLLLV-AAVSPSQA:<font color = red>VV</font>YSATAYWMPAEKTVQVKN-VMDRNGDAYGFYNNSVKTTGWGILEIRAGYGSQTLSNDITMFVAGFLEGYLTAL
>PLBD1_felCat MARRSRDGRPGLSAPPTPPLLPLLLL---AAAVSPSLA:<font color = red>EV</font>HYATVYWMPAEKTIQVKN-VLDRNGDAYGFYNDSVKTTGWGVLEIRAGYGSQALSNEIIMFVAGFLEGYLTAP
>PLBD1_canFam MPRRARDARLEPCPPLLPLLLLLL-----AAAVPQGRA:<font color = red>EV</font>YYATAYWIPDEKTIQVKN-VLDRNGDAYGFYNDSVKTTGWGILEIRAGYGSQILSNEITMFVAGFLEGYLTAP
>PLBD1_pteVam MSRRSLDGRLGLPATSAPPLLLLLLL---AAAVPPSLA:<font color = red>ev</font>yYATAYWMPAEKTVNVKN-LLDKNGDAYGFYNNSMNTTGWGILEIKAGYGSQTLSNDIIMFVAGYLEGYLTAP
>PLBD1_eriEur MSRRSRDGRLGLLLSPPLLLLLLLL-----AAAPPSLQ:<font color = red>EI</font>YYATAYWMPEEEEIQVKN-VLDKNGDAYGFYNDSMLTTGWGILEIKAGYGSHQLSNDVVMFVAGFLEGYLTAP
>PLBD1_sorAra MARGGGDGPPALLPLPLLSLLLALL----AAAVPPSLA:<font color = red>EV</font>HYATAYWMPDEQRVEIKT-TLDKKGDAYGYYNDSVLTTGWGILEIRAGYGSQDLTDEITMFVAGALEGYLTAP
>PLBD1_loxAfr MSSRSRGRHHGPAPQLPQLLLLLLLLLLVAAAAPPSLA:<font color = red>EV</font>HYATVYWMSSEKTMQVKD-VLDKKGDAYGYYNDSVLTTGWGVLEIKAGYGSQALSNDIIMFAAGYLEGYLTAL
>PLBD1_proCap MCSRSV--PCRLSPPLSPPLSLPLLLLLLAAAAPPSLA:<font color = red>EV</font>HYATVYWMSSEKTMQVKD-TLDKNGDAYGFYNDSMQTTGWGVLEIKAGYGSQGLSNDVIMYAAGYLEGYLTAp
>PLBD1_echTel MSTHSRGGR--PAPPLSPSLSLTPLLLL-AALVAPSLA:<font color = red>EI</font>HYATAYWMSSEKTIQIKD-VLDKSGDAYGFYNDSVNATGWGILEIRAGYGSQNLSNDIIMFAAGFLEGYLTAP
>PLBD1_choHof MSRSCQAERLGPVPRRRLLLLLL-----VASAAPPSVA:<font color = red>EV</font>FYATAYWIPSEKKIVVKD-ILDQNGDAYGFYNDSMKTTGWGILEIKAGYGSHIPSNEIIMFTAGFLEGYLTAE</font>
<font color = green>>PLBD1_triVul MSRRSRDGRLGLPAPPAPLLLLLLL----AAAVPPSLA:<font color = red>GV</font>YYATAYWMPTEKRMLVKN-VLDRNGDAYGFYNDSMKTTGWGILEIRAGYGSQSLSNNIIMFAAGYLEGYLTAP
>PLBD1_monDom MTRFSCFGRLQLW--PLQVLLLLLL----TFGAPVTQA:<font color = red>GI</font>HYATVYWNSSTSSAEVKD-SLDPDGDAYGFYNDTIQTTGWGILEIRAGYGANSLTDEIIMFVAGFLEGYLTAQ
>PLBD1_ornAna MSRTCRGGRSGPPQPAPTPAGLLLLLL--TVASPLLQS:<font color = red>HV</font>RYATAYWESATQTVRVKD-VLDWDGDAYGFYNHTVQTTGWGTLEIRAGYGAQALSDEVVMFVAGFLEGYLTAP</font>
<font color = blue>>PLBD1_taeGut MARAGGGVCRCCCWALVLLWAAAGGRA-----------:<font color = red>EL</font>RYATVYWNRAEKILQVKN-TLDRSGDAYGFYNNSLQTTGWGVLEIRAGYGSQTLSNEDIMYVAGFLEGYLTAP
>PLBD1_galGal MARLGGGALCCCWGLVLLWAVAGGRA------------:<font color = red>EM</font>RYATLYWNKAQKILQVKN-ILDRSGDAYGFYNNTVQTTGWGVLEIKAGYGHQTLSNEDIMYAAGFLEGYLTAP
>PLBD1_melGal MARLGGGPLCCCWGLVLLWAVAGGRA------------:<font color = red>EM</font>RYATLYWNKAQKILQVKN-ILDRSGDAYGFYNNTVQKTGWGVLEIKAGYGHQTLSNEDIMYAAGFLEGYLTAP
>PLBD1_sisCat MIRFGNPSSSDTRRQRCRSWYWGGLLLLWAVAETRA--:<font color = red>DI</font>HYATVYWLEAEKSFQIKD-VLDKNGDAYGYYNDTIQSTGWGILEIKAGYGNQPISNEILMYAAGFLEGYLTAS
>PLBD1_ambMex MGGLRQLLPLCALLLLQPLGAR----------------:A<font color = red>IR</font>YATVYWTD-RKTVLVKE-VLDKGGDAYGFYNDTIQSTGWGVLEIRAGYAPTSRTNEEIMFAAGYLEGYLTAL</font>
<font color = brown>>PLBD1_takRub MFLLTSTCAFVLLTLPATSST<font color = red>AD</font>G--------------:GTAAATVYWDPQHKTVLLKEGVLEQEGDAYGYFNDTLSSTGWSVLEIRAGYGTTPETDEVIFFLAGYLEGFLTAQ
>PLBD1_danRer MPDFSFCVLFLIGFLFSSRS<font color = red>D-----------------:K</font>LK-ATVYWDATHKSAVLKQGVLDPAGASYGYYDNVLLSTGWGVLEVRAGYGDTTQTDDITMFTAGYLEGFLTAP
>PLBD1_ictPun MTEFMVCVCMFLCAVIAVRT<font color = red>DS</font>----------------:VHK-ATAYWDPDSKTVLLKDGVLEDTGDAYGFYNDSFSETGWGVMEVRAGYGQTPRADERTFFLAGYLEGFLTAR
>PLBD1_perFla MEKQSIKLCVLLSTLAASVQT<font color = red>Y----------------:Q</font>LQEATVYWDGAQKSVILKEGVMETEGGAYGYFNDTLLLSGWGVLEICAGHGGITQEDETTFFLAGYLEGYLTAG
>PLBD1_gasAcu MFLEKTLYVLLLCSVSTTSSA<font color = red>D----------------:K</font>MTAATVYWDPQHKVVLLKEGVLEKEGDAYGYLNDTLSSTGWSVLEIRAGYGETPETDEVTFFLAGYLEGFLTAQ
>PLBD1_oryLat MKLEVFLLLHVIATFASS<font color = red>Q-------------------:K</font>LTAATVYWDAQHKLVLLKEGVLETEGDAYGYLNNTLSTSGWSILEIRAGYGKTPEDDEITFFLAGYLEGFLTAQ
>PLBD1_pimPro MDTNSICVLLLLCSVSTTSSA<font color = red>D----------------:K</font>MTAATVYWDPQHKVVLLKEGVLEKEGDAYGYLNDTLSSTGWSVLEIRAGYGETPETDEVTFFLAGYLEGFLTAQ
>PLBD1_dicLab MPLVTRLYVFLLFTVVTSFASA<font color = red>D---------------:K</font>MTAATVYWDPLHKLVKLKEGVLETEGDAYGYLNDTLSSSGWSILEIRAGYGKTPETDELTFFLAGYLEGYLTAQ
>PLBD1_salSal MKRVCLLFFFYVAASFASA<font color = red>D------------------:E</font>MKAATVYWDATHKTVQLKEGVIEKEGDAYGYLNDTLSQTGWSVLEIRAGYGETLEHDEVTYFLAGYLEGFLTAP</font>
Difference alignment of exon 1 from placental mammals:
<font color = blue>PLBD2_homSap  MVGQMYCYPGSHLARALTRALALALVLALLVGPFLSGLAGAIPAPGGRWARDGQVPPASRSRSVLLDVSAGQLLMVDGRHPDAVAWANLTNAIRETG
PLBD2_panTro  .......S.............................................P...........................................
PLBD2_ponAbe  ......GSS.....----...................................P.T...........A......L......................
PLBD2_rheMac  .......SS..P....................L.................H..P.T..........HAAT....L....Q...........S.H...
PLBD2_papHam  .......SS..P....................L.................H..P.T...........AAT....L......................
PLBD2_calJac  ...K..SS.S.R..Q...............A.L.....S..............S..SG.G....V..AA.....L..................H...
PLBD2_otoGar  ...P..GS..GR..................I.L...C......P..SGR....LIT.....S.....ATTD..RL..............S...H...</font>
<font color = brown>PLBD2_musMus  .AAPVDGSS.GWA....R.....TSL..S.T.LL...P...L.TL.PG.Q.QNPD..V..T..L...AAS...RLE..F..................
PLBD2_ratNor  .AAP.DRTH.GRA....R.....----.S.A.LL.......L.TL.PG.R.QNPE.....T..L...AAS...RLEY.F..................
PLBD2_dipOrd  .AAPP.GSR.GRP.GS.S...V.----.V...LSP..P...V.S..D..G.HKPE..V.......V.AAS...RL...L..G...............
PLBD2_cavPor  .AAPT.VSLDGRPV..--......PA.C....LS.GR....V....P.G....P..A.--C......AAS...RL...LQ.G...........P...
PLBD2_oryCun  ..APRDGCA.GR.....A--------....T.LL.G.....A.....GEQ..PPS....CC..A...AAT...RL..................H...
PLBD2_ochPri  .AATRDSSA.CR...V.......---...PT.L....P.....VR.DGEE.GRPA.SG..C....V.AES...RL......A...........H...</font>
<font color = green>PLBD2_turTru  ..DP..GC..GR....................L.....T....T.R.HRGPGRP......C......PET...RL......................
PLBD2_bosTau  ..AP..GS..GR....V...............L.....T....T.R.QRG.GMP......C..L...PET...RL......................
PLBD2_oviAri  ..AP..GS..GR....V...............L.....T....T.R.QRG.GMP......C..L...PET...SL......................
PLBD2_susScr  ..AP..GS..GR....................L.....TS...T.K.YRGSGRS.............TET...RL..................H.N.
PLBD2_ursAme  .AAP..GS..GR....................L.....T....IS.RQ.GPN.P...D.........AET...RL......E...............
PLBD2_myoLuc  ..APPSRS..GR.TP..S..P...PG....A.L....WT....T.RDP.GPN.P..........V..ART...QL....Q.............H...
PLBD2_pteVam  ..AP.DRS..GR..G....T.E.T....P.A.L....RTS..QT..S..GSE.P.S...........PQT...RL..................H...
PLBD2_eriEur  ..AP.CGS..GRP...........PA......L...S......P.EDN.G.N.SF..V..C......SET...RL..............S...H...</font>
<font color = purple>PLBD2_loxAfr  ..APV.GS..GR......Q...V.........L.....T...SLT.H..GP..PA............TAT...RL......................
PLBD2_echTel  ..ATE.GS..GR........P....M......L.....T...SPA...REPN.R.....S...A...PAT...RLA.....E...........H...</font>
<font color = darkorange>Consensus      MVAPMYGSPGGRLARALTRALALALVLALLVGLFLSGLAGAIPaPGGRWGRDGPVPPASRSRSVLLDAATGQLRLVDGRHPDAVAWANLTNAIRETG</font>
 
Alignment of first two exons of PLBD1 from vertebrates showing onset of conservation:
 
[[Image:PLBD1exon12.jpg]]
<br clear="all">
 
== Understanding conserved residues in PLBD1 and PLBD2 ==
 
Although the gene duplication creating these paralogs took place in early unicellular eukaryotes with PLBD1 quite diverged in primary sequence from PLBD2 today, it is nonetheless instructive to compare individual residues and residue patches that are still conserved, given the folds have diverged rather little. Here we wish to exploit the situation that more is known about the maturation and substrates of PLBD1 whereas excellent crystallographic structures exist for PLBD2 and certain of its ancient homologs.
 
Localization of conserved residues within compared secondary structures: s = beta sheet, h = alpha helix
38    PTGVHCATAYWSPESKKVEIKTVLDKNGDAYGYYNDSIKTTGWGILEIRAGYGSQVLSNEIIMFLAGYLEGYLTALHMYDHFTNLYPQLIKN----PSIV PLBD1         
61    PPVSRTRSLLLDAASGQLRLEDGFHPDAVAWANLTNAIRETGWAYLDLST---NGRYNDSLQAYAAGVVEASVSEELIYMHWMNTVVNYCGPFEYEVGYC PLBD2         
        ssssssssss    sssssss    ssssssssss  ssssssssss      hhhhhhhhhhhhhh  hhhhhhhhh              hh         
        ssssssssss    sssssss    ssssssssss  sssssssss  s    hhhhhhhhhhhhhh  hhhhhhhhhh          hhhh         
 
134  KKVQDFMEKQEMWTRQNIKAQKDDPFWRHTGYVVTQLDGLYLGAQKRASEE-KIKPMTMFQIQFLNAVGDLLDLIPSLSPTKSSSMMKFKIWEMGHCSAL PLBD1         
158  EKLKNFLEANLEWMQREMELNPDSPYWHQVRLTLLQLKGLEDSYEGRLTFPTGRFTIKPLGFLLLQISGDLEDLEPALNKTN----------GSGSCSAL PLBD2         
      hhhhhhhhhhhhhhhhhhhh    hhhhhhhhhhhhhhhhhhhhh                      hhhhhhhhh                    sss         
      hhhhhhhhhhhhhhhhhhhh    hhhhhhhhhhhhhhhhhhhhh                      hhhhhhhhh                    sss         
 
233  IKVLPGFENIYFAHSSWYTYAAMLRIYKHWDFNIKD------KYTLSKRLSFSSYPGFLESLDDFYILSSGLILLQTTNSVYNKTLLKQVVPK-TLLAWQ PLBD1         
253  IKLLPGGHDLLVAHNTWNSYQNMLRIIKKYRLQFREGPQEEYPLVAGNNLVFSSYPGTIFSGDDFYILGSGLVTLETTIGNKNPALWKYVQPQGCVLEWI PLBD2         
      sssss  sssssssssssss    ssssssss              sssssss          ssss sssssssss    hhhh          hh         
      sssss  sssssssssssss    ssssssss sss      sss  sssssss          ssss sssssssss    hhhh          hh         
 
326  RVRVANMMAEGGKEWAQIFSKHNSGTYNNQYMVLDLKKVTINRSL-DKGTLYIVEQIPTYVEYSDQTNV-LRKGYWASYNIPFHKTIYNWSGYPLLVHKL PLBD1         
353  RNVVANRLALDGATWADVFKRFNSGTYNNQWMIVDYKAFLPNGPSPGSRVLTILEQIPGMVVVADKTAELYKTTYWASYNIPYFETVFNASGLQALVAQY PLBD2         
      hhhhhhhh  hhhhhhhh        sssssssss            ssssssss  sssssss hh    sssss      hhhhhh  hhhhhhh         
      hhhhhhhh  hhhhhhhh        sssssssss            ssssssss  ssssssshhhhhhhsssss      hhhhhh  hhhhhh           
 
424  GLDYSYDLAPRAKIFRRDQGNVTDMASMKYIMRYNNYKEDPYSKGDPC-------STICCREDLNGAS---------PSPGGCYDTKVADIFLASQYKAYAISGPTVQDGLPPFNWNRF--NETLHRGMPEVFDFNFVTMK -         
453  GDWFSYTKNPRAKIFQRDQSLVEDMDAMVRLMRYNDFLHDPLSLCEACNPKPNAENAISARSDLNPANGSYPFQALHQRAHGGIDVKVTSFTLAKYMSMLAASGPTW-DQCPPFQWSKSPFHSMLHMGQPDLWMFSPIRVPWD
                hhhhhhh        hhhhhhhhh                          sss                ssssssssss hhhhh  ssssss        sss              sss    sss         
                hhhhhhhh      hhhhhhhhh        sss    sss      sss                ssssssssss hhhhh  ssssss        sss              sss    sss
 
=== Shared conserved residues: defining specializations of phospholipases ===
 
Suppose a phylogenetically broad set of curated PLBD1 and PLBD2 are aligned together. After careful consideration of gap placement, a restricted number of residues will prove very deeply conserved in both proteins throughout eukaryotes. Of these, some are universal localizational, modificational, structural, or catalytic features basic to the entire NTN clan of 12 protein families and so not particular to phospholipases.
 
This class of residues must be found by structural alignment of crystallographic structures, as primary sequences are too diverged for these to be accurately located by ClustalW or similar tools. Since the fold of PLBD2 was originally recognized by the fold comparison (via Dali) to all of PDB, these are known already: the autocatalytic cysteine residue at the N-terminus of the 40 kDa fragment and the three active site residues noted above.
 
An additional 6%-14% of structurally equivalent amino acids (themselves only half of the chain) are identical as amino acids, with IMPC (inosine monophosphate cyclohydrolase) being the highest but PVA (penicillin acylase V) and CBAH (conjugated bile acid hydrolase) also significant. Dali provides a primary sequence multiple alignment and so the super-invariant amino acids (plus those with narrow reduced alphabets, while eliminating accidental matches) not specific to phospholipases PLBD1 and PLBD2. These largely lie within the beta strands of the core αββα sandwich as they are better conserved than alpha helices within NTN hydralases but it is fair to say that this fold class is not understood until an explanation can be given for each of the universally conserved residues.
 
1oqz CA  cephalosporin acylase
3pva PVA  penicillin V acylase
1k5s PGA  penicillin G acylase
2bjf CBAH choloylglycine hydrolase
2ntm IMP  cyclohydrolase
1ryp ---  a chain among 28 of yeast proteasome
Dali report on 3fgr vs 1oqzB: AyaA is a candidate super-invariant region but only second A occurs in both PLBD1 and PLBD2
DSSP  leeeeeeeeelllleeeeeelllllLLEEEEEEEEhhhhleEEEEEEELllllhhhHHHHH
Query vsrtrsllldaasgqlrledgfhpdAVAWANLTNAiretgwAYLDLSTNgryndslQAYAA  61
ident                                                          A  A
Sbjct ---------------pqapiaaykpRSNEILWDGY------GVPHIYGV-------DAPSA  33
DSSP  ---------------llllllllllLLLEEEEELL------LLEEEELL-------LHHHH
DSSP  HHHHHHHHHHHHHHHHHhhLLLL--LLLL------LLLH-HHHH--HHHHHHHHHHHHH
Query GVVEASVSEELIYMHWMntVVNY--CGPF------EYEV-GYCE--KLKNFLEANLEWM  109
ident                                                           
Sbjct FYGYGWAQARSHGDNIL-rLYGEarGKGAeywgpdYEQTtVWLLtnGVPERAQQWYAQQ   91
DSSP  HHHHHHHHHHHHHHHHH-hHHHHhlLLHHhhhlhhHHHHhHHHHhlLHHHHHHHHHHLL
DSSP  hhhhhhllllhhhhhHHHHHHHHHHHHHHHHL---------llllllllllLLLLlLHHHHL-HHHH-HHHHhhlLLL
Query qremelnpdspywhqVRLTLLQLKGLEDSYEG---------rltfptgrftIKPLgFLLLQI-SGDL-EDLEpalNKT
ident                      L                                   
Sbjct ---------------SPDFRANLDAFAAGINAyaqqnpddispdvrqvlpvSGAD-VVAHAHrLMNFlYVAS---PGR
DSSP  ---------------LHHHHHHHHHHHHHHHHhhhhlhhhllhhhhlllllLHHH-HHHHAHrLMNFlYVAS---PGR
 
In effect, these residues must be 'subtracted off' the larger set of phylogenetically invariant residues between PLBD1 and PLBD2 to reach conserved residues defining what is specifically important to phospholipases. These sites developed subsequent to the divergence of phospholipases from generic NTN hydralases but prior to the gene duplication giving rise to PLBD1 and PLBD2 because convergent evolution later is implausible. They are evidently critical to structure/function, being retained in both paralogs in all surviving species up until the present day. Several active site residues fall into this category as described earlier.
 
=== Conserved residues determining mannosylation ===
 
PLBD1 and PLBD2 are among the 2500 human proteins carrying signal peptides targeting them initially to the endoplasmic reticulum and subsequently to the golgi, where they are sorted and packaged according to final destination (lysosome, plasma membrane, extracellular secretion). Many of these proteins are initially glycosylated via a [http://themedicalbiochemistrypage.org/glycoproteins.html#lysosomes limited repertoire] but few of the soluble members are ultimately targeted to the lysozome (about 50 of 2500).
 
These receive an additional post-translational modification in the golgi of terminal mannose residues to mannose 6-phosphate making them recognizable to the two lysosomal mannose receptors ([http://en.wikipedia.org/wiki/Insulin-like_growth_factor_2_receptor IGF2R] and [http://en.wikipedia.org/wiki/Cation-dependent_mannose-6-phosphate_receptor M6PR]). Bizarrely, this simple phosphorylation is not accomplished by a kinase but rather requires 3 separate gene products, GNPTAB (UDP GlcNAc-1-phosphotransferase, internally cleaved into α,β catalytic and recognition subunits), GNPTG (γ regulatory subunit of the α2β2γ2 hexamer) and NAGPA (N-acetylglucosaminidase, or uncovering enzyme).
 
Overall, trafficking is very imperfect, with some protein molecules [eluding the uncovering enzyme yet http://www.ncbi.nlm.nih.gov/pubmed/20615935 still ending up in the lysosome] and while others even with M6P markers are improperly secreted.
 
An alternate pathway exists: β-glucocerebrosidase (of Gaucher disease) localizes to the lysozyme via  the coiled coil of the[http://www.cell.com/abstract/S0092-8674%2807%2901290-1 LIMP2] protein encoded by SCARB2.) This presumably continues despite mutations (inclusion-cell disease) in GNPTAB, GNPTG, NAGPA, IGF2R and M6PR that disable the main pathway. Conversely, mutations in SCARB2 (causing action myoclonus renal failure syndrome) do not appear to affect proteins using the M6P lysosomal trafficking route.  The SORT1 gene product sortilin may also let proteins [http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf sidestep the M6P pathway] to the lysosome.
 
PLBD1 and PLBD2 likely [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2739207/?tool=pubmed use the conventional M6P pathway], nonetheless raising difficult bioinformatic issues. The 50 proteins using it have unrelated folds. They share the generic glycosylation NxT/S sequence motif but this is completely insufficient to differentiate them from proteins not targeted to the lysozome. There is no support for a larger motif enveloping NxT/S or a supplementary contiguous motif elsewhere.
 
Recognition must reside within individual GNPTAB/GNPTG/NAGPA subunits but their [http://www.ncbi.nlm.nih.gov/pubmed/19955174 roles] are complex, providing no clear bioinformatic handle, only two separated lysines forming a critical triad with M6P at 34 angstrom distance.  
 
However in homologous lysosomal sulfatases ARSA and ARSB, the two identified lysines did not occupy homologous positions, perhaps because sulfatases duplicated and diverged prior to the evolutionary appearance of the lysosome.
 
This then illustrates convergent evolution rather than conservation of homology. This applies too to the many signal peptides and NxT/S motifs that must have arisen independently a great many times (as no such mobile elements could have provided them).
 
PLBD2 targeting is then fairly complex from a predictive standpoint as its 40 lysines and 6 M6P would yield a great many candidate pairs, depending on the softness of the 34 angstrom requirement. As PLBD1 and PLBD2 may have diverged later than sulfatases (ie, after recruitment to the lysosome), the lysine pairs might still be homologous. Looking at human gene alignment, this still gives an unwieldy set of 7 homologous pairs (some poor quality because of gap uncertainty).
 
However these lysines need to be invariant within phylogenetically dispersed PLBD2 and PLBD1 orthologs. Under that assumption, only 3 of the lysines are strong candidates (red), 1 is so-so (magenta) and the rest are implausible (blue). The lysine in CSALIK may be part of the autocleavage motif, leaving two candidates to be compared to the six M6P sites for the required 34 angstrom geometry.
 
PLBD2:  189 LKGLEDSYEGRVSFPAGK-FTIKPLGFLLLQLSGDLEDLELALNKTK---IK--PSLGSG 242
              + GL    + R      K  T+  + FL    GDL DL  +L+ T<font color = blue>K</font>  +<font color = blue>K</font>      G
PLBD1:  169 IDGLYVGAKKRAILEGTKPMTLFQIQFL--NSVGDLLDLIPSLSPTKNGSLKVFKRWDMG 226
PLBD2:  243 SCSALIKLLPGQSDLLVAHNTWNNYQHMLRVIKKYWLQFREGPWGDYPLVPGNKLVFSSY 302
              CSALI<font color = red>K</font>+LPG  ++L AH++W  Y  MLR+ <font color = red>K</font> +W  F      D      ++L FSSY
PLBD1:  227 HCSALIKVLPGFENILFAHSSWYTYAAMLRIYK-HW-DFNVID-KD---TSSSRLSFSSY 280
PLBD2:  303 PGTIFSCDDFYILGSGLVTLETTIGNKNPALWKYVRPRGCVLEWVRNIVANRLASDGATW 362
              PG + S DDFYIL SGL+ L+TT    N  L <font color = blue>K</font> V P  +L W R  VAN +A  G  W
PLBD1:  281 PGFLESLDDFYILSSGLILLQTTNSVFNKTLLKQVIPE-TLLSWQRVRVANMMADSGKRW 339
PLBD2:  363 ADIFKRFNSGTYNNQWMIVDYKAFIPGGPSPGSRVLTILEQIPGMVVVADKTSELYQKTY 422
              ADIF ++NSGTYNNQ+M++D <font color = magenta>K</font>  +    S    L I+EQIP  V  +++T  L +<font color = blue>K</font> Y
PLBD1:  340 ADIFSKYNSGTYNNQYMVLDLKK-VKLNHSLDKGTLYIVEQIPTYVEYSEQTDVL-RKGY 397
PLBD2:  483 DFLHDPLSLCKACNPQPNGENAISARSDLNPANGSYPFQALRQRSHGGIDVKVTSMSLAR 542
              ++  DP S    CN        I  R DLN  N S P        G  D <font color = red>K</font>V  + LA
PLBD1:  458 NYKKDPYSRGDPCN-------TICCREDLNSPNPS-P--------GGCYDTKVADIYLAS 501
 
The phylogenetic emergence of these features could time the onset of targeting to the lysozyme during the evolutionary history of phospholipases PLBD1 and PLBD2. Mutations localizing at M6P receptor targeting sites would lead to non-recognition, non-arrival, and perhaps lysosomal storage disease (as the proteins would then be secreted).
 
The [http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0854.2010.01044.x/full evolution of the lysozyme itself], preposterously claimed a vertebrate innovation as late as 2006, instead uses components that readily trace back to slime mold. These surface as the eight loci responsible for flawed lysosome related organelle biogenesis (Hermansky-Pudlak syndrome).
 
=== Ragged carboxy terminus in PLBD1 ===
 
The deuterostome orthologs of PLBD1 display an unusual pattern of extensions and contractions of the carboxy terminus. These do not correspond to subclades, implying numerous separate events affecting the stop codon in fairly recent times (which is supported by read-through past the stop codon, lower case). The precise location of the stop codon may not be at all important but conservation does continue on to the proline near the end (which is strongly invariant. Proline is a common helix terminating residue but here the structural model suggests it is terminating a beta strand.
 
PLBD2 is much more orderly at its carboxy terminus, though some extensions are seen in early deuterostomes. Again an almost-terminal proline is conserved. However past it, a tryptophan is also universally conserved. The xray structure fortunately includes both residues -- the goal for every protein is to provide an explanation for each conserved residue (for rational interpretation of possible disease SNPs) though here the focus initially is on activation and catalysis.
 
PLBD1_homSap VADIYLASQYTSYAISGPTVQGGLPVFRWDRFNKTLHQGMPEVYNFDFITMKPILKLDIK*
PLBD1_panTro VADIYLASQYTSYAISGPTVQGGLPVFRWDRFNKTLHQGMPEVYNFDFITMKPILKLDIK*
PLBD1_ponAbe VADIYLASQYTSYAVSGPTVQGGLPVFRWDRFNKTLHQGMPEVYNFDFITMKPILKLDIK*
PLBD1_rheMac VADIYLASQYTSYAISGPTVQGGLPVFHWNRFNKTLHQGMPEVYNFDFITMKPILKRDMK*
PLBD1_papHam VADIYLASQYTSYAISGPTVQGGLPVFHWNRFNKTLHQGMPEVYNFDFITMKPILKRDMK*
PLBD1_calJac VSDIYLASQYTSYAISGPTVQGGLPVFRWNRFNKTLHQGMPEVYNFDFITTKPILK*hkmk
PLBD1_tarSyr VADIYLASQYTAYAISGPTVQDGLPVFHWNRFNKTLHQGMPEVYNFDFVTMKPILKLDIK*
PLBD1_micMur VsDIFPASQFTGHAINGPTVPSGlPVFYRPPFNKTPHQGIAEAYHFDFISKKPILKPDIK*
PLBD1_otoGar VADIYLASQYTAYAISGPTVQGGLPVFHWHRFNKTLHHGMPEAYNFDFITMKPVLKLDIK*
PLBD1_tupBel VADIYLASQYTAYAISGPTVQDGLPVFHWNRFNRTVHQGMPEAYNFDFITMKPVLKLDIK*
PLBD1_musMus VADIFLASQYKAYAISGPTVQDGLPPFNWNRFNDTLHRGMPEVFDFNFVTMKPILS*dkk*
PLBD1_ratNor VADIFLASQYKAYAISGPTVQNGLPPFNWNRFNDTLHQGMPDVFDFDFVTMKPILT*dkn*
PLBD1_perMan VADIFLAFQYTAYAISGPTVQDGLPAFDWKHFNKTLHEGMPDVFNFDFVTMKPILTEDIK*
PLBD1_dipOrd VSDIFLASKYIAYAISGPTVQDGLPAFSWRLFNKTLHQGMPEIYNFDFVLMKPFFND*qk
PLBD1_cavPor VADIHLASEYTAYAISGPTVQGGLPVFRWNRFNDTLHQGMPEVYNFDFITMKPILKPNVKRRRKMRE*
PLBD1_speTri VSDIYLASQYTAYAISGPTVQGGLPVFRWNRFNTTLHQGMPEAYNFDFITMKPVLKIDIK*
PLBD1_oryCun VSDIYLASRYTAYAISGPTVQGGLPVFHWNRFNKTLHQGMPEVYNFDFITTKPILKLDKR*
PLBD1_ochPri VSDVHLASQYTAYAISGPTVQGKLPVFHWSQFNKTLHQGMPDAYNFDFITMKPILKKMREDEAEGNRMK*
PLBD1_vicPac VADIYLASQSTAHAISGPTAEDGLPVFHWNRFNKTLHSGMPEVYNFDFITMKPIL*ldik*
PLBD1_turTru VADIHLASAYTAYAISGPTVQGGLPVFHWSRFNKTLHEGMPEAYNFDFITMKPIL*ldmk
PLBD1_bosTau VADIYLASKYKAYAISGPTVQGGLPVFHWSRFNKTLHEGMPEAYNFDFITMKPIL*ldik
PLBD1_oviAri VADIYLASKYKAYAISGPTVQGGLPVFHWSRFNKTLHEGLPEAYNFDFITMKPIL*ldik
PLBD1_lamPac VADIYLASQSTAHAISGPTAEDGLPVFHWNRFNKTLHSGMPEVYNFDFITMKPIL*LDIK
PLBD1_susScr VADIHLASTYTAYAISGPTVQDGLPVFHWNHFNKTLHEGMPEAYNFDFITMKPTL*LD
PLBD1_equCab VADIYLASKYTAYAISGPTVQGGLPVFHWNRFNKTLHEGMPEAYNFDFITMKPILKPYVKGRR*
PLBD1_ailMel VADIYLASEYTAYAISGPTVQGGLPIFHWNRFNKTLHKGMPETYDFDFITMKPILKRDKK
PLBD1_felCat VADIYLASAYTAHAISGPTVQDGLPVFHWNRFNKTLHQGMPETYNFDFIIMKPILKQDIK*
PLBD1_canFam VADIYLASEYTAYAISGPTTQGGLPVFHWNRFNKTLHKGMPEIYNFDFMTMKPILKHDRK*
PLBD1_myoLuc VADMYLALEYTAHAISGPTVQGGLPVFHWKRFNKTLHEGMPEAYNFDFITMKPILKPDIK*
PLBD1_pteVam VADIYLASQYTAHAISGPTVQGALPVFHWNQFNKTLHEGMPEAYNFDFVTMQPILKPDKK*
PLBD1_eriEur VADFYLTFKYTAYAISGPTVQDGLPAFHWNRFNKTLHKGMPEVYNFDFVTMKPVL*ldrk
PLBD1_sorAra VADIYLAAKFTAYAISGPTVQGGLPVFRWDPFNKTLHRGMPESFDFDFITVKPTL*qdkk
PLBD1_loxAfr VADMYLASEYTAYAISGPTVQNGLPVFHWNRFNKTLHHGMPEAYNFDFVTMRPILKPDRN*
PLBD1_proCap VSDMFLASEFIAYAISGPTVQNGLPVFHWNNFNKTLHQGMPEAYNFDFVTMQPILKLDRKL
PLBD1_echTel VADMWLASKYRAYAISGPTVQDGLPVFRWGSFNKTVHQGMPEAYNFDFTHMKPILT*gr*
PLBD1_dasNov VADIYLASQYTAYAISGPTVQGGLPVFHWNRFNKTLHEGMPEAYNFDFITMKPSLNSDIK*
PLBD1_choHof VADIYLASQYTAYAISGPTVQGGLPVFHWNRFNKTLHRGMPETYNFDFITMKPILT*ne*
PLBD1_monDom VADMFLASQFTAYAINGPTVDDGLPVFEWKKFNETIHKGLPEAYNFDFVTMKPLLEFCELHKEKKKRCGKQVRRWKRRN*
PLBD1_ornAna VSDMALAARLTAHAISGPTVQGGLPVFRWSRFNGTVHRGLPEAYDFDFVTMRPVLRPPWPREAGGR*
PLBD1_galGal VSDFRLASAFTATAINGPPVQGGLPVFTWRRFNNTRHQGLPESYNFKFVTMRPIL*
PLBD1_taeGut VSDFRLAAAFTASAINGPPVQGGLPAFSWRRFNRTRHQGLPESYNFDFVTMRPIL*
PLBD1_anoCar VADINMAMKFTSYAINGPPVEEGLPIFTWSRFNQTKHQGLPDSYNFDFITMKPVL*
PLBD1_botJar VADISMAAKFTAYAISGPTVEKGLPVFSWVHFNKTKHQGLPESYNFDFVTMKPVL
PLBD1_ambMex VSDFYLAATYTAHAINGPPVADGLPPFSWSPFHETIHEGLPEHYNFSFILTKPVL
PLBD1_tetNig VTDFLMAGKFRAEAINGPTTQSGLPPFVWDRFGSVSHQGLPQSYNFTFVPMQPLLF*
PLBD1_takRub VTDFFMAGKFRAEAINGPTTQNGLPPFAWDGFGNISHEGLPKYYNFTFVQMQPILFQP*
PLBD1_gasAcu VTDFHMAGDFRAEAVNGPTTQDGLPPFFWDKFSSMSHQGLPQFYNFTFIRMQPVLFEP*
PLBD1_oryLat VTDFFMAGDFTAEAVNGPTTQDGLPPFYWDKFSSISHQGLPRFYNFTFVTMKPLMFKP*
PLBD1_salSal VTDFHMAQEFRAEAVNGPTTQGDLPPFSWEDFNSTAHQGLPDHYDFPFISMQPALFMP*
PLBD1_danRer VADYRMAQMFTAEAVNGPTSQNGLPLFSWSRFNRTAHQGLPQTYNFTFITMQPLLFAFRDQAKTER...
PLBD1_ictPun VTDLNMAQQFVSEALNGPSTDGDLPPFTWDAFNRTTHQGLPRLYNYTFVTMHPVLFSP
PLBD1_petMar VADMRMAKKFMTSAVNGPTVEGKLPAFSWSPFDNIKHEGLPNTYKFPFVTMQPTLFTIP*
PLBD1_braFlo VSDYYLARNLTSFAINGPTLGTGLEPFSWSDKFKISHIGLPKVYNFSFVTMTPAEL*
PLBD1_strPur VTNLAMAAKQTSFVINGPTRGDGSLPPFKWVAPFTGWSHVGLPTVYDFNFVEMCPKEL*
PLBD2_homSap VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD*
PLBD2_gorGor VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD*
PLBD2_ponAbe VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD*
PLBD2_nomLeu VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD*
PLBD2_papHam VTSMSLARILGLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD*
PLBD2_calJac VTSMSLAKILSLLAASGPTWDQVPPFQWSTSPFSTLLHMGQPDLwKFCAPKVSWD*
PLBD2_micMur VTSMSLAKALSLLAVSGPTWDQVPPFQWSASPFSSLLHMGQPDLWKFSPIRVWWQ*
PLBD2_otoGar VTSMSLAKALSLLAVSGPTWDQVPPFQWSTSPFSSLLHMGHPDLWKFLPIEVWWD*
PLBD2_tupBel VTSMSLAKTLSLVAASGPTWDQVPPFQWSTSAFSHLLHMGHPDLWRFSPIQVSWD*
PLBD2_musMus VTSFTLAKYMSMLAASGPTWDQCPPFQWSKSPFHSMLHMGQPDLWMFSPIRVPWD*
PLBD2_ratNor VTSVALAKYMSMLAASGPTWDQLPPFQWSKSPFHNMLHMGQPDLWMFSPVKVPWD*
PLBD2_dipOrd VTSMSLAKALGLLAVSGPTWDQVPPFQWSSSPFPDVLHMGQPDLWKFLPVEVLWGL
PLBD2_cavPor VTSMSMAKTLSLQAVSGPTWDQVPAFQWSTSPFRDMLHMGHPDLWKFAPVEVSWG*
PLBD2_oryCun VTSKSLAKAMSLLAASGPTWDQVPPFQWSTSPFRDQLHMGHPDLWKFLPIRVLWD*
PLBD2_ochPri VTSMSLAKALSLLADSGSTWDQVPPFQWSASPFRDKLHMGHPDLWKFLPFKVLWD*
PLBD2_vicPac VTNMALAKALRLLAASGPTWDQLPPFQWSTSPFSRLLHMGQPDLWKFSPIDVWWD*
PLBD2_turTru VTSTALAKALRLLAVSGPTWDQLPPFQWSSSPFSSLLHMGQPDLWKFSPIEVWWD*
PLBD2_bosTau VTSTALAKALRLLAVSGPTWDQLPPFQWSTSPFSGMLHMGQPDLWKFSPIEVSWD*
PLBD2_oviAri VTSTALAKALRLLAVSGPTWDQLPPFQWSTSPFSGMLHMGQPDLWKFSPIEVSWD*
PLBD2_lamPac VTNMALAKALRLLAASGPTWDQLPPFQWSTSPFSRLLHMGQPDLWKFSPIDVWWD*
PLBD2_susScr VTSMALARVFGLLAASGPTWDQLPPFQWSTSPFSHLLHMGQPDLWKFSPIEVSWD*
PLBD2_felCat VTSMALAKAFQLVAASGPTWDQLPPFQWSASPFSGLLHMGQPDLWKFSPIEVRWD*
PLBD2_canFam MTSMALAKAFHIIAVSGPTWDQVPPFQWSSSPFSGLLHMGQPDVWKFLPIETWWD*
PLBD2_ailMel VTSMALARAFHIIAVSGPTWDQLPPFQWSSSPFSSLLHMGQPDLWKFSPIEVWWD* 
PLBD2_myoLuc VTSMALAKALRLVAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFSPVKVSWD*
PLBD2_pteVam VTSAALAKALRFLAASGPTWDQLPPFQWSTSPFSGLLHMGQPDLWKFSPIEVWWD*
PLBD2_eriEur VTNVSLVRALGLLAVSGPTWDQLPPFQWSTSPFSGLLHMGQPDLWKFspievwwd*
PLBD2_loxAfr VTSLAMAKALRLLAVSGPTWDQLPPFQWSTSPFRSLLHMGQPDLWKFLPIEVWWD*
PLBD2_proCap VTNLAMAKALRLRAVSGPTWDQLPPFQWSTSPFQSLLHMGQPDLWKFLPIEVWWN*
PLBD2_echTel VTSSGLAKSLRLWAVSGPTWDQLPPFQWSSSPFHNLLHMGQPDLWKFSPVEFGWD*
PLBD2_macEug VTSYELSKDLRLIAVSGPTWDQLPPFQWSSSPFDKLLHMGHPDLWKFFPIKVSWE*
PLBD2_ornAna VTSSQLAKDFRFVAASGPTWDQVPAFRWSSSPFKGLVHMGHPDLWRFSPVHVRWD*
PLBD2_galGal VTSFGMARTFGLVAASGPTWDDVPPFRWSTSPCSHLLRMGHPDLWRFPPVKVRWD*
PLBD2_taeGut VTSSAMVPTFGLVAVSGPAWDDVPPFRWSASPCSSLLHMGHPDLWTFPPVKVHWD*
PLBD2_anoCar VTSFEMAKLYSFVATSGPTWDDLPAFEWSSSPYRNLLHMGHPDLWRFSPIQVHWG*
PLBD2_xenTro LTSYEMAKKYEMVVVNGPTWDQVPPFQWSTSPFSSLMHMGHPDLWKFSPITIRWH*
PLBD2_ranCat VTSYEFAKEYMMFAVNGPTWDQVPPFQWSTSPFSNLMHMGHPDLWKFDPILIRWK*
PLBD2_cynPyr VTSFNMARVYGMVAVSGPTWDDLPPFQWSTSPFSVQLHMGHPDLWQFDPVEVLWWQ*
PLBD2_tetNig LTSYQMFRDYAMIAVSGPTWDQVPPFQWSTSPYKDLLHMGHPGTWTFKPVKVTWKP*
PLBD2_takRub LTSYKMFRDYGLIAVSGPTWDQVPPFQWSTSPYKDLLHMGHPDTWTFKPITVIWTP*
PLBD2_gasAcu LTSFEMFRDYAMLAVSGPTWDQVPPFQWSTSPYSDLMHMGHPDSWAFKPVKVSWNP*
PLBD2_oryLat MTSFGMFKEYGMIAVSGPTWDQLPPFQWSTSPYKDLVHMGHPDVWNFKPIKVTWTP*
PLBD2_salSal VTSYGLWREFGFLAASGPTWDQVPAFQWSSSPYSDLMHMGHPDTWAFTPIHVTWST*
PLBD2_danRer MTSSSMFRQWELLAASGPSCEQTPVFQWSRSPYSSLMHMGQPDRWDFPTVHVRWAT*
PLBD2_ictPun MTSYGLFKQYELLAVSGPTWDQVPAFEWSTSPYSSLTHIGHPDRWDFPTVHIRWSE
PLBD2_squAca VTSFELHSTYQMIAVNGPTWDEVPAFQWSKSPFSSLMHMGHPDLWRFLPVMVQWK*
PLBD2_calMil VTSSEMYKTFEMIAISGPAWDQVPPFQWSKSSYSGLIHMGHPDLWKFPPVMVRWS*
PLBD2_petMar ITSKAMVPRLEMVAQSGPTWDQQPPFQWSKSPFSSLSHVGQPDLWSFLPEHISWCKHSGQ*
PLBD2_braFlo MTSYSMHESHQMMAVSGPTHDQQQPFQWSTSDYDKQFYHLGHPDLFNFDPIHVIWFDQSDN*
PLBD2_cioInt VVGYSMMKNFEILAECGPTHDQQPPFVWSKSPFSHVSHKGMPDKYDFKPTLIIWDKFSPLKMLDKIHKSVNL*
PLBD2_halRor VTSYTLHKTLQMVAEAGPTHDQQPVFQWSTSPYASKSHEGHPDRFDFLPVLIKWDGEILPK*
PLBD2_strPur VTTSSMVKSLSMVAVCGPTTDQQPPFQWSKSDFNQTLHLGHPDLFNFKPINVQWYD*
PLBD2_sacKow LTNYVMHKDLSFVAISGPTQDQQPVFQWSTSPYLDVLHLGHPDKFDFGPVQVNWKND*
PLBD2_aplCal LISYDLFKSLSFLAISSPTYDDLPPFQWSKSDYNYMSHLGHPDVWKFPRILFKGTDPLA*
PLBD2_creFor LTSAEMVKDLTFIAVGGPSWDQQPPFQWSKSDFKSTSHIGQPDLWKFPPVLFNISLIF*
PLBD2_eupSco LTNSSLARDLQFVAIAGPTYDQVTPFQWSKSDFKNTVSHIGHPDIFKFEPYIFGDDVMKFE*
PLBD2_craGig LTNSAMFKAMQFVAISGPTYDQFAPFQWSKSDFKDNTPHMGHPDTFKFDPVVFDGTTDFKPFQR*
PLBD2_helRob ITSYSMFKNFQFLAVSGPTRDQVAPFQWSKSDLKDTIRHAGHPDLWVFDPVQF*
PLBD2_capCap ITSYSMFKNFQFLAVSGPTRDQVAPFQWSKSDLKDTIRHAGHPDLWVFDPVQF*
PLBD2_limPol LTTFGLSQKFEFVAIGGPTHDPLPPFQWSKSDFSKDLPHYGHPDLWVFKPVTHHWK*
PLBD2_ixoSca VTNFALFERQVFYAVSGPTSDDQRPFRWSTSGFDNVSHAGHPDLWDFDPILAQWRY*
PLBD2_derVar LTTYQQFKEQQFFAISGPTWSQQPVFQWSTSGFNDSHVGHPDRWEFGPVLNYWGSCR*
PLBD2_dapPul VTSHQLMTSLDFIAVGGPTFDSLPAFRWSESDFVNMSHIGHPDLWKFEPVQTEWTL*
PLBD2_calCle VTSFDLLLRGSFIAGGGPTYDSVEPFQWSKADFEKDTPHFGHPDKWDFKPMRVEWDNVL*
PLBD2_lepSal ITSFDLFMKGSFIAGGGPTYDSVEPFQWSKTDFGKTTPHFGHPDKWAFKPIKVDWENAFNRDEDVV*
PLBD2_hydMag ATSFQLSKLMSQFIVGGPTYDQQPPFQWSKTEWNRPLGHPDIFKFNPELLDWRKEEWIYSKLNI*
PLBD2_nemVec ITSFELFQKFQCIAVSGPTHDQQPAFQWSTSEWEKPLGHPDKFDFEPVKVSWDNKD*
PLBD2_merSen ITSSELFKKFQCQAVSGPTHDQQPVFQWSKSDWKRPLGLPDKFDFSPVMVSWENEE*
PLBD2_acrPal ITNSEMVKSLECVAVSGPTHDQQPVFKWSASGWDTPLGHPDAWDFEPIVVKWQEN*
PLBD2_porAst ATSHQLVQQLSTIATCGPTHAQQPVFKWSESGETKPLGHPDAFDFPTVQIKWNKQ*
PLBD2_monFav VTNSELIKELQCMAVSGPTHDQQPVFKWSTSGWKRPTGHPDAWDFEPIKVTWE*
PLBD2_ampQue LTNSEMVKSLSCLATSGPTHSQQPVFKWSTSGFQDTPPLGHPDEFDFAPIVIKWGEIN*
PLBDa_dicDis VVSADMVAALLVNAQSGPSHDNETPFTWNSQWNQKYTYAGQPTTWNFDWMTMSLQSMKPASPSSDSSSDSTTFN*
 
=== Signature conservation in the final exon ===
 
Using the above collection of sequences for the final exon of PLBD1 and PLBD2, it is possible to compare the same 45 vertebrate species for both genes. When these are [http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_multalin.html aligned] retaining phylogenetic order and allowing 0,1,2,...,10 departures from absolute invariance at each site, the two tables below result.
 
From them, residues conserved in PLBD1 but not PLBD2, conserved in PLBD2 but not PLBD1 or conserved in both can be extracted. The former serve quite well to classify phospholipases in the very earliest diverging eukaryotes which otherwise might have poor overall Blast scores to their mammalian ortholog. That classification is usually supported by an indel location: PLBD2 has lost one residue relative to PLBD1 just before the conserved SGPT motif and PLBD1 has lost two residues relative to PLBD2 further down.
 
The two proteins have been conserved equally well over the 500 million years of vertebrate evolution, quantitated simply by a dot count (.) in the two tables relative to the total number of residues (45 spp x 55 sites = 2475). Oddly, this conservation often takes place at non-homologous (different) sites in the two proteins despite the overwhelming similarity in their folds. At two sites near the carboxy terminus, a residue is conserved but it differs for the two protein (F/P and P/W). Otherwise 11 residues are conserved in both proteins -- this conservation may date back to the original gene duplicated (and perhaps earlier in NTN hydratases).
 
Vertebrate conservation of last exon of PLBD1 with increasing tolerance for exceptions:
  0 V.D..........A..GP.....LP.F....--F....H.G........F....P..
  1 V.D...A......A..GP.....LP.F.W..--F....H.G.P....F.F....P..
  2 V.D...A......A..GP.....LP.F.W..--F....H.G.P..Y.F.F....P.L
  3 V.D...A......A..GP.....LP.F.W..--F....H.G.P..Y.F.F....P.L
  4 V.D...A......A..GP....GLP.F.W..--F..T.H.G.P..Y.F.F..M.P.L
  5 V.D...A......A..GPT...GLP.F.W..--FN.T.H.G.P..Y.F.F..M.P.L
  6 V.D...A......AI.GPT...GLP.F.W..--FN.T.H.G.P..YNF.F..M.P.L
  7 V.D...A......AI.GPT.Q.GLP.F.W..--FN.T.H.G.P..YNF.F..M.P.L
  8 V.D...A....a.AI.GPT.Q.GLP.F.W..--FN.T.H.G.P..YNF.F.TM.P.L
  9 V.D..LA....a.AI.GPTVQ.GLP.F.W..--FN.T.H.G.PE.YNFDF.TM.P.L
10 V.D..LA....a.AI.GPTVQ.GLP.F.W..--FN.T.H.G.PE.YNFDF.TM.P.L
    <font color = brown>V.D...A...............gL.......--Fn.t............F..m.P.L conserved PLBD1 not PLBD2</font>
    <font color = red>.............A..GPt.....P.F.W.........H.G.P..w.F........  conserved PLBD1 and PLBD2</font>
    <font color = blue>.Ts............S...-WDq......S.SP......m...D.....P..v.W.. conserved PLBD2 not PLBD1</font>
Vertebrate conservation of last exon of PLBD2 with increasing tolerance for exceptions:
  0 .T..............G..-....P.F.WS.S........G.P..W.F......W..
  1 .T..............G..-....P.F.WS.S........G.P..W.F......W..
  2 .T...........A.SGP.-WD..P.F.WS.SP.....H.G.PD.W.F......W..
  3 .T...........A.SGPT-WD..P.F.WS.SP.....HMG.PD.W.F.P....W..
  4 .TS..........A.SGPT-WDQ.P.F.WS.SP.....HMG.PD.W.F.P....W..
  5 .TS..........A.SGPT-WDQ.P.FQWS.SP.....HMG.PD.W.F.P..V.W..
  6 .TS..........A.SGPT-WDQ.P.FQWS.SP.....HMG.PD.W.F.P..V.WD.
  7 .TS..........A.SGPT-WDQ.PPFQWS.SP.....HMG.PD.W.F.P..V.WD.
  8 .TS..........A.SGPT-WDQ.PPFQWS.SP....LHMG.PDLW.F.P..V.WD.
  9 VTS..........A.SGPT-WDQ.PPFQWS.SP...LLHMG.PDLW.F.P..V.WD.
  10 VTS..........A.SGPT-WDQ.PPFQWS.SP...LLHMG.PDLW.F.P..V.WD.
 
=== Lysosomal storage disease: allele prediction ===
 
[[Image:Plbd2CpG.png|left]]
 
Experience has shown that it is fairly simple to predict about half of the common mutations that will eventually surface as disease alleles. These are comprised of (1) rare polymorphisms in the population that occur at phylogenetically conserved sites and and (2) CpG hotspot non-synonymous substitutions at similarly conserved residues. Here there will also be replication slippage errors in the regions of anomalous GC compositions such as the signal peptide; their outcome is difficult to predict but would lead to frameshifts 2/3 of the time (if not 3n).
 
The reason for this is both PLBD1 and PLBD2 are autosomal, meaning that both alleles must be affected. This occurs in the consanguineous case mainly for adverse polymorphisms that have managed to attain a reasonable frequency in the population. These can otherwise combine with the dominant de novo mutations, namely CpG mutations resolving to TpG or CpA. Indeed the Q54P discussed earlier for the reference genome arose as a transversional CpG mutation CCG (pro) -> CAG (gln), as can be seen from chimpanzee sequence.
 
The significance of a given substitution can then be read off the 46-way vertebrate alignment -- a departure from the evolutionarily tested reduced alphabet is rarely an adaptive innovation but rather almost always an unacceptable change reducing fitness (what else could account for observed conservation?).
 
As a practical matter, the [http://www.1000genomes.org/ 1000 Human Genome Project] provides rare alleles to the 1% level for every gene (though currently these are difficult to extract for a particular gene). These are supplemented with previously observed SNPs. Consequences of CpG mutations are obtained by in silico substitution, followed by Blastx against wildtype to flag differences (55 for CpA, 50 more for less frequent TpG), followed by evaluation of the change, using mammals from the 46-way with [http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_multalin.html MultAlin] set to maintain input order.
 
Neanderthal has 4 substitutions (three arising from CpG) relative to human, none of which are predicted to affect protein function:
 
human genome location      #reads change  N  H  comment
chr12:112280948-112280949  1C>0A(CCG>CAG) P 54Q  reference genome has a mutation at an ultra-conserved site
chr12:112294980-112294981  0G>3C(GCT>CCT) A182P  human has a phylo-acceptable change
chr12:112297191-112297192  0C>2T(CGG>TGG) R286W  human has a change at an unselected residue
chr12:112309977-112309978  0G>3A(GAC>AAC) D496N  human has a change at an unselected residue
 
To these should be added fatal CpG disruptions of splice donors, which PLBD2 has in 3 consecutive exons, all of phase 2 which give rise in the CpG hotspot effect either to a phylogenetically non-conservative change in 2nd letter of codon -- shown at left: T373M, P405L, P429L for CpG mutating to TpG -- and/or loss of GT-AG splice donor soon leading to a premature stop codon for for CpG transitioning to CpA:
 
<font color = blue>ag</font>GTGACACTGGAGACCACCATTGGCAACAAGAACCCAGCCCTGTGGAAGTATGTGCGGCCCAGGGGCTGTGTGCTGGAGT
GGGTACGCAACATCGTGGCCAACCGCCTGGCCTCGGATGGGGCCACCTGGGCAGACATCTTCAAGAGGTTCAACAGCGGCA<font color = red>Cg</font><font color = blue>t</font> 2 T373M
<font color = blue>ag</font>GTATAACAACCAGTGGATGATCGTGGACTACAAGGCGTTCATCCCGGGTGGGCCCAGCCCCGGGAGCCGGGTGCTTACCATCCTGGAGCAGATCC<font color = red>Cg</font><font color = blue>t</font> 2 P405L
<font color = blue>ag</font>CGGCATGGTGGTGGTGGCTGACAAGACCTCGGAGCTCTACCAGAAGACCTACTGGGCCAGCTACAACATAC<font color = red>Cg</font><font color = blue>t</font> 2 P429L
 
No stop codons result from CpG mutations in PLBD2 but numerous non-conservative changes occur at invariant sites. A sampler of these is shown flanking the GSGSCSALIK cleavage motif below:  E153K, R183Q, E197K, R199H, R272H, R282Q, G294S are all predicted to be disruptive. Dashes indicate lack of data rather than deletions -- input sequences could be greatly improved by curation as is done below -- and at much greater phylogenetic depth -- for the terminal exon.
 
        160      170      180      190      200      210      220      230      240      250      260      270      280      290      300
        |        |        |        |        |        |        |        |        |        |        |        |        |        |        |
CpG2CpA YCKRLKSFLEANLEWMQEEMESNPDSPYWHQVQLTLLQLKGLEDSYKGHVSFPAGKFTIKPLGFLLLQLSGDLEDLELALNKTKIKPSLGSGSCSALIKLLPGQSDLLVAHNTWNNYQHMLHVIKKYWLQFQEGPWGDYPLVPSNKLVFS
homSap  ..E.............................R.............E.R........................................................................R.........R...........G......
panTro  ..E.....................Y.A.....R.............E.R........................................................................R.........R...R.......G......
gorGor  ..E.......................A.....R.............E.R.....................................................................Y..R.........R...RSFETVFNASG.QAL
ponAbe  ..E.......................A.....R.............E.R........................................................................R.........R...R.......GS.....
rheMac  ..E.......................A.....R.............E.R........................................................................R.........R...Q..S....G......
papHam  ..E.......................A.....R.............E.R........................................................................R.........R...Q..S....G......
calJac  ..E.................Q.....A.....----------------------------------------------------------------------------------------------------------------------
  micMur ..E.......V.......QVAL.K..V.....R.............E.R.N..TE.............I........P......V.AA..A.......................HS.....R......F..R...RD.D....G..V...
tupBel  ..E..................R.Q..T....KTKV.-------------------------------------------------------..GCS....---.RH.VATR----W.....R....CL.-IHQ..QE.S...........
musMus  ..EK..N..........R...L..........R.............E.RLT..T.R............I........P.....NT..................GH..........S..N..RI....R...R...QEE....AG.N....
ratNor  ..EK.............R...LS.........R.............E.RLT..T.R.N..........I........P.....NT...V..............SH..........S..N..RI....R...R...QEE...IAG.N.I..
dipOrd  ..EK.........A...A...QQVN.T.....R.............E.R.T..TE.......X--............P....SNS..V....A....V.....HR..........A..S..R.........R...---------------
cavPor  ..E.......T......QQ..LH..C......R.............E..L...T.R.S..........I.............S.TRAT.........L......R..........A..Y..R.L...Q...RV..RE.F..A.G..V...
speTri  ..EK..H..........R..QWDQ..G...K.R.............E.R....T..............I........P.....RTRAV............M...R.....L.--S.S.N..-I....TS.I--..QK.AL...G.QM...
oryCun  ..E...R..............R.K..T.....R.............E.R.T..T.R............I........P...R..TQAA...............HR..........S.....R......F..R...QE......G.RVI..
ochPri  ..E...RY.........A.....RG.M.C...R.............E.R....M.R............I........P......T.AV................Q..........S.....RI.....F..RKD.Q..S....G.RVI..
turTru  ..E...K..........K...L.NG.A.....R.............E.S.T..T..............I...............T.HAT.........................HS..Y..RIM....F..R...QEQSTRA.G.RVI..
bosTau  ..E...N..........K...L.NG.A.....R.............E.S.A..T....V.........I........V......TNHAM...............R.........HS..Y..RIM....F..R...QAESTRA.G..VI..
equCab  ..E......DT............K..A.....R.............E.S.A..T.R............I....G...P.....EARRT..A.A....V.....HR..........S..S..RI.....--------S.A.PA.G.R....
felCat  ..EK..N...T.......Q.QR.KG.A----.R.............ESS.T..T.R.............A...G...P......S.HVM...................I.....SS..N..RI.....F..R.D.QVNS..A.G......
canFam  ..E..MR...T..D..L.LIKL.K.FA..--.R.............E.SMT..T.R.................G...P......T.HIM...................I.....SS..N..RI.....F..R.D.QENS..A.G......
myoLuc  ..E...G..........KQV.L.K..A.....R.............E.S.A..T..............I...............T.HT...............H...........S..N..RI.....F......QE.S..A.G......
pteVam  ..EK.................L.K..A.....R.......-.....E.S.T..T..............I...............TRHT..A....V.......HG..........S..N..R.....SF..RQ..QE.S..A.G......
loxAfr  ..EK.................L.R..A.....R.............G.RMT..T.......................P....S.T.RA................R..........S.....R.V...SFH.H...RENSL.A.G.SM...
proCap  --------------...K...L.R..A.....R......-......G.RMT..T.N.....................P......T.RA................R..........S.....R.L...SFH.R...QE.SV.A.G.T....
echTel  ..EK......T..........R.K..T.....R.A...........G...T..T.R.....................P......T.R.M...............R..........S.....RIV...SF..H...QD.SL.I.G...I..
dasNov  ..E...R..........R..ALS.HDA.....R.............E.R.T.......F.........V---------------------------------------------------------------------------------
macEug  ..EK..NYI.L..A...KQIA.SK.DE.....E.A...........Q.RIA..SKN...T.F....F..........P.....EQRRVM.............NN.E...S.D...S.....RI....DFR.RTL.K---..I.G..Q...
monDom  ..EK...YI.M..A...KQ.A.GK.AE.....E.A...........Q.RIA..SKN...T.F....F..........P.....AHRRVM.............NRKE...S.D..SS.....RI....KFH.RTL.Q.EA..I.G.EQ...
A serious CpG mutation -- G551S -- could occur via G -> A in the terminal exon whereas other substitutions are nearly neutral:
                                +                  -            -
CpG2CpA        VTSMSLARILSLLAASSPTWDQVPPFQWSTSPFS-SLLHMGQPDLWKFTPVKVSWD
PLBD2_homSap  ................G..................G............A.......
PLBD2_gorGor  ................G..................G............A.......
PLBD2_ponAbe  ................G..................G............A.......
PLBD2_nomLeu  ................G..................G............A.......
PLBD2_papHam  ..........G.....G..................G............A.......
PLBD2_calJac  .......K........G..................T............CAP.....
PLBD2_micMur  .......KA.....V.G............A..................S.IR.W.Q
PLBD2_otoGar  .......KA.....V.G........................H......L.IE.W..
PLBD2_tupBel  .......KT...V...G..............A...H.....H....R.S.IQ....
PLBD2_musMus  ...FT..KYM.M....G.....C......K...H..M.........M.S.IR.P..
PLBD2_ratNor  ...VA..KYM.M....G.....L......K...H.NM.........M.S....P..
PLBD2_dipOrd  .......KA.G...V.G............S...P.DV...........L..E.L.G
PLBD2_cavPor  .....M.KT...Q.V.G.......A........R.DM....H......A..E...G
PLBD2_oryCun  ...K...KAM......G................R.DQ....H......L.IR.L..
PLBD2_ochPri  .......KA.....D.GS...........A...R.DK....H......L.F..L..
PLBD2_vicPac  ..N.A..KA.R.....G.....L............R............S.ID.W..
PLBD2_turTru  ...TA..KA.R...V.G.....L......S..................S.IE.W..
PLBD2_bosTau  ...TA..KA.R...V.G.....L............GM...........S.IE....
PLBD2_oviAri  ...TA..KA.R...V.G.....L............GM...........S.IE....
PLBD2_lamPac  ..N.A..KA.R.....G.....L............R............S.ID.W..
PLBD2_susScr  ....A...VFG.....G.....L............H............S.IE....
PLBD2_felCat  ....A..KAFQ.V...G.....L......A.....G............S.IE.R..
PLBD2_canFam  M...A..KAFHII.V.G............S.....G........V...L.IETW..
PLBD2_ailMel  ....A...AFHII.V.G.....L......S..................S.IE.W..
PLBD2_myoLuc  ....A..KA.R.V...G..................G............S.......
PLBD2_pteVam  ...AA..KA.RF....G.....L............G............S.IE.W..
PLBD2_eriEur  ..NV..V.A.G...V.G.....L............G............S.IE.W..
PLBD2_loxAfr  ...LAM.KA.R...V.G.....L..........R..............L.IE.W..
PLBD2_proCap  ..NLAM.KA.R.R.V.G.....L..........Q..............L.IE.W.N
PLBD2_echTel  ...SG..KS.R.W.V.G.....L......S...H.N............S..EFG..
PLBD2_macEug  ...YE.SKD.R.I.V.G.....L......S...D.K.....H......F.I....E
PLBD2_ornAna  ...SQ..KDFRFV...G.......A.R..S...K.G.V...H....R.S..H.R..
PLBD2_galGal  ...FGM..TFG.V...G....D....R.....C..H..R..H....R.P....R..
PLBD2_taeGut  ...SAMVPTFG.V.V.G.A..D....R..A..C........H....T.P....H..
PLBD2_anoCar  ...FEM.KLY.FV.T.G....DL.A.E..S..YR.N.....H....R.S.IQ.H.G
PLBD2_xenTro  L..YEM.KKYEMVVVNG....................M...H......S.ITIR.H
PLBD2_ranCat  ...YEF.KEYMMF.VNG..................N.M...H......D.ILIR.K
PLBD2_cynPyr  ...FNM..VYGMV.V.G....DL............VQ....H....Q.D..E.L.W
PLBD2_tetNig  L..YQMF.DYAMI.V.G...............YK.D.....H.GT.T.K....T.K
PLBD2_takRub  L..YKMF.DYG.I.V.G...............YK.D.....H..T.T.K.IT.I.T
PLBD2_gasAcu  L..FEMF.DYAM..V.G...............Y..D.M...H..S.A.K......N
PLBD2_oryLat  M..FGMFKEYGMI.V.G.....L.........YK.D.V...H..V.N.K.I..T.T
PLBD2_salSal  ...YG.W.EFGF....G.......A....S..Y..D.M...H..T.A...IH.T.S
PLBD2_danRer  M..S.MF.QWE.....G.SCE.T.V....R..Y....M......R.D.PT.H.R.A
PLBD2_ictPun  M..YG.FKQYE...V.G.......A.E.....Y....T.I.H..R.D.PT.HIR.S
PLBD2_squAca  ...FE.HSTYQMI.VNG....E..A....K.......M...H....R.L..M.Q.K
PLBD2_calMil  ...SEMYKTFEMI.I.G.A..........K.SY..G.I...H......P..M.R.S
PLBD2_petMar  I..KAMVPR.EMV.Q.G.....Q......K.......S.V......S.L.EHI..C
PLBD2_braFlo  M..Y.MHESHQMM.V.G..H..QQ.......DYDKQFY.L.H...FN.D.IH.I.F
PLBD2_cioInt  .VGY.MMKNFEI..ECG..H..Q...V..K.....HVS.K.M..KYD.K.TLII..
PLBD2_halRor  ...YT.HKT.QMV.EAG..H..Q.V.......YA..KS.E.H..RFD.L..LIK..
PLBD2_strPur  ..TS.MVKS..MV.VCG..T..Q......K.D.N.QT..L.H...FN.K.IN.Q.Y
PLBD2_sacKow  L.NYVMHKD..FV.I.G..Q..Q.V.......YL.DV..L.H..KFD.G..Q.N.K
PLBD2_aplCal  LI.YD.FKS..F..I....Y.DL......K.DYN.YMS.L.H..V...PRILFKGT
PLBD2_creFor  L..AEMVKD.TFI.VGG.S...Q......K.D.K..TS.I........P..LFNIS
PLBD2_eupSco  L.NS....D.QFV.IAG..Y...T.....K.D.KNTVS.I.H..IF..E.YIFGD-
PLBD2_craGig  L.NSAMFKAMQFV.I.G..Y..FA.....K.D.KDNTP...H..TF..D..VFDGT
PLBD2_helRob  I..Y.MFKNFQF..V.G..R...A.....K.DLKDTIR.A.H....V.D..QF---
PLBD2_capCap  I..Y.MFKNFQF..V.G..R...A.....K.DLKDTIR.A.H....V.D..QF---
PLBD2_limPol  L.TFG.SQKFEFV.IGG..H.PL......K.D..KD.P.Y.H....V.K..THH.K
PLBD2_ixoSca  ..NFA.FERQVFY.V.G..S.DQR..R....G.D.NVS.A.H....D.D.ILAQ.R
PLBD2_derVar  L.TYQQFKEQQFF.I.G...S.Q.V......G.-.NDS.V.H..R.E.G..LNY.G
PLBD2_dapPul  ...HQ.MTS.DFI.VGG..F.SL.A.R..E.D.V.NMS.I.H......E..QTE.T
PLBD2_calCle  ...FD.LLRG.FI.GGG..Y.S.E.....KAD.EKDTP.F.H..K.D.K.MR.E..
PLBD2_lepSal  I..FD.FMKG.FI.GGG..Y.S.E.....KTD.GKTTP.F.H..K.A.K.I..D.E
PLBD2_hydMag  A..FQ.SKLM.QFIVGG..Y..Q......KTEWN.--RPL.H..IF..N.ELLD.R
PLBD2_nemVec  I..FE.FQKFQCI.V.G..H..Q.A......EWE.--KPL.H..KFD.E.......
Four serious mutations could arise from C -> T mutations at CpG sites: A548V, T553M, P559L, and S565L
                            +    +    +    +                -
CpG2TpG        VTSMSLARILSLLVASGPMWDQVPLFQWSTLPFS-GLLHMGQPDLWKFVPVKVSWD
PLBD2_homSap  .............A....T.....P.....S.................A.......
PLBD2_gorGor  .............A....T.....P.....S.................A.......
PLBD2_ponAbe  .............A....T.....P.....S.................A.......
PLBD2_nomLeu  .............A....T.....P.....S.................A.......
PLBD2_papHam  ..........G..A....T.....P.....S.................A.......
PLBD2_calJac  .......K.....A....T.....P.....S....T............CAP.....
PLBD2_micMur  .......KA....AV...T.....P....AS....S............S.IR.W.Q
PLBD2_otoGar  .......KA....AV...T.....P.....S....S.....H......L.IE.W..
PLBD2_tupBel  .......KT...VA....T.....P.....SA...H.....H....R.S.IQ....
PLBD2_musMus  ...FT..KYM.M.A....T...C.P....KS..H.SM.........M.S.IR.P..
PLBD2_ratNor  ...VA..KYM.M.A....T...L.P....KS..H.NM.........M.S....P..
PLBD2_dipOrd  .......KA.G..AV...T.....P....SS..P.DV...........L..E.L.G
PLBD2_cavPor  .....M.KT...QAV...T.....A.....S..R.DM....H......A..E...G
PLBD2_oryCun  ...K...KAM...A....T.....P.....S..R.DQ....H......L.IR.L..
PLBD2_ochPri  .......KA....AD..ST.....P....AS..R.DK....H......L.F..L..
PLBD2_vicPac  ..N.A..KA.R..A....T...L.P.....S....R............S.ID.W..
PLBD2_turTru  ...TA..KA.R..AV...T...L.P....SS....S............S.IE.W..
PLBD2_bosTau  ...TA..KA.R..AV...T...L.P.....S.....M...........S.IE....
PLBD2_oviAri  ...TA..KA.R..AV...T...L.P.....S.....M...........S.IE....
PLBD2_lamPac  ..N.A..KA.R..A....T...L.P.....S....R............S.ID.W..
PLBD2_susScr  ....A...VFG..A....T...L.P.....S....H............S.IE....
PLBD2_felCat  ....A..KAFQ.VA....T...L.P....AS.................S.IE.R..
PLBD2_canFam  M...A..KAFHIIAV...T.....P....SS.............V...L.IETW..
PLBD2_ailMel  ....A...AFHIIAV...T...L.P....SS....S............S.IE.W..
PLBD2_myoLuc  ....A..KA.R.VA....T.....P.....S.................S.......
PLBD2_pteVam  ...AA..KA.RF.A....T...L.P.....S.................S.IE.W..
PLBD2_eriEur  ..NV..V.A.G..AV...T...L.P.....S.................S.IE.W..
PLBD2_loxAfr  ...LAM.KA.R..AV...T...L.P.....S..R.S............L.IE.W..
PLBD2_proCap  ..NLAM.KA.R.RAV...T...L.P.....S..Q.S............L.IE.W.N
PLBD2_echTel  ...SG..KS.R.WAV...T...L.P....SS..H.N............S..EFG..
PLBD2_macEug  ...YE.SKD.R.IAV...T...L.P....SS..D.K.....H......F.I....E
PLBD2_ornAna  ...SQ..KDFRFVA....T.....A.R..SS..K...V...H....R.S..H.R..
PLBD2_galGal  ...FGM..TFG.VA....T..D..P.R...S.C..H..R..H....R.P....R..
PLBD2_taeGut  ...SAMVPTFG.VAV...A..D..P.R..AS.C..S.....H....T.P....H..
PLBD2_anoCar  ...FEM.KLY.FVAT...T..DL.A.E..SS.YR.N.....H....R.S.IQ.H.G
PLBD2_xenTro  L..YEM.KKYEMV.VN..T.....P.....S....S.M...H......S.ITIR.H
PLBD2_ranCat  ...YEF.KEYMMFAVN..T.....P.....S....N.M...H......D.ILIR.K
PLBD2_cynPyr  ...FNM..VYGMVAV...T..DL.P.....S....VQ....H....Q.D..E.L.W
PLBD2_tetNig  L..YQMF.DYAMIAV...T.....P.....S.YK.D.....H.GT.T.K....T.K
PLBD2_takRub  L..YKMF.DYG.IAV...T.....P.....S.YK.D.....H..T.T.K.IT.I.T
PLBD2_gasAcu  L..FEMF.DYAM.AV...T.....P.....S.Y..D.M...H..S.A.K......N
PLBD2_oryLat  M..FGMFKEYGMIAV...T...L.P.....S.YK.D.V...H..V.N.K.I..T.T
PLBD2_salSal  ...YG.W.EFGF.A....T.....A....SS.Y..D.M...H..T.A.T.IH.T.S
PLBD2_danRer  M..S.MF.QWE..A....SCE.T.V....RS.Y..S.M......R.D.PT.H.R.A
PLBD2_ictPun  M..YG.FKQYE..AV...T.....A.E...S.Y..S.T.I.H..R.D.PT.HIR.S
PLBD2_squAca  ...FE.HSTYQMIAVN..T..E..A....KS....S.M...H....R.L..M.Q.K
PLBD2_calMil  ...SEMYKTFEMIAI...A.....P....KSSY....I...H......P..M.R.S
PLBD2_petMar  I..KAMVPR.EMVAQ...T...Q.P....KS....S.S.V......S.L.EHI..C
PLBD2_braFlo  M..Y.MHESHQMMAV...TH..QQP.....SDYDKQFY.L.H...FN.D.IH.I.F
PLBD2_cioInt  .VGY.MMKNFEI.AEC..TH..Q.P.V..KS....HVS.K.M..KYD.K.TLII..
PLBD2_halRor  ...YT.HKT.QMVAEA..TH..Q.V.....S.YA.SKS.E.H..RFD.L..LIK..
PLBD2_strPur  ..TS.MVKS..MVAVC..TT..Q.P....KSD.N.QT..L.H...FN.K.IN.Q.Y
PLBD2_sacKow  L.NYVMHKD..FVAI...TQ..Q.V.....S.YL.DV..L.H..KFD.G..Q.N.K
PLBD2_aplCal  LI.YD.FKS..F.AI.S.TY.DL.P....KSDYN.YMS.L.H..V...PRILFKGT
PLBD2_creFor  L..AEMVKD.TFIAVG..S...Q.P....KSD.K.STS.I........P..LFNIS
PLBD2_eupSco  L.NS....D.QFVAIA..TY...TP....KSD.KNTVS.I.H..IF..E.YIFGD-
PLBD2_craGig  L.NSAMFKAMQFVAI...TY..FAP....KSD.KDNTP...H..TF..D..VFDGT
PLBD2_helRob  I..Y.MFKNFQF.AV...TR...AP....KSDLKDTIR.A.H....V.D..QF---
PLBD2_capCap  I..Y.MFKNFQF.AV...TR...AP....KSDLKDTIR.A.H....V.D..QF---
PLBD2_limPol  L.TFG.SQKFEFVAIG..TH.PL.P....KSD..KD.P.Y.H....V.K..THH.K
PLBD2_ixoSca  ..NFA.FERQVFYAV...TS.DQRP.R...SG.D.NVS.A.H....D.D.ILAQ.R
PLBD2_derVar  L.TYQQFKEQQFFAI...T.S.Q.V.....SG.-.NDS.V.H..R.E.G..LNY.G
PLBD2_dapPul  ...HQ.MTS.DFIAVG..TF.SL.A.R..ESD.V.NMS.I.H......E..QTE.T
PLBD2_calCle  ...FD.LLRG.FIAGG..TY.S.EP....KAD.EKDTP.F.H..K.D.K.MR.E..
PLBD2_lepSal  I..FD.FMKG.FIAGG..TY.S.EP....KTD.GKTTP.F.H..K.A.K.I..D.E
PLBD2_hydMag  A..FQ.SKLM.QFIVG..TY..Q.P....KTEWN.--RPL.H..IF..N.ELLD.R
PLBD2_nemVec  I..FE.FQKFQCIAV...TH..Q.A.....SEWE.--KPL.H..KFD.E.......
>CpG2CpA 55 changes for PLBD2
MVGQMYCYPSSHLAQALTQALALALVLALLVRPFLSSLAGAIPAPGGHWAHNGQVPPASHSHSVLLDISAGQLLMVDRHHPDTMAWANLTNTIHKTGWAFLELGTSGQYNDSLQAYAASV
VEAAVSEELIYMHWMNTVVNYCSPFKYEVSYCKRLKSFLEANLEWMQEEMESNPDSPYWHQVQLTLLQLKGLEDSYKGHVSFPAGKFTIKPLGFLLLQLSGDLEDLELALNKTKIKPSLG
SGSCSALIKLLPGQSDLLVAHNTWNNYQHMLHVIKKYWLQFQEGPWGDYPLVPSNKLVFSSYPSTIFSCNNFYILGSGLVTLETTIGNKNPALWKYVQPRGCVLEWVHNIMANHLASDGA
TWADIFKRFNSSTYNNQWMIMDYKAFIPGGPSPRSQVLTILEQIPSMVVVADKTSELYQKTYWASYNIPSFKTVFNASGLQALVAQYGDWFSYDRSPQAQIFQQNQSLVQDMDSMVRLMR
YNDFLHDPLSLCKACNPQPNGENAISTHSNLNPANGSYPFQALHQHSHGGINVKVTSMSLARILSLLAASSPTWDQVPPFQWSTSPFSSLLHMGQPDLWKFTPVKVSWD*
>CpG2TpG 50 changes for PLBD2
MVGQMYCYPGSHLAWVLMWVLVLALVLALLVGLFLSGLVGVIPVLGGCWVCDGQVPPASCSCLVLLDVLVGQLLMVDGCHPDAVAWANLTNAICETGWAFLELGTSGQYNDSLQAYAAGV
VEAAVLEELIYMHWMNMVVNYCGPFEYEVGYCERLKSFLEANLEWMQEEMESNPDSPYWHQVWLTLLQLKGLEDSYEGCVSFPAGKFTIKPLGFLLLQLSGDLEDLELALNKTKIKPSLG
SGSCSALIKLLPGQSDLLVAHNTWNNYQHMLCVIKKYWLQFWEGPWGDYLLVPGNKLVFSSYPGTIFSCDDFYILGSGLVTLETTIGNKNPALWKYVWPRGCVLEWVCNIVANCLALDGA
TWADIFKRFNSGMYNNQWMIVDYKVFILGGPSPGSWVLTILEQIPGMVVVADKTLELYQKTYWASYNILSFETVFNASGLQALVAQYGDWFSYDGSPWAQIFWWNQSLVQDMDSMVRLMR
YNDFLHDPLSLCKACNPQPNGENAISACSDLNLANGSYPFQALCQCSHGGIDVKVTSMSLARILSLLVASGPMWDQVPLFQWSTLPFSGLLHMGQPDLWKFVPVKVSWD*
 
== Homologs in model organisms ==
 
In this section, species are discussed that have an existing (or potential) phospholipase experimental literature for either PLBD1 or PLBD2. Their relevance to the human situation varies greatly depending on divergence and other complications. Drosophila LAMA -- despite two early papers will prove completely irrelevant. Nematodes have a reasonably comparable protein and could be quickly studied by genetic techniques. Oddly slime mold may be the most useful of the three because it has been used for decades as a model system for lysosomal targeting.
 
=== Slime molds (Dictyostelium) ===
 
Although slime molds seem an improbable model system for mammals given the immense time since divergence from the last common ancestor, Dictyostelium works fairly well for the lysosomal sorting process via the M6P pathway, establishing its great antiquity, with 220 on-topic papers dating back decades. A phospholipase B, presumably from lysosomes, was [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133800/?tool=pubmed characterized] in 2004. Called PLBDa here, it is only one of seven found in the newly sequenced [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1352341/ Dictyostelium genome].
 
It is not immediately clear how this gene family expansion relates to mammalian PLBD1 and PLBD2. In one scenario, a single gene  in the ancestral stem  had expanded to the two PLBD1 and PLBD2 clades at the time of the last common ancestor. No further expansion occurred in the lineage leading to human but each clade expanded further in slime molds. This would cause the seven Dictyostelium genes to classify clearly with one branch or another of the gene tree. The data below do not support this.
 
[[Image:PLBDa dicDisPDB.png|left]]
 
A second scenario envisions a single gene at the time of species divergence. This duplicated subsequently in the lineage leading to human and expanded independently in Dictyostelium. This would give rise to the gene tree ((PLBD1, PLBD2),(dicDis subtree)). The data fit this better; it predicts a slightly later diverging species can be found in which the PLBD1/PLBD2 duplication had already occurred and the Dictyostelium-type genes are missing. The choanoflagellate Monosiga fits this description. It also predicts earlier diverging species than Dictyostelium will only have ambiguously classifying PLBDx (assuming ancestral features do not predominate in PLBD1 or PLBD2, respectively).
 
Note all seven genes are perfectly conserved for the six active site residues, meaning this had been established earlier. Thus any or all of them could be used as a generic structural model for mammalian PLBD1 or PLBD2, an important consideration in view of the similar processing, activation, and known substrate.
 
It can be seen from Blastp against SwissProt that all of the Dictyostelium proteins have been assigned accession numbers. This means they might have already been modeled by SwissModel using the mouse PLBD2 structure as template but in fact [http://swissmodel.expasy.org/repository/?pid=smr01&uid=&token=&zid=async have not], though modeling requests can be submitted using the 3FGW template. Note PLBDd and PLBDg are the most favorable for PLBD1; whereas PLBDb, PLBDc, and PLBDa have better matches to PLBD2. Only PLBDa has its substrates determined.
<br clear=all>
   
  [http://www.uniprot.org/uniprot/Q550U9 Q550U9]  PLBLA_DICDI  PLBDa_dicDis  SwissModel+ [http://swissmodel.expasy.org/workspace/index.php?func=additional_astex_page&userid=tom@cyber-dyne.com&prjid=P000006&pic=img_model_pic&model=1&token=&key=e8c4d8ab8db0f4c04227f5ebfdce3ca0 interactive]
  [http://www.uniprot.org/uniprot/Q55BJ6 Q55BJ6]   PLBLB_DICDI  PLBDb_dicDis  SwissModel-
  [http://www.uniprot.org/uniprot/Q54M94 Q54M94]  PLBLC_DICDI  PLBDc_dicDis  SwissModel-
  [http://www.uniprot.org/uniprot/Q54PS7 Q54PS7]  PLBLD_DICDI  PLBDd_dicDis  SwissModel-
[http://www.uniprot.org/uniprot/Q54ZI6 Q54ZI6]  PLBLE_DICDI  PLBDe_dicDis  SwissModel-
[http://www.uniprot.org/uniprot/Q554H5 Q554H5]  PLBLF_DICDI  PLBDf_dicDis  SwissModel-
[http://www.uniprot.org/uniprot/Q55FN1 Q55FN1]  PLBLG_DICDI  PLBDg_dicDis  SwissModel-
 
<font color = blue>PLBD1_homSap Homo sapiens (human) FLJ22662            2968  1.2e-313 100% blastp query PLBD1</font>
<font color = blue>PLBD1_musMus Mus musculus (mouse)                      2321  4.5e-245  79%</font>
<font color = blue>PLBD1_braFlo Branchiostoma floridae (lancelet)        1369  3.4e-144  50%</font>
<font color = blue>PLBD1_nemVec Nematostella vectensis (anemone)          1349  4.5e-142  51%</font>
<font color = blue>PLBD1_strPur Strongylocentrotus purpuratus (urchin)    1122  5.1e-118  47%</font>
<font color = blue>PLBD1_monBre Monosiga brevicollis (choanoflagellate)  1089  1.6e-114  51%</font>
PLBDd_dicDis Dictyostelium discoideum (slime_mold)      822  3.1e-86  37% d
PLBDg_dicDis Dictyostelium discoideum (slime_mold)      746  1.2e-84  36% g
<font color = brown>PLBD2_triAdh Trichoplax adhaerens (trichoplax)          721  1.2e-82  38%</font>
<font color = brown>PLBD2_braFlo Branchiostoma floridae (lancelet)          722  2.0e-80  36%</font>
PLBDb_dicDis Dictyostelium discoideum (slime_mold)      762  7.2e-80  34% b
PLBDc_dicDis Dictyostelium discoideum (slime_mold)      733  8.5e-77  35% c
PLBDa_dicDis Dictyostelium discoideum (slime_mold)      717  4.2e-75  33% a
PLBDe_dicDis Dictyostelium discoideum (slime_mold)      717  4.2e-75  35% e
<font color = brown>PLBD2_acyPis Acyrthosiphon pisum (aphid)                667  4.7e-75  37%</font>
<font color = brown>PLBD2_homSap Homo sapiens (human) LOC196463            639  2.0e-72  36%</font>
<font color = brown>PLBD2_musMus Mus musculus (mouse)                      631  6.0e-71  37%</font>
PLBDf_dicDis Dictyostelium discoideum (slime_mold)      640  6.1e-67  32% f
<font color = brown>PLBD2_monBre Monosiga brevicollis (choanoflagellate)    547  3.3e-66  32%</font>
<font color = magenta>PLBDx_droMel Drosophila melanogaster (fruitfly)        142  5.5e-19  32%</font>
<font color = brown>PLBD2_homSap Homo sapiens (human) LOC196463            3142  0.0      100%  blastp query PLBD2</font>
<font color = brown>PLBD2_musMus Mus musculus (mouse)                      2571  1.4e-271  81%</font>
<font color = brown>PLBD2_braFlo Branchiostoma floridae (lancelet)        1528  4.8e-161  54%</font>
<font color = brown>PLBD2_triAdh Trichoplax adhaerens (trichoplax)        1293  3.9e-136  50%</font>
<font color = brown>PLBD2_acyPis Acyrthosiphon pisum (aphid)              1129  9.2e-119  47%</font>
PLBDb_dicDis Dictyostelium discoideum (slime_mold)      873  1.2e-91  39% b
<font color = brown>PLBD2_monBre Monosiga brevicollis (choanoflagellate)    835  1.3e-87  38%</font>
PLBDc_dicDis Dictyostelium discoideum (slime_mold)      819  6.5e-86  39% c
PLBDa_dicDis Dictyostelium discoideum (slime_mold)      805  2.0e-84  38% a
<font color = blue>PLBD1_nemVec Nematostella vectensis (anemone)          702  1.6e-78  38%</font>
<font color = blue>PLBD1_musMus Mus musculus (mouse)                      688  5.9e-77  38%</font>
PLBDd_dicDis Dictyostelium discoideum (slime_mold)      731  1.4e-76  37% d
<font color = blue>PLBD1_homSap Homo sapiens (human) FLJ22662              639  2.0e-72  36%</font>
PLBDg_dicDis Dictyostelium discoideum (slime_mold)      685  1.0e-71  36% g
PLBDe_dicDis Dictyostelium discoideum (slime_mold)      661  3.6e-69  32% e
<font color = blue>PLBD1_braFlo Branchiostoma floridae (lancelet)          652  3.2e-68  36%</font>
<font color = blue>PLBD1_monBre Monosiga brevicollis (choanoflagellate)    646  1.4e-67  38%</font>
PLBDf_dicDis Dictyostelium discoideum (slime_mold)      640  6.1e-67  35% f
<font color = blue>PLBD1_strPur Strongylocentrotus purpuratus (urchin)    534  7.7e-61  35%</font>
<font color = magenta>PLBDx_droMel Drosophila melanogaster (fruitfly)        218  2.9e-38  30%</font>
Alignment of PLBDa_dicDis with conserved residues of PLBD1 or PLBD2 shows no clear classification:
VTDLNMAQQFVSEALNGPSTDGDLPPFTWDA--FNRTTHQGLPRLYNYTFVTM best PLBD1 match
V  +M    +  A +GPS D + P FTW++  +N+ T+ G P  +N+ ++TM
VVSADMVAALLVNAQSGPSHDNETP-FTWNSQWNQKYTYAGQPTTWNFDWMTM PLBDa_dicDis
+ S  MV  L + AQSGP+ D + P F W+      ++ GQP  W+F
ITSKAMVPRLEMVAQSGPTWDQQPP FQWSKSPFSSLSHVGQPDLWSFLPEHI best PLBD2 match
V.D...A...............gL.......--Fn.t............F..m.P.L conserved PLBD1 not PLBD2
.............A..GPt.....P.F.W.........H.G.P..w.F........  conserved PLBD1 and PLBD2
.Ts............S...-WDq......S.SP......m...D.....P..v.W.. conserved PLBD2 not PLBD1
 
=== Arthropods (Drosophila) ===
 
The standard fruitfly has a very odd PLBD2-type gene product LAMA (lamina ancestor) that has been the subject of [http://www.ncbi.nlm.nih.gov/pubmed/8892229,16077094 two publications]. Note first that a poor choice of name has lead to widespread confusion: this protein has nothing whatsoever to do with the important vertebrate protein laminin (whose homolog in fly is called LamA). Instead it has to do with an obscure neuroanatomical structure in insects called the lamina.
 
The gene, call PLBD2x here, is so diverged from conventional phospholipases that a number of hypotheses have to be considered:
 
* Fraud: It is not unusual for annotational hoaxes to be perpetrated in the scientific literature, complete with bogus submissions to GenBank. Here however simple tBlastn searches show that the most recent assembly of the drosophila genome does contain a letter-perfect match to the LAMA entry and further that this protein has a more or less a full length match to conventional vertebrate PLBD2. This is beyond anyone's ability to manipulate.
 
* Pseudogene: This is a large protein of almost 600 amino acids. Should it have lost its function, rapid divergence would result (as seen), with accrual of frameshifts and stop codons (not seen). The divergence would occur equally rapidly at ultra-conserved residues as at ones not under selection (not seen), either in D. melanogaster or one of the other 11 drosophilids with sequenced genome (not seen). However recent pseudogenization in certain lineages cannot be ruled out as comparative genomics becomes insufficient.
 
* Horizontal transfer: The LAMA protein might fail to nest within the gene tree as expected from the fruitfly position in the species tree. This happens rarely in metazoan from gene transfer from other species, notably parasites, commensals, and intra-cellular symbionts. In this case, tBlastn would locate the appropriate clade, even if the exact species had no data because genomic coverage is so pervasive today. However the LAMA gene has no outside affinities.
 
* Role change: If this gene lost its original function and acquired another unrelated one, then the selection profile could change radically across the gene. For example, if the catalytic function were redirected, then the six active site residues might experience relaxed constraint. The fold itself might be conserved to assure structural stability but otherwise rapid evolution to perfect the new role might alter many otherwise conserved residues. Here the change is so rapid and so drastic that a simple change to a new substrate can be ruled out -- indeed, the two experimental papers provide evidence for a regulatory role.
 
* Role loss: Along the lines of [http://en.wikipedia.org/wiki/Gene_sharing Piatigorsky], a single gene product may have multiple roles, sometimes quite distinct (eg aldehyde dehydrogenase ALDH3A1 also serving as a lens crystallin). Should the gene subsequently be duplicated, these pre-existing roles can be split up in the two copies (rather than one copy continue with the parental role and the other copy scramble to acquire a new adaptive function). Selection initially acts on all the functions in proportion to their importance.
 
[[Image:DroMelPLDB2X.png|left]]
 
In this last scenario, phospolipase activity was lost, leaving selection to act solely on the residual regulatory function. However within the 12 drosophilid genomes, the PLBD2 are quite diverged, with percent identity quickly ranging from 75% down to 45%. This suggests the protein is not yet well adapted to what was initially a secondary function or that few of its features are needed.
 
This raises the question whether PLBD2 also serves a cryptic non-catalytic function in other species (eg mammals). It is thus important to date the onset of loss of phospholipase activity (coupled with retention of within the PLBD2 locus) within arthropods because this also provides some dating of the secondary function origin. This can be dated to the last tens of millions of years because only a few other insects (eg tsetse Glossina morsitans, [http://www.ncbi.nlm.nih.gov/nucleotide/289740208 EZ422576]) outside drosophilids carry a comparable gene.
 
Within insects, some species such as Acyrthosiphon carry both a normal and derived PLBD2. However the normal form has been lost in other insects with derived PLBD2. Although no chelicerate has the derived PLBD2, Ixodes has multiple copies of normal PLBD2, some closely related and others fairly diverged but still clearly classifying as  PLBD2. Molluscs, annelids, and trematodes have PLBD2 but not PLBD1 or PLBD2x.
 
The loss of active site residues characteristic of NTN hydratases and more specifically those shared by both PLBD1 and PLBD2 phospholipases is taken here as a proxy for loss of phospholipase (and indeed all) catalytic activity.
 
Active site residues are lost in derived PLBD2 though the first disulfide is retained:
                <---- human active site ----> <-- human disulfides --> <-- glycosylation-->
<font color=blue>PLBD2_homSap  C244 H261 W264 T325 N427 R458  C142-C152 C492-C495        N436S N515S</font>
PLBD2x_droMel  A216 <font color=blue>H238</font> A241 G305 G438  ?      <font color=blue>C98-C108</font>
PLBD2x_glomor  A210 <font color=blue>H231</font> A234 G298 G424  ?     <font color=blue>C94-C104</font>
PLBD2x_acrPis  A233 Q250 S253 V315 S413 V436  <font color=blue>C132-C139</font>
<font color=blue>PLBD2_acrPis  C208 H225 W228 T288 N390 R421  C112-C119 C434-C437        N399S N477T</font>
 
Unless there is some counterpart to the secondary function, the tempo and mode of evolution of the LAMA protein makes it largely irrelevant to its human ortholog and lysosomal storage diseases. Indeed, fruitflies have in effect lost both PLBD1 and PLBD2, proving they are non-essential to lysosome function.
 
Drosophila PLDB2x at SwissModel: little of the original fold remains:
PLDB2x    45        CATAL WTKQV-GFQI ENWKQQNDLV NIPTGVGRIC YKDSVYENGW
3fgrA    63    vsr--trsll ldaasgqlrl ed-----g-- fhpdavawan ltnairetgw
                      ssss ss    ssss s              sssss sssss  ss
                sss  sssss ss    ssss ss    s        sssss sssss  ss
PLDB2x    89    AQIEVETQRT YPDWVQAYAA GMLEGSLTWR NIYNQWSNTI SSSCERDEST
3fgrA    104  ayldlstngr yndslqayaa gvveasvsee liymhwmntv vnycgpfeye
                ssssssss    hhhhhhhh hhhhhh  h hhhhhhhh           
                ssssssss    hhhhhhhh hhhhh  hh hhhhhhhh           
PLDB2x    139  QKFCGWLRDL LTTNYHRLKR QTEKAENDHY WHQLHLFITQ LEGLETGYKR
3fgrA    154  vgyceklknf leanlewmqr emelnpdspy whqvrltllq lkgledsyeg
                hhhhhhhhhh hhhhhhhhhh hhhh    hh hhhhhhhhhh hhhhhhhhh
                hhhhhhhhhh hhhhhhhhhh hhhh    hh hhhhhhhhhh hhhhhhhhh
PLDB2x    189  GASRARSDLE EEIPFSDFLL MNAAADIQDL KIYYENY -- -       
3fgrA    204  rltfptgr-- ftikplgfll lqisgdledl epalnktgsg s       
                                    hh h  hhhhhh hhh                 
                                    hh h  hhhhhh hhh
 
=== Nematodes (C. elegans) ===
 
Potentially a better model organism than fruitfly, C. elegans has 3 phospholipase B genes, with the best match to human PLBD2 having 44% identity. These appear to be a lineage-specific expansion of PLBD2, whereas PLBD1 has been lost from this and other nematodes. (The C. elegans genome is assuredly complete and the detection methods are sufficiently sensitive.)
 
>PLBD2a_caeEle Caenorhabditis elegans (nematode) NP_499668 Y37D8A
MNWIFIFLAAAVAIGCEARQERTYTVCQKPEGDLHYFKEGRKTDEELCAKRLATAYFHDEVNQTGWAFLEVDVISPKIPHYLQGYAAGFAEGRATRHLID
LHIINTVNGYCDGAKHFCDELGEFMVDNLKWMEQEIRENPEDEYWQQVNLTVNQLFGLIHGYENQLGAEIDFKQIAVHPIFMIQIAGDLEDLAMKFKKPE
NPKKVFSGPGHCSALVKLLPKNEDILFSHVTWSSYGTMLRINKKYSFKTGDPGQIYSFSSYPASITSTDDFVLTSAKLAILETTIGNYNEKSLDLITPNT
VLTWIRAEIAHRTASSGLQWAEAFGRHNSGTYNNEWVVVDYKQFHRGKEVQPETGIIHVVEQMPGHIVHSDKTAHLFRETYWPGYNQPYYKQIIRFSDTD
KMVEKFGDWYSYDKTPRALIFKRDHNTVTDMSSMIALMRSNNYTKDPLSKCDCNPPYSAENAIACRSDLNPLNGTYPFKSLGFRDHGAIDVKVTNSKLIN
SLQFTAVSGPPGGVTKDVPIFDWKTSPLREKVPHFGQPDRWNFAPVTYKWRKDAHRHYHLYQKLHKELSSL*
>PLBD2b_caeEle Caenorhabditis elegans (nematode) NP_510509 F09B12
MTRLIRSKKQFLIRSLHSVFYYLGSLLHSTFEMNVFIGLLLATVVASQSSEGRDESYTYKQLCIVDDKPQVLDGFDCRNQVAVARWQNAVNTTGWTFLEV
ETKENYCPQLQAYSAGYLEGLLSKTVLTYHLKNAQEDYCKNFTGYCSRLSDFLTENQKWIQSSLETVAPDDLYWGAVNRTYHQVSGLIDAYEGREFKPRI
TYELHPILYLNLNGDFYDLEKKLNKTRDPAFEQTGGKCSGLIKVAPGNADLFISQVTMSGFQNMLRVIKLYKFGYDRQFYPGYASSFSSYPGLLYSSDDF
ALQTSGLAVIETTISVFNTSLFENTKPVGQLPTWIRAIVSNQLARDAREWCKLYSLYNSGTYNNQWAVLDYKKFKPNQPLPKNGLFYVLEQMPGKIVYSD
LTWFVEKYSYFPSYNIPFFKEITEISGFIGQAAKMGDWFKWGASPRAKIFERDHGNVHDLDSLTALMRYNDYKNDEFSKCKCNPPYSAEAGISARGDLNP
ANGTYEFPGQGHVNHGALDYKGTNVELMKKLQFVAQGGPTWGKVPSFKWSEFDFKDKVNHVGHPDEWKFNTLVHKWETEINA*
>PLBD2c_caeEle Caenorhabditis elegans (nematode) NP_497570 Y54F10AM
MKLLFFLFGLIFAVEQEKPYLDNNRVPVEQILNDHSSAKFDYTYVSVCVNSTDETLLDIVYAKECKNAASRVALGKYSNQVNTTGWGILEIETFASHSYD
VQAYGAGVAEGELTRLQIYYHYRNTIETMCNNHTLFCKRLYIYLQQNLDWMRSQVQANPPTDPFWRQVNLTFAQLTGIYDAYSKRNLTPEIGFDLHPIYM
  MQLAGDMFDLNKLLNKTADPMEYPEGGRCSGFVKLAPGNKDMFMAHVSMSSLSWMQRVLKIYKFGYDVNEVPGHIVTFSGYPGVLISTDDYTITSAGLTS
IETTIAIFNQTLYTDKFMKPEGQVHCWIRSMISNLLSRTGKQWVDMFGRYNSGTYNNQWTVLDWKQFTPEKELPDKDVLWISEQTPGYYETRDMTWYLKK
YTYFASYNIPFLPKVSEISGFDNKARQFAWFDWGGSPRARIFDRDHSKVTDIDSLTKLMRYNDYTHEEFARCKCTPNPYTGEGGISARGDLNTPGGTYEV
ESMGFRDHAGLDFKGTNYEMFKKMRFRAWGGPPYDPLPVFDWNHTNLTNVRHFGQPDVWNFTYVDLEWQLAAQVQLTPYDN*
 
== Reference sequences ==
 
These two genes are well represented at GenBank though some predicted gene models are erroneous. A genomic alignment of 46 species of vertebrates at UCSC (under protein fasta section of the gene details page) allows this large set of orthologs to be collected as needed. These sequences -- which are no better than the underlying assemblies -- also contain extensional errors at the termini. Insertions relative to human also are not shown. However these sequences provide a strong starting point for correction at the wgs contig division of NCBI blast.
 
Below, only a few key sequences are shown -- ones with an experimental literature and some from deeply diverged pre-bilaterans. These have been intronated by blast of protein against genome assemblies.
 
A separate section holds anomalous PLBD2x insect sequences. These resulted from a duplication of PLBD2, followed bu severe specialization of one copy and often loss of the original. These nonetheless will retain most features of the fold, though use of SwissModel here would require considerable intervention after the fact. Despite an experimental literature, these are largely irrelevant to human considerations.
 
In the last section, sequences have been trimmed to the region covered by the xray structural determination. Species with one-off insertions that confuse gap placement have been adjusted. This helps avoid artifacts in alignment.


=== PLBD1 reference sequences ===
=== PLBD1 reference sequences ===
Line 36: Line 969:
  2 NYKKDPYSRGDPCNTICCREDLNSPNPSPGGCYDTK 0
  2 NYKKDPYSRGDPCNTICCREDLNSPNPSPGGCYDTK 0
  0 VADIYLASQYTSYAISGPTVQGGLPVFRWDRFNKTLHQGMPEVYNFDFITMKPILKLDIK* 0
  0 VADIYLASQYTSYAISGPTVQGGLPVFRWDRFNKTLHQGMPEVYNFDFITMKPILKLDIK* 0
>PLBD1_musMus Mus musculus (mouse) NM_025806 note earlier stop codon
0 MCHRSPGRSLRPPSPLLLLLPLLLQPPWAAGAASQSDPT 1
2 GVHCATAYWSPESKKVEIKTVLDKNGDAYGYYNDSIKTTGWGILEIRAGYGSQVLSNEIIMFLAGYLEGYLTAL 2
1 HMYDHFTNLYPQLFKNPSIVKKVQDFME 2
1 KQEMWTRQNIKAQKDDPFWRHTGYVVTQLDGLYLGAQKRASEEKIK 0
0 PMTMFQIQFLNAVGDLLDLIPSLSPTKSSSMMKFKIWEMGHCSALIK 0
0 VLPGFENIYFAHSSWYTYAAMLRIYKHWDFNIKDKYTLSKRLSFSSYP 1
2 GFLESLDDFYILSSGLILLQTTNSVYNKTLLKQVVPKTLLAWQRVRVANMMAEGGKEWAQIFSKHNS 1
2 GTYNNQYMVLDLKKVTINRSLDKGTLYIVEQIPTYVEYSDQTNVLRK 1
2 GYWASYNIPFHKTIYNWSGYPLLVHKLGLDYSYDLAPRAKIFRRDQGNVTDMASMKYIMRYN 1
2 NYKEDPYSKGDPCSTICCREDLNGASPSPGGCYDTK 0
0 VADIFLASQYKAYAISGPTVQDGLPPFNWNRFNDTLHRGMPEVFDFNFVTMKPILS* 0
   
   
  >PLBD1_braFlo Branchiostoma floridae (lancelet) XM_002595538
  >PLBD1_braFlo Branchiostoma floridae (lancelet) XM_002595538
Line 51: Line 997:
  0 VSDYYLARNLTSFAINGPTLGTGLEPFSWSDKFKISHIGLPKVYNFSFVTMTPAEL* 0
  0 VSDYYLARNLTSFAINGPTLGTGLEPFSWSDKFKISHIGLPKVYNFSFVTMTPAEL* 0
   
   
>PLBD1_strPur Strongylocentrotus purpuratus (urchin) XM_001192029
0 MANKFRMFKILTAFLVLVLVNLST 1
2 GELLQGTVYKQEDGTFTVSSGIIDKQGVAYGSYNNTLFQTGWGELHLFAGYSTADNVALSDADRMYAAGILEGALTAK 2
1 QISQTLRNINVTFFSAESDPEIWRRVADFFETQDAWMKGMIIERADEDPFWEGVGLVLAQFEGLIKGYEMSQFSNAST 0
0 SNGFLAMQVLNSCGDLLDLKSAVMPSLIPDWDKLTKKEFLKFIRTSGHCSALVK 0
0 ICAALVKVGRFAPPFQSLLYSIS 0
0 SYFKSQAILKLNSPSCQLFGIE 1
2 GFLESLDDFYIMSSGLSMLQTTNNIFNKTLYKYVKPQSLLAWQRVRVANMMARSGKDWARIVARYNS 1
2 GTYNNQYMVIDRTKIKPNVAILDDALWVVEQVPTLVASGDQTNILRA 1
2 GYWPSYNVPFYEEIYNISGYPEYAYKGGADISYQLAPRAKIFRRDQGNVVDMESFKKIMRFN 1
2 DYKNDPYSEGDPSKSICMRGDLMTSPMPNGCYDTK0
0 VTNLAMAAKQTSFVINGPTRGDGSLPPFKWVAPFTGWSHVGLPTVYDFNFVEMCPKEL* 0
>PLBD1_nemVec Nematostella vectensis (anemone) XM_001638165
0 MTLIRNSVMITVTFVLILFVFGCHGSQKSATVYYNRGQG 2
1 YSLKFGVVDKLMGVAYGTFEDSLNTTG 2
1 WYELNIVSGTGIEPYNDDVIMHAAGYLEGALTAS 2
1 QINDNYANLYGVFFKSEDDPMVAKVEKFFIEQ 0
0 DIWMRKMIALKSSNSSFWRQMGNIIAQFD 1
2 GLVEGYQKYPATDK 0
0 ALGVFAFQMLNGVGDLLDLTKALMPERMADWDHMTEKEILEK 0
0 VAMDGHCSALIKVLPAYENVFASHVS 2
1 WFTYSAMLRVYKHYHLNLKDETT 1
2 AAQRMSFSSYPGFLESLDDFYIMDS 2
1 KLVMLQTTNNVFNKSLYEQVVPESLFSWQRVRLANLVASSGRQWADIVGQYNS 1
2 GTYNNQYMVLDLKLIQLNNTIQDNALWVVEQIPT 2
1 LVASGDQTAILRAGYWPSYNVPFYEL 0
0 VYNLSGYPDFVARHGVQFSHELAPRAKIFRRDQSM 0
0 VHDLDSMKHIMRYNDFQHDPYSQGNPMNAICSRGDLIADGPRASGCYDGK 0
0 VTDFTMAQSLISHAINGPTHE 0
0 QQVPFHWSQYQFKNKHEGQ 0
0 PDLFNFDFVEMKPKF* 0
>PLBD1_monBre Monosiga brevicollis (choanoflagellate) XM_001745398
MSSLNNGIPEPLLKFLAAQFNWTRSQVAANQDDVFWQQVGLIMA
QYDGLRAGYGANVYDKHVLPEFAFQLLNGNGDFFDIIPKAVDVTKMSSREFHDWRMRN
GRCSALIKLTGDFSDLFMSHSAWYIYQAMNRIYKHCASYNFQATITHAKKISFSSYPG
YLESLDDFYLMSSGLVMLQTTNNVFNTDLQQYIQPESLQSWIRIRTATALAQTSEDWA
ELAGRHNSGTYNNQYMVMDLNKFTPGQPLLDGTLYVAEQIPGTWEYADVTKMLSLGYW
PSYNVPFFEKIYNLSGYPAVVKQHGTDDSYELAPRAKIFRRDQTTVVDLDSFKAIMRY
NDYKNDPYAKGDPYNAICSRGDLESDSPSPGGCYDTKVTTYSMALKLQSQVINGPTTS
HGLPPFSWSQFPNASHLGMPEVFNFTFETMDAGW*
=== PLBD2 reference sequences ===  
=== PLBD2 reference sequences ===  


  >PLBD2_homSap Homo sapiens (human) PMID: 19706171,19237744,17007843
  >PLBD2_homSap Homo sapiens (human) LOC196463 PMID: 19706171,19237744,17007843,17105447
  0 MVGQMYCYPGSHLARALTRALALALVLALLVGPFLSGLAGAIPAPGGRWARDGQVPPASRSRSVLLDVSAGQLLMVDGRHPDAVAWANLTNAIRETG 2
  0 MVGQMYCYPGSHLARALTRALALALVLALLVGPFLSGLAGAIPAPGGRWARDGQVPPASRSRSVLLDVSAGQLLMVDGRHPDAVAWANLTNAIRETG 2
  1 WAFLELGTSGQYNDSLQAYAAGVVEAAVSEE 0
  1 WAFLELGTSGQYNDSLQAYAAGVVEAAVSEE 0
Line 66: Line 1,055:
  1 YNDFLHDPLSLCKACNPQPNGENAISARSDLNPANGSYPFQALRQRSHGGIDVK 0
  1 YNDFLHDPLSLCKACNPQPNGENAISARSDLNPANGSYPFQALRQRSHGGIDVK 0
  0 VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD* 0
  0 VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD* 0
>PLBD2_musMus Mus musculus (mouse) NM_023625
0 MAAPVDGSSGGWAARALRRALALTSLTTLALLASLTGLLLSGPAGALPTLGPGWQRQNPDPPVSRTRSLLLDAASGQLRLEDGFHPDAVAWANLTNAIRETG 2
1 WAYLDLSTNGRYNDSLQAYAAGVVEASVSEE 0
0 LIYMHWMNTVVNYCGPFEYEVGYCEKLKNFLEANLEWMQREMELNPDSPYWHQ 0
0 VRLTLLQLKGLEDSYEGRLTFPTGRFTIKPLGFL 2
1 LLQISGDLEDLEPALNKTNTKPSLGSGSCSALIKLLPGGHDLLVAHNTWNSYQNMLRIIKKYRLQFREGPQ 1
2 EEYPLVAGNNLVFSSYPGTIFSGDDFYILGSGL 0
0 VTLETTIGNKNPALWKYVQPQGCVLEWIRNVVANRLALDGATWADVFKRFNSGT 2
1 YNNQWMIVDYKAFLPNGPSPGSRVLTILEQIP 2
1 GMVVVADKTAELYKTTYWASYNIP 2
1 YFETVFNASGLQALVAQYGDWFSYTKNPRAKIFQRDQSLVEDMDAMVR 0
0 LMRYNDFLHDPLSLCEACNPKPNAENAISARSDLNPANGSYPFQALHQRAHGGIDVK 0
0 VTSFTLAKYMSMLAASGPTWDQCPPFQWSKSPFHSMLHMGQPDLWMFSPIRVPWD* 0
   
   
  >PLBD2_braFlo Branchiostoma floridae (lancelet) XM_002612057
  >PLBD2_braFlo Branchiostoma floridae (lancelet) XM_002612057
Line 80: Line 1,083:
  0 MTSYSMHESHQMMAVSGPTHDQQQPFQWSTSDYDKQFYHLGHPDLFNFDPIHVIWFDQSDN* 0
  0 MTSYSMHESHQMMAVSGPTHDQQQPFQWSTSDYDKQFYHLGHPDLFNFDPIHVIWFDQSDN* 0
   
   
  >PLBD2_droMel Drosophila melanogaster (fruitfly) U57314 retinal lamina neuron ancestor (lama) PMID: 16077094,8892229
  >PLBD2_acyPis Acyrthosiphon pisum (aphid) XM_001948827
  MERPEYDGTYCATALWTKQVGFQIENWKQQNDLVNIPTGVGRICYKDSVYENGWAQIEVETQRTYPDWVQAYAAGMLEGSLTWRNIYNQWSNTISSSCERDESTQKFCGWLRDLLTTNYHRLKRQT
0 MLSIRCILLSLLFVWALQCSATQKNQTLLAVKTDNNRITIQPKHYSVKDKEIIIGKGKFIDRINSTG 2
  EKAENDHYWHQLHLFITQLEGLETGYKRGASRARSDLEEEIPFSDFLLMNAAADIQDLKIYYENYELQNSTEHTEEPRTDQPKNFFLPSATMLTKIVQEEESPQVLQLLFGHSTAGSYSSMLRIQK
1 WAYLEIRTSQKAKDEDQAYGAGYLEGTLTADLIYSYWFNTAKGYCTDRPNVCQQLKDYMTTNKNWIKSKLNESDPYWYQ 0
  RYKFHYHFSSKLRSNTVPGVDITFTGYPGILGSTDDFYTIKGRHLHAIVGGVGIKNENLQLWKTVDPKKMVPLVARVMAANRISQNRQTWASAMSRHPFTGAKQWITVDLNKMKVQDNLYNVLEGD
0 VGLYYKQLDGLYDGYMRGKSPSTPDLTWDDLY 2
  DKHDDAPVVLNEKDRTAIQQRHDQLRDMVWIAEQLPGMMTKKDVTQGFLVPGNTSWLANGVPYFKNVLELSGVNYSEDQQLTVADEEELTSLASVDKYLRTHGFRGDLLGSQESIAYGNIDLKLFS
1 WLNALDDLGDLSIALYPSDISNRVLGSGSCSALIKLMPDNKDILVSHATWSG 2
  YNARLGISDFHAFAGPVFLRFQHTQPRTLEDEGQDGGVPPAASMGDERLSVSIEDADSLAEMELITERRSVRNDMRAIAMRKIGSGPFKWSEMSPVEEGGGHEGHPDEWNFDKVSPKWAW*
1 YETMLRIQKRYSLRFRKSKKSNKLIRGFDMSFSSFPGGIQSGDDFYLISSGLTTMETTIENYNDSLWSNVKPVGQ 0
0 VLEFVRAMVANRLADNPTDWANLFKLHNSGTYNNQWMILNYAAFQPGSPLPPRDVLHVLEQIPGHVMHDDFTGHLINRTYWASYNVPYFPFIFNVSGNYEMEQIYGSW 2
1 FSYSETPRARIFARDHVKIHCDKCMLHLMRSNNYTRDPESRCDCSPPYSAENAISSR 2
1 NDLNPANGTYPIRALGHRSHGATDVKVTSSQLFQQLQFKAIAGPTQGSNNSLGPFCWSKSDFNDKVSHLGHPDCFNFKPVLHQWSL* 0
>PLBD2_lepSal Lepeophtheirus salmonis (crustacean)
  MEILKGSIFPIFLLISTRALGTSSLKHSLSYDGKDFTLLPGHVSGSNILLHGSFVNLINQTGWSILKINTMSHFPDNIQAYGAGYIEAYMTKDLIYNHWR
NTIVGYCDGKEKICEGIQGFLSKNLNYIRSMTTNSKREKSSYWHHVALFYDQIRGITEGYNKYAPPGRSISAEDIFFMNVMGDVEDLEQVFSHRLNISLP
  DKVLGSGSCSALIRLLPGNRDLYAAHDTWNSYQSMLRIIKKYKFGLHWVLQKFPEKDDKHTIPGRSMSFSGYPGTVFSMDDFNVIPSSGLVSIETTIGNG
  NASLWQYVQPIGTVLEGIRATVANRLARSGGHWVSVFSKRNSGTYNNQWMVVDYKRFKPGEKIIRPGLLWVLEQIPGRIHYTDVTGVLKKIGYWPSYNSP
  YFKDIFNMSGGPEAVAKFGNWFSYEKTPRALIFKRDAPKVHNMGSMMKIMRYNDFRHDPISRCNCTPPYSAENAISARCDLNPKNGTYPFGALGHRSHGG
  TDMKITSFDLFMKGSFIAGGGPTYDSVEPFQWSKADFGKTTPHFGHPDKWAFKPIKVDWENAFNRGEDVV*
   
   
  >PLBD2_acyPis Acyrthosiphon pisum (aphid) XM_001948827
  >PLBD2a_ixoSca Ixodes scapularis (chelicerate) XM_002413831
  MLSIRCILLSLLFVWALQCSATQKNQTLLAVKTDNNRITIQPKHYSVKDKEIIIGKGKFIDRINSTGWAYLEIRTSQKAKDEDQAYGAGYLEGTLTADLIYSYWFNTAKGYCTDRPNVCQQLK
  MKNSYAPRFPSHVLGGSGSEFFCSKLNVYCSSTHRNMFVTLAVSALLALQAAAETPGDASCQYAWASLGPSGGDLHIHDGKPGQGAVAWGTFQNDIRFSG
  DYMTTNKNWIKSKLNESDPYWYQVGLYYKQLDGLYDGYMRGKSPSTPDLTWDDLYWLNALDDLGDLSIALYPSDISNRVLGSGSCSALIKLMPDNKDILVSHATWSGYETMLRIQKRYSLRFRKS
  WAFLDLQSNASYADDVQAYASGAVEAHLTRDLIEKHYNNMYSRYCDGQSEYCERLSKFLQENIKYSNDQEHLYYSTDPYWHMVHLQMKQLAGLSDHLENK
  KKSNKLIRGFDMSFSSFPGGIQSGDDFYLISSGLTTMETTIENYNDSLWSNVKPVGQVLEFVRAMVANRLADNPTDWANLFKLHNSGTYNNQWMILNYAAFQPGSPLPPRDVLHVLEQIPGHVMHD
  ALNTSNEYLNVTRALYLNLDGDLMDLEAYLRRVGDENSIFQTNACSVLIKVLPDNEDILFAHNTWFLYRSMLRIEKKYTFPWHVTSESSEIIPGHTISMP
  DFTGHLINRTYWASYNVPYFPFIFNVSGNYEMEQIYGSWFSYSETPRARIFARDHVKIHCDKCMLHLMRSNNYTRD
  SYPGKLLSLDDFYIASTGLAITETSLENNNDSLRNLIKPENAPLTWVRCMVATRLAVTGSQWVEYFGKLNSGTLNNQWMVLDYKLFTPGAAIGKDTFWIL
  PESRCDCSPPYSAENAISSRNDLNPANGTYPIRALGHRSHGATDVKVTSSQLFQQLQFKAIAGPTQGSNNSLGPFCWSKSDFNDKVSHLGHPDCFNFKPVLHQWSL*
  EQMPNTTKTKDVSEYLQNQKYWASYNIPFLPEIFNLSGQPQLVKKYGNYYSHDMCPRAQLFRREQGKVEDVDSMTALMRYNDYTHDPVSGCNCTPPYNAI
YAISSRYDLLDPEGSYDMPNMARRAVGATDMKLTNYGMSRRLEFIAINGPTYTNDSSVPPFQWSTSGFQDLHEGHPDKWTFGPTHHRWDSCPNF*
>PLBD2b_ixoSca Ixodes scapularis (chelicerate) XM_002405205 frag
98 TGWSSIVVHTNEFYLDDQQAFAAGLVEGRLTRTLIQEQFTNEFNKYCEEDPQFCAELFKYLHANIESMVANVAEYSSTDPYWHQVRKTAFYDKPTEQRNK
ACFGHSNQPVNEFFYTKRLLNLHGDLSDLETKFNRTNSRKINPTGSGSCSAIVKLAPGSRDVYFAHATWTHYNSMNRILKKYRLNYHTAPMSKDLIPGSS
IVFSGYPARIFSGDDFYLINTGLAVMETTIGNENSDLWKKVTPIDTVPTSIRNMVANRLSLTGQHWTNTFARFNSGTYNNEWMVLDYKRFESTSQKLNRG
FLWILEQIPGLVMSKDVTPILEEQGYWASYNSPYFTEIFNESGLPALVEKYGDWYTYDKSPRALMFKRDQEKAVDLRSVMKLMRYNDFTNDPLSRCTGCD
PPYSAENAISARSDLNPANGTYPFKALGRRAHGGTDAKVTNFALFERQEFYAVSGPTSDDQRPFRWSTSGFDNVSHAGHPDLWDFDPILAQWRY*
>PLBD2_plaOce Placobranchus ocellatus (mollusc) HP171048 
MAIIMNSRLCVQLFVAISIFSLCNAAVTKLWVTYNTESSIFEVSDQQATDYVAVASFDNTVNQTGWAKLDVITQAGPKRKYNDSVQAYAAGFVEGHITKS
LMTMHWANTGAWVCPEPLTSQCIQIKKFLESNLKWVLENIKTFSTTSPFWHHVRLFLEQTAGLQDGFAGMKGQLNLDIDVMSVYMWQVGGDMETLLNVFT
GSEDNQYKTPDPLAMGMGHCSALLRLLPDYSDLYVAQDTWSDFSSMLRVLKRYSFEMLKSPQYPDIPAPGNTMTFSSYPGVLFSGDDFYIMSSGLVSQET
TIGYANSSLNQYIKPTSVLEGVRTMVANRLAASGREWAQIFSLINSGTYNNEWMIVDYKLFRPGQVIPVPGLFTVLDQIPGTIVYDDLTLHLYKYKYFGS
YNTPFFPEVFNKSGQPALVAKFGDWFTHDKTPRALIFKRDAPKVNDLNSMIKLMRYNDFKHDPLSRCNCTPPYSAENAIAARCDLNPANGTYPFGALGHR
PHTATDMKVTTFEMAKSLSFRAVSSPPwDDLPVFQWSTSSF*
>PLBD2_aplCal Aplysia californica (mollusc) est frag
YPGPLMSVDDFYAISSGLVSQETTIGFSNAELVKYITSEAVMEGFRSMVANRLASSGGEGSKLFAQYNSGTYNNQWMIVDYKRFVPSRETGVPGTLYLIE
QIPGTIEYADLTDYLYEKSYFGSYNVPYFPDIFNKSGLPPLVKKLGDWFTYDKNPRALIYKRECSKVTDLDSMTKLMRYNYFKNDPLSRCNCTPPYSAEN
AISCRDDLNPANATYPFGALGH
>PLBD2_helRob Helobdella robusta (annelid) EY564899 frag
FAYLEIQTNEKVSDIEQAYTAGLAEGLLTSDLIKLHWYNTFQDYCKAPLNEFCAKLQNAMEANFNWMANQIFLEANLNPFWHQVELILVQLHGLIDGYKF
EQRPRDTLTKFVFKPDVTGLLMLFLSGDISDLESVLGGNKFNVYKPFDHLSGPNTCSALIKLLPNNKDLLISQTTRSNYGTMLRILKKYDFGYHTVTGGQ
LVPAKVISFSSYPGVQFSVDDFYLLSSGLVAQETTIGNSNADRWKLVKPESLLEVIRNMVANRLATTGEEWADLFVRYNSGTYNNQWMVLNYKLFTPGAK
EIKDGLLYVVEQIPGLTVGQDVTFVLRNQTYWPSYNSPYFKEIFNASGCLENVKKYGDWFTYEKTPRALIFKRDHSKVVDMNSMMRLMRYNDFTHDPLSA
CNCTPPYSAENAIAARCDLNPKDGKYPFDALGHRGHGATDVKITSYSMFKNFQFLAVSGPTRDQVAPFQWSKSDLKDTIRHAGHPDLWVFDPVQF
>PLBD2_alvPom Alvinella pompejana (annelid) frag
YCRIKVWILGLFSFFISISTAVDVYVKWDTVSNNFQISEKRLSDPVAWATFGDEIAKTGWSYLEVYTNGAYDDSKQAYAAGLAEGYITQDLITKHWYNTY
KGFCTKPLNANCKKLQAFIDKNLKWMASKIATEAHKDSYWHQVQLFLEQQAGLLDGYSGQPQFPSNYSMDVTGIALLQLSGDIKDLQTALGIASANKLWFGTKFGIRGHK*
>PLBD2_schJap Schistosoma japonicum (trematode) FN318739
MYFTVVFTIVISFIEIHTQHPSKALILANSHKPYLYFDYVSDDNILQNPHIIAMAVFKEEINTTGWSSLTVSTSSDFPDYLQAYWAGFLETNLTFSLTAS
QWANTVRDMCPLPLSKDCQALRKYLSENMAYMLNEAYKNDKHSSFWYHVALQLWQLKGMSDAFDKRFIKRADLLNRNYLDELVDNVMGIYLLQLNGDLGD
LVSALSLPTLKEGCNQNGHPFIASSSCSALIKVVDSNVYLSHVTWSPYSIMLRVLKHYNFPWKIVNNTGSQKIPGFAITFSSYPTYTSSVDDFYITSANL
TITETTNNVYNKTLWEIVRNGSKNAVLTFMRGMVASRLAKTGEEWITYFKYNNSGTYNNQWMIFDAKQWPKNKGSLLIAEQLPGIVSSLDVTKILKINGY
WASYNLPFIGDIYTLSGTEEMAKMFGDWYVHNKTARAKIFRRDHHKVVDFPSMLSLMRYNDFMNDPLSTCPCKPPYTSNSAISARDELNDPKGQYPIPSW
SYRLHGGTDAKIVDLSMINQLNMIAISGPTYDDLPPFRWSLIKDVKKPLMHPDKWQFPPVITDFIDYPKANSNISARFLSFESLF*
   
   
  >PLBD2_triAdh Trichoplax adhaerens (trichoplax) XM_002107718 introns largely conserved
  >PLBD2_triAdh Trichoplax adhaerens (trichoplax) XM_002107718 introns largely conserved
Line 118: Line 1,172:
  1 GPTNGPPPNQPVFNWSTSYYKDTRHRGMPEAFDFDWVNFNWPF* 0
  1 GPTNGPPPNQPVFNWSTSYYKDTRHRGMPEAFDFDWVNFNWPF* 0
   
   
  >PLBD2_dicDis Dictyostelium discoideum (slime_mold) AAFI02000019 AF411829 introns both novel
  >PLBDa_dicDis Dictyostelium discoideum (slime_mold) AAFI02000019 AF411829 introns both novel
  0 MRVIRSLLLLTIAIIGSVLSQSSIDDGYTVFYSQPDNYYVKPGTFSNGVAQAIFSNEMMTTGWSFMSISSSEGLYPNDIIAAGAGYLEGYISQEMIYQNWMNMYNNEYHNVIGSD
  0 MRVIRSLLLLTIAIIGSVLSQSSIDDGYTVFYSQPDNYYVKPGTFSNGVAQAIFSNEMMTTGWSFMSISSSEGLYPNDIIAAGAGYLEGYISQEMIYQNWMNMYNNEYHNVIGSD
  VENWIQENLQYLQTMIDSAPSNDLYWQNVETVLTQITYMQRGYNQSVIDNGVDASQSLGITEFFLMNMDGDMIDLGPALNLTNGKQVTSPATATSPKQAFKEFMRRTGHCSALIKMTDDLSDLFSGHTTW 2
  VENWIQENLQYLQTMIDSAPSNDLYWQNVETVLTQITYMQRGYNQSVIDNGVDASQSLGITEFFLMNMDGDMIDLGPALNLTNGKQVTSPATATSPKQAFKEFMRRTGHCSALIKMTDDLSDLFSGHTTW 2
Line 124: Line 1,178:
  0 WIIVDYNKFIKGYGALDGTLYILEQVPDYVEYGDQTAILRTGYWPSFNIPFYENIYGLTGFNETYAQFGNWFSYQASPRSMIFKRDANNIHSLTQFQAMLRYNNWQNDPFSQGNAGN
  0 WIIVDYNKFIKGYGALDGTLYILEQVPDYVEYGDQTAILRTGYWPSFNIPFYENIYGLTGFNETYAQFGNWFSYQASPRSMIFKRDANNIHSLTQFQAMLRYNNWQNDPFSQGNAGN
  QISSRFDLVTADDPNNQYLDPDAFGGIDSKVVSADMVAALLVNAQSGPSHDNETPFTWNSQWNQKYTYAGQPTTWNFDWMTMSLQSMKPASPSSDSSSDSTTFN* 0
  QISSRFDLVTADDPNNQYLDPDAFGGIDSKVVSADMVAALLVNAQSGPSHDNETPFTWNSQWNQKYTYAGQPTTWNFDWMTMSLQSMKPASPSSDSSSDSTTFN* 0
>PLBDb_dicDis Dictyostelium discoideum (slime_mold) XM_640726 2 exons
0 MNKLKSNFILNIVILFTILIFNINFINCENQKQQQHQQQQQQQQQQSSSTTTTLPIYSIKFSSETGFTIYSGNDSTSIAQSGFSNEMMTMGW
AYLTITTNSQFEDSLQAEAAGYIEGYLTFEMIWQCWYNILVNEYQNQTIPNQVLNWANENIAYMKQQVATNENDPYWINIGLVLTQLSGMVD
GYNAANQDPSRQLSFLDFILINMDADLGDISSTFNQSTSFSEISNFKKSMDHIKKTDHCSGLIKLTDDLTELYSA 2
1 HTSWSSYINMLRIFKSYNFKFSSITNIKSKLTLFSGYPATIASLDDFYLLDTKLVVLETTNGLNNNDLYYLIKPESVLTWMRVIIANRLANGGQSWCETFERENSGTYNNQWMI
VDYNKFVPGVKVRDGTLFVLEQVPGYIEFADVTNVLRTGYWPSYNIPYFETIFNMSGFNDELTDSSDYEAYEEDARSQIFRRDANKVYSLTDFQAIMRYNNFQNDPLSHGDAANQI
SSRFDLNSPDSQDYDAFGGVDSKVTSFSLVNQLLVIAQSGPTHDQEPPFQWSSANWSNIYPSIGMPNLYDFGWVNFTDISYNY*
>PLBDc_dicDis Dictyostelium discoideum (slime_mold) XM_632848
MNKIIILISLFLNFLFGYVVCLNENNQKSQNLIIPTYSIKLKSDGEYLVYSGNDTLSIAQASYTNEMMSIGWGYISITTNPKYNDSLQIEAAGYLEGYLSYE
MIWQNWNNMMVNQNANNSFGNDIISWAKENILYMNQQIQLNQNDPYWINVNLVLQQLNGLTNGYSDANQNPDRQLSLMDFILLNMNVEIYDIMNSLKNNSSSFYQQPNNNSFDNNQ
HCSALIKLTDDLTELYTGHTTWSDYYQMVRMIKSYNFRFSKLVAAKSNTTMFSGYPGVLMSVDDFYMLDSKLVVLETTNGIKDNDSELFKLIKPQSVLTWIRIIVTNRIAHSGKSW
CEIFEKENSGTYNNQWMIVDYNKFIKGVRVQDGTLYVFEQLPGYVEYADVTNILRTGYWPSFNVPYFETISNMSGFNYQSSSSDSSSSSGSIAYEQYPRSQIFRRDSNKVYSISDF
QAFMRYNDFQNDPLAYGDPGNQISSRFDLITPQNNASAAGGIDSKVTSLELINQFLMIAQSGPTHDQEPPFSWSSENWKNKYPTIGQPDTFDFEWVTFSTTSFGSFPSASNEKNY*
>PLBDd_dicDis Dictyostelium discoideum (slime_mold) XM_633485
MIIFKNLLKLLIILLTIKLYFCIEIKREEHLTILNELNENSDVIQYSILPGNNEEYEIVKGIQEDAIVYGYYMSNVEVNGWAYLSLVSNDKYNDSTQSRAFG
YLEGYLTKDLIWNSKVNYYKNAFNSSEIPNKLDDWLTENIESIHTFIVNNRKSRYWNQITLVMDQINGMLDGYNEANTNSSETLSLHDFFVLNMFGDLFDLMPALNLDKEYKYFQK
DLNDIQDWFKRSQHCSALIKVSSDYSELYSGHTTWSGYYTMLRIFKSYNQQFSSDVSGTLSKRNIFSSYPGALISVDDFYLLGDTRMVVIETTNSLVTNDLYHLIRPTTVLSWMRV
IVSNRMSTNGKEWCENFQRYNSGTYNNQWMIVSYNLFVPYNELKDGALYVLEQIPGYIEFSDQTQALRQGWWNSYNIPFYETIYDASGYNNYTANNYSDSTIYYMSYQTCPRAEIF
RNFAGYVESLEDFQSLLRYNDFEYDPLSHKLPFYAIASRYDLSKKNPSPFGATDTKVTCNSMIDQNTIVAISGPTTSNGQPIFEWNSKIDFMESTSHLGCPEKYNFPWVSFSDTTFRNL*
>PLBDe_dicDis Dictyostelium discoideum (slime_mold)
MKLFILLIVIVFLISNSYSLSSSDSSSDSGSDVQYYSLTSQFQVVQGKQIPGSIAWGYFKDEMNKDGWGKLSIETVSTVSDNIAFKAAGYLEGYLTWEYIYK
FSGNYFNSFFNTSNIKEIPTETLTFVSDNWEYMMERVNSSSTTDPYWIQIRNAMSQQIGLYEGYNAAAGEDYQKTFIEIYMINLYGDMGDIVTLTTTPNNEFIPMDRKEVEQLMAT
TGHCTSIIKLTNNCSDLMSAHTSWADFSVMIRIYKRINIPVASTPYGSETLFSSYPGLLVSIDDFYQIRPSKLHLTETLNTILNQTLYQQINAQSFMYWVRNLVANRLANNGFQWV
SIFVENNSGTNNIQFVVLDYKLFTPYSTELQSDLLWIVEQYPGGYQAADVTLTLWEQGYWPSYNRPYFEEVFDILGYPYYVEKFGDLFTYEYNPRANIFRRDHSKLETLQDMMNII
DYNQYKTDPFSMGYPGNSINARFDIKGGSLPSGNPIYSWFYHGTHGGIDGKAINYDMVNSFTAVARNGPTVTSDCPPFNWNDWSLISHQYMPQIYNFTWISINI
>PLBDf_dicDis Dictyostelium discoideum (slime_mold) XM_633485
MKIINSFVFIFVLLFVFNTNAIKLSEDEKYSIEEKPEWYSIDSNFAVSPGKISDALGWGYYKNEILKDGWGKLYVEMNKFMTPENSSIYYEATGYLEGYLTW
STTWNYSQAYFQNYMNGTNESDIPTPLVEFLKINYDWMTSTFSNRDESVYDTQVSNVIYQFEGFARGYQQAADSDKQLTTLQLLLLQYAGDLEDVAGYLEYEMAQNKTEYINRVKS
LKEIETLFAVKGRCSGLVRITPDYGELFISHTTWGSYFTAGYRIFKRIIIPDPTVPGNEILFASYAGVLTSDDDFFMIPSTEMVIIETTNDILNTSLYQYVTPNSLLYFVRSIIAN
RLSNTAQEWTNNFIQYNSGTYSNQWMIVDYKLFTPYQPLQPNTFWIIEQLPGGFMSADMTEVLALGNWPSFNRPFFPEIYDAMGYSYYEKLYGDIISYDLNPRSKMFRRDVPNVMS
LDGMQQIMTQNNYKSDPFSGGFPGNAIAARYDLGGGPAEPLSWSFIGLHGAIDSKITSYSLLQQNQAIAINGMTVTPDCPPFTWNSNWSTVSAHYGSPETFDFDWISITITDK
>PLBDg_dicDis Dictyostelium discoideum (slime_mold)
MIKSYYLFFIILIFLIFINNFILCENNNNNKYNNNNNYPFINVDKIIYTNSSNSIYLVWSNEQFNLILQSSYNEEFDDIVMIGEYFDKIETTGWGELNITFN
SNSATIQISDNEQAYFTGFIESVLTGERINQMYNNFAASEFTNSDHTPSPKLIDFLNTQMEFVRDQVFENNGSSQYWYSTGLIMSQFDGLVNGYQQSPFPQLSEIQLYILTSAGDL
ETLVTLFPSSSSSSSSTTNENKNKNIKISSIKPNFKDELTDCSGFIRILPDYSDVYFGHTTWRYYYALLRIYKFINLQFNFQDTPMEYKVSFSSSPGFISSKDDFYITGNKLAIME
TTNNIYNESLYQYTIPQSVLVWQRAMIANMIATNSSDWVKIFSEFNSGTYQNQWMVFDYKLFIPNKQQSSSSLPPNTFWIAEQIPGQVKTADLTNILNEQGYWKSYNIPYFESIYN
ISGYSEKTDLPPSYQYSYEKCPRSLIFSRNASDVLNFEDMKSLMQFNNYKTDPLSYSSPLNSISSRGDLLTIENGNRSAVAFGGVDSKITSFNQVLTLSCTAISGPSTNGGTLPPF
TWSQSPLFQNITHIGVPETFNFDWQEMGPYPN*
=== Derived PLBD2 sequences ===
>PLBD2x_droMel Drosophila melanogaster (fruitfly) Q9VRK8 retinal lamina neuron ancestor (lama) PMID: 16077094,8892229
0 MERPEYDGTYCATALWTKQVGFQIENWKQQNDLVNIPTGVGRICYKDSVYENGW 0
0 AQIEVETQRTYPDWVQAYAAGMLEGSLTWRNIYNQWSN 2
1 TISSSCERDESTQKFCGWLRDLLTTNYHRLKRQTEKAENDHYWHQLHLFITQLEGLETGYKRGASRARSDLEEEIPFSD
FLLMNAAADIQDLKIYYENYELQNSTEHTEEPRTDQPKNFFLPSATMLTKIVQEEESPQVLQLLFGHSTAGSYSSMLRIQK
RYKFHYHFSSKLRSNTVPGVDITFTGYPGILGSTDDFYTIKGRHLHAIVGGVGIKNENLQLWKTVDPKKMVPLVARVMAANRI
SQNRQTWASAMSRHPFTGAKQWITVDLNKMKVQDNLYNVLEGDDKHDDAPVVLNEKDRTAIQQRHDQLRDMVWIAEQLPGMMTKK
DVTQGFLVPGNTSWLANGVPYFKNVLELSGVNYSEDQQLTVADEEELTSLASVDKYLRTHGFRGDLLGSQESIAYGNIDLKLFS
YNARLGISDFHAFAGPVFLRFQHTQPRTLEDEGQDGGVPPAASMGDERLSVSIEDADSLAEMELITERRSVRNDMRAIAMRKIGSGP
FKWSEMSPVEEGGGHEGHPDEWNFDKVSPKWAW* 0
>PLBD2x_gloMor Glossina morsitans (tsetse) EZ422576 59% PLBD2_droMel V for iMet, C confirmed by ests
VYCATAFWTKQFGFLVRLSIEYWKQQNDLLNIPKGAARICYKDSIYENGWAQIEIETQRTSPDWVQAYGAGVIEGSLTWNQIYNQWLNTI
MSSCDRDENAQHFCTWLRELLENNFNEIKETAASKSEHDHYWHQINLYFIQLKGLETGYRQGAKRARSDLEGAIPWSDFLLMNSAADIHDLKIYYENYIPNG
INRTTDETEPKNFFLPSATMITKIYPTGNASTWQLLFGHSSAGSYTSMLRIQKRYKFHYHIASNQNSNTVPGMDIAFTGYPGILASTDDFYIIKGRQVHAIVAGVGIKNENLELWQ
TVNVQKAMPLAARVMAANRLAQSRRSWTKALSRHPFTGSKQWITIDLNKLSPLESLYELDENNLELDEEAASIERQNHLKGLVWIAEQLPGRMHMRDVSALFLETVDNSTTWLASG
IPYYKEILEASCVNNEAYITNLTAAESRELNNLEAIDKFLRTRGFRGDLLDDEQSVAYGNIDIKLFAYNARLGMSDYHAFAGPVFIRLQHVQTRSLPAAMGYRGDLAPASLIRDDR
LSVVVDDAAELAELQVITERRIVRDDMRAVAMSQISSSPFKWSSVAEKMATLQHAGHPDEWNFGKVSPKWAW*
>PLBD2x_aedAeg Aedes aegypti (mosquito) XM_001658072
MDRPNYSGTYCATAYWARNSGFRVEFWGQREELGEVPPGAVRACFKHSLMESGWSQLELESQPEYPDSIQAFAAGMLEGTLTWNNIYLHWSNTIESECNR
DDQSEEFCHWLRRIISTNVETVKKMADMKGKNDHYWYQIGLFFDQLDGLEFGFRKGVRRSRMDYEIPMEDFLLMNSAVDIRDLKAYYMNFLDSESGMHVE
PNKGMMLLKLVESVTGKVNILLGHTSDGSYASMLRMMKKYTLNYHFAEESIVERVVPNTNIVFTGYPAVLASLDDFYMLSGKHHRMVVGGIKIKNDNLNL
WTKIDLVRSVSLAPRVMACNRLAHSGRVWSKYFARSPSTGAKQWLLVDLTRLNRENELLDDSAEVEEMLLSHAEYDDPKAMRKAQSTDYVEVDFEGIVRK
MTSIGKISDGGLFWVVDQLPGRLHAEDMTEKIVRDGYWLGTGIPTFEELADIGQVKTNGTDSQRHSIQEKILNNITDLEGLAKFIRQSAYRGDLDQQHPT
AFGNIDMKLFVETNASNGTGIFQAYSGPLYDPLTDGQAVKRSVENDGEVEAEAIPVAAAKKKRVKPFDWDSAKKLDVPHQGQPTLWNFRRQSPKWAWI*
>PLBD2x_anoGam Anopheles gambiaei (mosquito) XM_316013
MLKVVGASWHKTRIGSYILIGALMLAVAALFLADMERPSYNGTYCATTYWARNSGYRLEFWGQRNDLDQVPKGAVRACFRDSILTNGWSQLELESQSTYT
DTVQAYAAGIMEGALTWHNIYMHWSNTIDAVCSKDEESEEFCDWLRGLITTNVDTVKKMADMKGKHDYYWYQIALFYDQLDGLEIGFRKGVKRSRMDYEI
PKQDFLLMNAAVDIRDLKIYYTNFLMENSGLKPEPAKGIMLLKLLQQPNGQSKVLLGHTSDGSYSSMLRMVKKYTLRYHLSAETARTVVSAQAAEDEDRA
VVPGTNIVFTGYPGVLASLDDFYVISGRRQHRLVAAGVKMENEHVDLWRKIDLVRSVSLAPRVMAANRLGHSGRSWARYFARNPSTGVKQWLVIDMSRFV
GSNETEVELLATSPASVAVDQSVEDNDIAAEGGQDVVADGEHQRIVTKRMEDYVDDDFKEHLSAIRQVRMVTIPKAPEPVAGEELVVEPSVGHTFGSGMF
WVVDQLPGRLHAEDITDKIVSDGYWLTNGVPYFKEALDIGQATANATNTTAPSSTMEKILQNITDLETLARFFRQSAYRGDLDQDEPAAFGNIDLKMYTE
TADGTVHFRAYSGPLYDPARSTPGQAAKRSIVKSEQLHQSTATDQKSAPASHGDSATVASHPFDWSRILPFEVRHQGHPDVWDFEHITPEWAWI*
>PLBD2x_apiMel Apis mellifera (bee) HP560493
MLKVVGASWLQTRISTYILVAVALLGIGAIILGEFGHVEQDGIYSATVLWNRKGGYRIDFWGQGNDLTAVPLHAARAYYKTGIFEYGWSYIEIETSSKYP
DTVQAYAAGLLEGSLTWQLIHHHWYNTIRLECEAKPIECRKLMRYLRDNTAVNRKRAELKESTDSFWHMVRLFYAQLDGLEAGWKFAVRRSRVSASLESE
DFLWLALASDLCGFQQIFNISDSLVSSMIYFKSLPRENSGPLIAISHHTSASYTQMLRLLKKYTFGYHILPVTKTAALIPSRSIVMSSYPGALSSRDEFY
LINGENRELVVTGTSLLMTNRTEWSFLYPDDYVMLTVRLMAANRLATNSQSWFNTLSYQNDGASALQWIIFEPRSMTLLLVEQLPSITVPINYTEEFKKI
GFISYIGISNFKSTNNIVQPAKKNLNLWKTRLTRLQKNITTFEQFRNMMRGCSQEGCSAVNQKTDYLQELMFRGDLEDVPIPYGIIDTKILTVDIDGFKG
FEAISGPSQSRTPFKWSQSFPNISHVGHPDTFNFESVTPKWVWI*
>PLBD2x_pedHum Pediculus humanus (louse) XM_002424573
MLKVVGASWVKIRMSTWIIGIVLVFGIIVVILGQMGESEEDGNYSATAYWNEETGFEIKFWGQGLEKDKIPKGVARVYFQPHIDTTGWAIIDVETQSEYP
DWVQAYAAGMLEGSLSWQLIYWHWKNTVQSVCQNQTLFCKYIKKYLEENFKSVKELYENTDDPYWHQAYLFYIQLEGMQQGFEHGVKRKGLIIEEIEFID
FLWMNAASDIIDLEYKFYDSKPYSTTTTNSSSFSANNNNNSNLLLKKLIQGRSDNVESMEKLSFVGCGVTLGSALLNSFQNMRNYFSPIIPLDQTKLLVA
GTPITVFNKNLWNLVSPLKQLLSGVRVVVANRLSNNASDWGSYLSKYNSGTGNKQWLIIDVDKFKRKINDSSVGERENFFWVAEQLPGHFKIDDLTENLI
NSSYWASYGLPFYKDMYEMSGTNYMKENFGDAFSYENSPLAKIYRRDHNKSTSIKSILNLMRSNSFKRDPLSLGNPRCAIGARGDIPNKVSNSFPIGVID
SKIISLEKKLENVTSNDTTTNNNNNIIPFNNNVSNDELIIFKAVAGPAHSLHKNSENLKGNDEIRKEKNETVEVEGIGVGGGDDDTLKPFNWQNSVFNEF
PHNGHPNNWDFDAISLSP*
>PLBD2x_acyPis Acyrthosiphon pisum (aphid) XM_001948052
MLKVVGASWAQTQLSWCLIIAITLLGLLAFYFGGTIRNEYDGKYAATAFWSKEFGMRIDFCGQNNDPLTVRKGVARAYYRPDLSENGWAVLEVETQAEYP
DYVQAKAAGYLEGSLTWRMIYWHWKNTVENTCIGRRNFCERIRKYLEENAEEIKRMAKRRGESDPFWHQVNMFYTQLKALEDGWRFGVKRSRQDIDIPSV
DFLWMNIMPDLKNFEQKWNASKDFNPDKPPLSATLVKIISTNPIDFVLAQSASGYYGSMLRIQKRYNFGFHETQSDDSALVNGKIVEFTSYPGSIYSQDD
FYKVIKKGSKTETTVVGTELQNNNRQLWEKIMKTDQVLLGARIMAANRLASNSKKWYEVFSRNNSGTGNKQWLVISTNSTSIAFGVIEQMPGIVSYEELS
KTLLSTGYWVSNSNPCLKETVYMSGADGRDAADHPVANILRSGQLNVTDIGTLVDLMRGTEMSLIGRTDLLGVKTNAMFRRFANQSFVEQLTAANRLQSA
DATAPLKDDRTTIVTNRLPSSAASDDKLDVDKCFTGTVDLKVTSANMNGYYAASGPPFTMDRSEVEPFRWSESPIRHLPHYGHPDVWDFEVEGVVWVWK*
=== Trimmed alignable sequences ===
It is useful for various purposes to trim protein sequences to their conserved core and matured length. Here, since compilations of signal peptides have been previously considered, they can be discarded, greatly simplifying acquisition of reliably alignable sequence.
Note too that exon boundaries mostly differ between the two paralogs and so limit the matched pairs from a fixed species that be collected from genomic contigs using tblastn from a necessarily diverged query. However the final exons do match and are already provided above from a very large and phylogenetically disperse species set. In some cases, there is a benefit to removing single-species insertions that introduce a gap in the rest of the alignment because then other gapping can be done more accurately.
Finally, as the xray structural determination did not extend over the whole protein, flanking sequence needs be included only to the extent it is strongly conserved. Thus the boundaries of the protein are established, allowing comparative genomics on the alignable core.


[[Category:Comparative Genomics]]
[[Category:Comparative Genomics]]

Latest revision as of 14:03, 15 November 2010

Introduction

A surprising number of orphan human enzymes (unknown substrate) still exist ten years after the completion of the human genome project. PLBD1 and PLBD2 are semi-orphans in the sense of being probable phospholipases of B class but with uncertain physiological substrates and thus functionalities. This is especially important in the case of PLBD2 which localizes to the lysosome, as its absence could plausibly lead to a serious yet unrecognized lysosomal storage disease.

No bioinformatic algorithm or experimental protocol leads with any certainty to determination of function. The gene pair here has eight targeted publications but cases exist where protein function remains unknown after ten thousand papers (eg PRNP).

PLBD1 and PLBD2 constitute a small gene family (sequence homology class) within vertebrates though one that occurs expanded in some early diverging eukaryotes. However, the Pfam clan NTN (N-terminal nucleophile aminohydrolases) may have, among its ten family members, additional representatives in humans diverged beyond recognizability in primary sequence. These establish the great antiquity of the fold and certain of its features but are not likely to shed additional light on phospholipases specifically.

PLBD2 presents a special difficulty in that a sequence of post-translational steps are apparently necessary for its activation. Without these, potential substrates can hardly be assayed. These steps include removal of the signal peptide, mannosylation appropriate to the lysosome targeting receptor, and self-catalytic proteolytic activation (into 28k and 42k fragments which remain associated) to expose the substrate binding site as this becomes appropriate.

Because PLBD1 and PLBD2 are full length paralogs, the bioinformatic approach below considers both on an equal footing. PLBD1 has been more amenable to activation whereas PLBD2 has a high-resolution structural determination. Thus comparative genomics allows for annotation transfer, first from PLBD2 to a structural model for PLBD1 (already provided by the SwissModel pipeline), then perhaps transfer of PLBD1 experimental protocols to PLBD2.

However the gene duplication event occurred some 650 million years ago and the two genes are quite diverged today. It is not known whether substrates have diverged or merely their cell type of expression. Increased gene dosage per se is seldom an explanation. Yet certain core features remain conserved, including the fold, active site residues, signature motifs, certain glycosylation sites and even the fragmentation pattern, implying these are essential functional features under long-range strong selective pressure for their maintenance.

Disulfides are only separately conserved within each paralog but this fortuitously provides a reliable signature for assigning deeply diverged proteins from early eukaryotes to their orthology class. As the respective functions become better known, we can hope to understand how the gene duplication event contributed advantageously to increasing evolutionary complexity, leading to persistence of both enzymes in most species over immense time spans.

Conservation at critical sites

The six residues of PLBD2 associated with the active site are completely conserved within vertebrates to within genomic sequencing error. These same six residues are also completely conserved within PLBD1. Indeed 3 of the residues are conserved in the broader NTN hydrolase clan.

This is perhaps unsurprising since the active site was established a couple billion years earlier in the bacterial ancestor. However if PLBD2 and PLBD1 have different substrates, this establishes that these six residues are insufficient to distinguish the two active sites. Note H266 and T330 do not contribute their side chain, leaving them and W269 to separate phospholipases from the other NTN hydrolases.

The glycosylation sites are surprisingly conserved both within and between PLBD2 and PLBD1. Some of the motifs may be either recently acquired within later vertebrates or spurious glycosylation motifs with N and D both acceptable (or similar small amino acids) in the first slot of the NxS/T motif. Glycosylation is important in correct targeting of lysosomal proteins, more so than in generic endoplasmic reticulum proteins where motifs are often poorly conserved (as in sulfatases).

PLBD2 has two established disulfides. Strict sequence conservation of these throughout vertebrates (indeed, throughout metazoan) suggests both play an important role in protein structure and stability.

In PLBD1 however, the first disulfide is not a possibility and while an opportunity exists for a disulfide homologous to the the second disulfide of PLBD2, indels cloud the alignment and spacing would have to be different. There is additionally ambiguity given C...CC as to the cysteines involved. Indeed a second distal disulfide may occur utilizing C...CC.............C which has no counterpart in PLBD2. While cysteines can be conserved for many reasons other than disulfide (as in the nucleophile cysteine here), suitably proximity and side chain orientation in the SwissModel of PLBD1 would argue for disulfide. Comparative genomics suggests that C2 and C4 may form an ancient disulfide whereas C1 and C3 might represent a deuterostome innovation. 

homSap CNTICCREDLNSPNPSPGGC human PLBD1
braFlo CSAICCRKDLAKVGAKPDGC Branchiostoma floridae
strPur SKSICMRGDLM-TSPMPNGC Strongylocentrotus purpuratus XM_001192029 
nemVec MNAICSRGDLIADGPRASGC Nematostella vectensis XM_001638165 
monBre YNAICSRGDLESDSPSPGGC Monosiga brevicollis XM_001745398 

SwissModel coordinates for PLBD1 show the 2nd and 4th sulfur atoms separated by 2.03 angstroms:
ATOM   3552  SG  CYS   471      49.680 -13.769 -12.461
ATOM   3579  SG  CYS   475      49.273 -14.310  -4.881
ATOM   3585  SG  CYS   476      51.067  -9.716  -9.172
ATOM   3678  SG  CYS   490      50.737 -13.198  -5.750

The known human SNPs of PLBD2 are in some cases quite radical substitutions in terms of both physical qualities of the substituted amino acid and the degree of observed phylogenetic conservation at that site. These likely result in unstable and/or inactive enzyme. Both enzymes are autosomal so compensation might occur in the recessive state, or alternately, PLBD2 and PLBD1 could fill for each other to some extent. In either case, lysosomal storage disease might not be clinically observable.

Here Q54P may actually be a deleterious mutation in the reference sequence individual (with the SNP representing wildtype) as proline is highly conserved throughout mammals. The neanderthal genome also has proline at this location (UCSC track: 1C>0A(CCG>CAG)).In A204V, valine is quite a bulky substituent for a site normally restricted to small amino acids; R354C is definitely a serious mutation, no doubt attributable to a CpG hotspot; Q521K appears milder as does R524C.

The known human SNPs of PLBD1 can be analyzed similarly. P26Q and V30L may be inconsequential as they occur in the rather unconstrained primary sequence of the N-terminus; V265I occurs at an ILV reduced alphabet; V377A and P534A are much more serious despite the aliphatic nature of alanine and likely give rise to dysfunctional protein.

PLBD2activeSiteComp.png
Structural superposition of active sites from five NTN hydrolases
showing conserved side chains (*) and relevant main chains (....)
(adapted from Fig 6 of Lakomek et al. BMC Struct Biol.2009;9:56:)
                                            *         (*)       *    *
PLBD2 phospholipase B-like     gray   3FGR  C244 H261 W264 T325 N427 R458 human numbering
PLBD1 phospholipase B-like     ....   pred  C228 H245 W248 T303 N402 R433 human numbering SwissModel
PLBD2 phospholipase B-like     ....   ....  C225 H242 W246 T302 N401 R432 Dictyostelium numbering
Cephalosporin acylase          pink   1OQZ  S170 .... H192 .... N413 R443
Conjugated bile acid hydrolase green  2BJF  C2   .... D21  .... N175 R228
Penicillin V acylase           yellow 3PVA  C1   .... D20  .... N175 R228
Penicillin G acylase           orange 1K5S  S1   .... Q23  .... N241 R263

Human SNPs resulting in amino acid substitutions:
PLBD2:                PLBD1:
 Q54P   rs7965471    P26Q   rs1141509
 A204V  rs12231990   V30L   rs12296104
 R354C  rs56935204   V265I  rs7957558
 Q521K  rs17852787   V377A  rs2287541
 R524C  rs12425042   P534A  rs1600
PLBD2colored.png


PLBD1colored.png


PLDB2consSites.png


PLDB1consSites.png


Intron evolution

PLBD1 and PLBD2, being full length paralogs, clearly indicate an early gene duplication and subsequent divergence to the current low percent identity. Segmental duplications preserve any introns present at the time of the event and these generally persist in both position and phase into living species.

However PLBD1 and PLBD2 -- despite having similar numbers of introns -- exhibit very little in common in terms of location as the diagram below shows. One possibility is that a second copy arose as a retroprocessed gene (a mechanism erasing existing introns) and was subsequently intronated at random positions. This is unlikely here given that 10-11 relatively rare events would be needed.

The remaining possibility is that the gene duplication took place prior to the main era in early eukaryotes during which the bulk of introns were established. This fits the current state of high divergence despite fairly slow rates of evolution during metazoan times.

The last five amino acids of each PLBD1 exon are colored below. Then using an alignment of PLBD1 to PLBD2, the colors are mapped to the homologous five residues within PLBD2. There they fall on the ends of exons only when these correspond to those of PLBD1. The outcome here -- despite uncertainties in alignment gapping -- shows intron positions do not correspond with the exception of the terminal intron (which also is phase 0).

While this merely compares human PLBD1 and PLBD2, the collected reference sequences (intronated against their respective genome assemblies) confirm that introns in both genes are deeply conserved. 

PLBD1 introns do not correspond well to those of PLBD2:

>PLBD1_homSap Homo sapiens (human) first and last introns are not mappable
0 MTRGGPGGRPGLPQPPPLLLLLLLLPLLLVTAEPPKPA 1
2 GVYYATAYWMPAEKTVQVKNVMDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQTLSNEIIMFVAGFLEGYLTAP 2
1 HMNDHYTNLYPQLITKPSIMDKVQDFME 2
1 KQDKWTRKNIKEYKTDSFWRHTGYVMAQIDGLYVGAKKRAILEGTK 0
0 PMTLFQIQFLNSVGDLLDLIPSLSPTKNGSLKVFKRWDMGHCSALIK 0
0 VLPGFENILFAHSSWYTYAAMLRIYKHWDFNVIDKDTSSSRLSFSSYP 1
2 GFLESLDDFYILSSGLILLQTTNSVFNKTLLKQVIPETLLSWQRVRVANMMADSGKRWADIFSKYNS 1
2 GTYNNQYMVLDLKKVKLNHSLDKGTLYIVEQIPTYVEYSEQTDVLRK 1
2 GYWPSYNVPFHEKIYNWSGYPLLVQKLGLDYSYDLAPRAKIFRRDQGKVTDTASMKYIMRYN 1
2 NYKKDPYSRGDPCNTICCREDLNSPNPSPGGCYDTK 0
0 VADIYLASQYTSYAISGPTVQGGLPVFRWDRFNKTLHQGMPEVYNFDFITMKPILKLDIK* 0

>PLBD2_homSap Homo sapiens (human)
0 MVGQMYCYPGSHLARALTRALALALVLALLVGPFLSGLAGAIPAPGGRWARDGQVPPASRSRSVLLDVSAGQLLMVDGRHPDAVAWANLTNAIRETG 2
1 WAFLELGTSGQYNDSLQAYAAGVVEAAVSEE 0
0 LIYMHWMNTVVNYCGPFEYEVGYCERLKSFLEANLEWMQEEMESNPDSPYWHQ 0
0 VRLTLLQLKGLEDSYEGRVSFPAGKFTIKPLGFL 2
1 LLQLSGDLEDLELALNKTKIKPSLGSGSCSALIKLLPGQSDLLVAHNTWNNYQHMLRVIKKYWLQFREGPW 1
2 GDYPLVPGNKLVFSSYPGTIFSCDDFYILGSGL 0
0 VTLETTIGNKNPALWKYVRPRGCVLEWVRNIVANRLASDGATWADIFKRFNSGT 2
1 YNNQWMIVDYKAFIPGGPSPGSRVLTILEQIP 2
1 GMVVVADKTSELYQKTYWASYNIP 2
1 SFETVFNASGLQALVAQYGDWFSYDGSPRAQIFRRNQSLVQDMDSMVRLMR 2
1 YNDFLHDPLSLCKACNPQPNGENAISARSDLNPANGSYPFQALRQRSHGGIDVK 0
0 VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD* 0

Signal peptide compositional anomaly

The first exon of both PLBD1 and PLBD2 are ill-behaved in alignments. The explanation can be see in their compositional distortion (very high GC content) that specialized masking tools such as seg and gnu recognize. Such dna manifests itself at the protein level by high levels of the amino acids, such as GPL that use those codons in the three reading frames.

Such regions are prone to repeated expansions and contractions via replication slippage. Not only do we expect such alleles in human but also that inter-species comparisons will be difficult and alignments problematic (as homology by definition is lost even if the sequences still align).

This matters very little to the mature protein since this region is trimmed off during maturation but the question still arises as to how signal peptide variations continue to be recognized efficiently by the signal receptor complex. Indeed a class of mutations could exist in which the signal peptide cannot be processed correctly and the protein never reaches the lysosomal compartment, in effect a knockout mutation.

This compositional anomaly may have caused vertebrate-wide sequencing problems. Many assemblies had difficulty sequencing back to the initial methionine and alignment programs also fell short. A set of reliable sequences could only be obtained after careful hand-curation and only then from fewer species than usual in comparative genomics.

Even then the set of first exons raises more questions than it answers as it seems to be evolving quite chaotically in fish. Mammals also exhibit a peculiar conserved insertion as placentals diverged from marsupials. And using SignalP 3.0 separately on each sequence, it emerges that the marsupial signal peptide and those of earlier diverging species are much shorter. That isn't a problem per se because signal peptide lengths are quite variable.

PLBD1 also exhibits a shift in the location of the signal peptide cleavage site over evolutionary time, crossing the boundary into exon 2 in some clades. (Since the exon break is extremely conserved, this conclusion is independent of alignment gapping.) Here again this would be functionally irrelevant since the co-processing of nascent chain takes place well after mRNA splicing. However this does provide an interesting case of homologous residues not being functionally homologous and so evolving under the different functional constraints. 

PLBD1:  ATGAcccgcggcggtccgggcgggcgcccggggctgccacagccgccaccgcttctgctgctgctgctgctgctgccgctgttgTTAGTCACCGCGGAGCCGCCGAAACCTGCAG
         MTRxxxxxxxxxxxxxxxxxxxxxxxxxxVTAEPPKPA
         MTRGGPGGRPGLPQPPPLLLLLLLLPLLLVTAEPPKPA

PLBD2: ATGGTGGGCCAGATGTACTGCTACCCCGGCAGCCACCTGGCCCGGGCGCTGACGCGGGCGCTGGCGCTGGCCCTGGTGCTGGCCCTGCTGGTCGGGCCGTTCCTGAGCGGCCTGGCGGGGGCGATCCCAGCGCCGGGGGGCCGCT... 
        MVGQMYCYPGSHxxxxxxxxxxxxxxxxxxxGPFLSGLAGAIPAPGGR...
        MVGQMYCYPGSHLARALTRALALALVLALLVGPFLSGLAGAIPAPGGR...

Phylogenetic variation in first exon signal peptide of PLBD2:
              <------ signal peptide ----------------->             <---- start of 3FGW 3FGR 3FGT------------->
>PLBD2_homSap MVGQMYCYPGSHLARALTRALALALVLALLVGPFLSGLAGAIPAPGGRWARDGQVPPASRSRSVLLDVSAGQLLMVDGRHPDAVAWANLTNAIRETG
>PLBD2_panTro MVGQMYCSPGSHLARALTRALALALVLALLVGPFLSGLAGAIPAPGGRWARDGPVPPASRSRSVLLDVSAGQLLMVDGRHPDAVAWANLTNAIRETG
>PLBD2_ponAbe MVGQMYGSSGSHLA----RALALALVLALLVGPFLSGLAGAIPAPGGRWARDGPVTPASRSRSVLLDASAGQLLLVDGRHPDAVAWANLTNAIRETG
>PLBD2_rheMac MVGQMYCSSGSPLARALTRALALALVLALLVGLFLSGLAGAIPAPGGRWAHDGPVTPASRSRSVLLHAATGQLLLVDGRQPDAVAWANLTNSIHETG
>PLBD2_papHam MVGQMYCSSGSPLARALTRALALALVLALLVGLFLSGLAGAIPAPGGRWAHDGPVTPASRSRSVLLDAATGQLLLVDGRHPDAVAWANLTNAIRETG
>PLBD2_calJac MVGKMYSSPSSRLAQALTRALALALVLALLAGLFLSGLSGAIPAPGGRWARDGSVPSGSGSRSVVLDAAAGQLLLVDGRHPDAVAWANLTNAIHETG
>PLBD2_otoGar MvGPMYGSPGGRLARALTRALALALVLaLLIGLFLSCLAGAiPPPGSGRARDGLITPASRSSSVLLDATTDQLRLVDGRHPDAVAWANLSNAIHETG
>PLBD2_musMus MAAPVDGSSGGWAARALRRALALTSLLASLTGLLLSGPAGALPTLGPGWQRQNPDPPVSRTRSLLLDAASGQLRLEDGFHPDAVAWANLTNAIRETG
>PLBD2_ratNor MAAPMDRTHGGRAARALRRALA----LASLAGLLLSGLAGALPTLGPGWRRQNPEPPASRTRSLLLDAASGQLRLEYGFHPDAVAWANLTNAIRETG
>PLBD2_dipOrd MAAPPYGSRGGRPAGSLSRALV----LAVLVGLSPSGPAGAVPSPGDRWGRHKPEPPVSRSRSVLVDAASGQLRLVDGLHPGAVAWANLTNAIRETG
>PLBD2_cavPor MAAPTYVSLDGRPVRARALALA--PALCLLVGLSLGRLAGAVPAPGPRGARDGPVPAA--CRSVLLDAASGQLRLVDGLQPGAVAWANLTNAIPETG
>PLBD2_oryCun MVAPRDGCAGGRLARALALALL--------TGLLLGGLAGAAPAPGGGEQRDPPSPPASCCRSALLDAATGQLRLVDGRHPDAVAWANLTNAIHETG
>PLBD2_ochPri MAATRDSSAGCRLARVLTRALAL---LALPTGLFLSGPAGAIPVRGDGEERGRPAPSGSRCRSVLVDAESGQLRLVDGRHPAAVAWANLTNAIHETG
>PLBD2_turTru MVDPMYGCPGGRLARALTRALALALVLALLVGLFLSGLTGAIPTPRGHRGPGRPVPPASRCRSVLLDPEtGQLRLVDGRHPDAVAWANLTNAIRETG
>PLBD2_bosTau MVAPMYGSPGGRLARAVTRALALALVLALLVGLFLSGLTGAIPTPRGQRGRGMPVPPASRCRSLLLDPETGQLRLVDGRHPDAVAWANLTNAIRETG
>PLBD2_oviAri MVAPMYGSPGGRLARAVTRALALALVLALLVGLFLSGLTGAIPTPRGQRGRGMPVPPASRCRSLLLDPETGQLSLVDGRHPDAVAWANLTNAIRETG
>PLBD2_susScr MVAPMYGSPGGRLARALTRALALALVLALLVGLFLSGLTSAIPTPKGYRGSGRSVPPASRSRSVLLDTETGQLRLVDGRHPDAVAWANLTNAIHENG
>PLBD2_ursAme MAAPMYGSPGGRLARALTRALALALVLALLVGLFLSGLTGAIPISGRQWGPNGPVPPDSRSRSVLLDAETGQLRLVDGRHPEAVAWANLTNAIRETG
>PLBD2_musPut       GS-GGRLARALTRALALALVLALLVGLFLSGLTGAIPISGRQWGPKGPVPPDSRSRSVLLDAETGQLRLVDGRHPDAVAWANLTNAIRETG
>PLBD2_canFam ...................................SGLTGATPVSGRRWGPSGPVPPASRSRSVRLDPQTGQFQLVDGRNPDAVAWANLTNAIRDTG
>PLBD2_myoLuc MVAPPSRSPGGRLTPALSRAPALAPGLALLAGLFLSGWTGAIPTPRDPWGPNGPVPPASRSRSVVLDARTGQLQLVDGRQPDAVAWANLTNAIHETG
>PLBD2_pteVam MVAPMDRSPGGRLAGALTRTLELTLVLAPLAGLFLSGRTSAIQTPGSRWGSEGPVSPASRSRSVLLDPQTGQLRLVDGRHPDAVAWANLTNAIHETG
>PLBD2_eriEur MVAPMCGSPGGRPARALTRALALAPALALLVGLFLSSLAGAIPPPEDNWGRNGSFPPVSRCRSVLLDSETGQLRLVDGRHPDAVAWANLSNAIHETG
>PLBD2_loxAfr MVAPVYGSPGGRLARALTQALAVALVLALLVGLFLSGLTGAISLTGHRWGPDGPAPPASRSRSVLLDTATGQLRLVDGRHPDAVAWANLTNAIRETG
>PLBD2_echTel MVATEYGSPGGRLARALTRAPALALMLALLVGLFLSGLTGAISPAGGRREPNGRVPPASSSRSALLDPATGQLRLADGRHPEAVAWANLTNAIHETG
>PLBD2_macEug mVATMYQ--GGCLALGLALGLGLVLVLSLP--------------------QPSLPPPPSRTRSVVMDSATGQLNVVEGWEAGAIAWANLTNAIAETG
>PLBD2_monDom MVATMCQ--GSSLALGLALALGLALGLR-------------------PPQPSLPPPAPSRSCSVVLDEASGQLKVVEGAQAGAVAWANLTNAIGETG
>PLBD2_anoCar MAPAWLLRFFGLALLLARSPARR------------------------PPPFPDPAAVPTRSCSVVLEPGSAALKLVNGWAPGAVAWANLTEGIRQNG
>PLBD2_galGal MAVVRALLVAAAVAAWVPGVASGP-------------------------------TPPPRSASVLLEPGSGRLRVLPGRQPAAVAWAELTDHIQAVG
>PLBD2_melGaL MAVVRALLVAAAVAAWVPGVASGP-------------------------------TPPPRSASVLLEPGSGRLRVLPGRQPAAIAWAELTDHIQAVG
>PLBD2_xenTro MGAQLLLIFMLFSLGAAQQAV---------------------------------------VSVLFDPATGNITTVEEKRVVGAVAWAELKDSILENG
>PLBD2_xenLae MAPWQLFIFSLFCVGAAQQQA--------------------------------------VVSVLFDPATGNITTVAEKKVAGAAAWAELTDSIQENG
>PLBD2_oryLat MAFRQNKTVCAKMSTFMKSLLVLGLFWGCGRAEI---------------------------RSAVIDKGSGKLTVVEGYHEGFVAWANFTNDIETSG
>PLBD2_dicLab MASRLNKTSAVGGFSKVLNVLAVLSGLCLLFASVGAE-----------------------IRTAVIDKQTGQLSVVDGYREGFVAWANFTDDIKTSG
>PLBD2_hipHip masrlnktDGVQDKQDVFCGEFSSASVAFYVLCLTCVRAEI--------------------KSAVIDGQSGELSVVDGFQKDFVAWANFTDDIQTSG
>PLBD2_parOli MASRINKMGVEDKQDVSCVEFCVRAEI----------------------------------KSAVIDAQSGDLCVRDGFHQDLVAWANFTDDIQTSG
>PLBD2_gasAcu MASRQNTTVTLRHFKAVLSALFVMCACVQAEI-----------------------------RSAVIDKQTGKLSVVEGYREGFVAWSNFTDDINTSG
>PLBD2_oreNil MACRRNGADRVRSFTEVLGLLKMFLLLFCLFAVRAEI-----------------------SRTAVIDKQTGQLSVIEGYQEDFVAWANFTNDIETSG
>PLBD2_sebCau MASRHNKMFAVGRFKVALSVLSTLCFMCASVGAEV--------------------------RTAVVNKQTGQLSVVEGYREDFVAWSNFTDDIKTSG
>PLBD2_osmMor MAFRLLRLSTTLHLAVFLHVLFLSCSSIKAEI-----------------------------STIVLDEKTGQLTILEGYRDDYVAWANFTDDIEHSG
>PLBD2_onyTsh MADRRTQMSLTTEKMFMFSCVFYLSWTSVRAEI----------------------------PSKILDKQTGQLSLEEGFRDDYVAWANFTDDIKNSg
>PLBD2_salSal madrrtqMSVTTEKMFMFLCVFYLSWTSVGAEI----------------------------HSAVLDKQTGQLSLEEGFRDDFVAWANFTDDIKNSG
>PLBD2_danRer MAHLQLLVSAVCVLLSVCQAQI---------------------------------------YSAIYEEETAQLLLIEGARTHSVAEANFTDHINTTG
>PLBD2_calMil MCVGVRGQGLGLGLPLLLVLAAVGVSPSARGHL---------------------------LRSVVLDEHSGRLRVVGGLNPHSIAWANLTDRIRATG
>PLBD2_braFlo MAACRNIFCGRMLSCLLLFSFVFSAV-----------------------------SDGSKLASVRYDEAAKTYQITDKLDPSAAAWANFTDRISSTG
>PLBD2_acyPis MLSIRCILLSLLFVWALQCSATQK------------------------------NQTLLAVKTDNNRITIQPKHYSVKDKEIIIGKGKFIDRINSTG
>PLBD2_triAdh MAQCGKFLIYFSIFIITLATLCSCQS-------------------------------------GSVIYKDGLYTFSKGINKRAASYGTFTDKIASSG

The two paralogs do not align at all in the signal region and only poorly thereafter:
PLBD2_homSap:    67 DVSAGQLLMVDGRHPDAVAWANLTNAIRETGWAFLEL--GTSGQ-YNDSLQAYAAGVVEAAVSEE 128
                         Q+  V  ++ DA  + N  N+++ TGW  LE+  G   Q  ++ +  + AG +E  ++  
PLBD1_homSap:    50 AEKTVQVKNVMDKNGDAYGFYN--NSVKTTGWGILEIRAGYGSQTLSNEIIMFVAGFLEGYLTAP 112

Phylogenetic variation signal peptide location in first two exons of PLBD1:
              <------ signal peptide --------->      <-------------------------- second exon ---------------------------------->
>PLBD1_homSap MTRGGPGGRPGLPQPPPLLLLLLLLPLLLVTAEPPKPA:GVYYATAYWMPAEKTVQVKN-VMDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQTLSNEIIMFVAGFLEGYLTAP
>PLBD1_panTro MTRGGPGGRPGLPQPPPLLLLLLLLPLLLVTAEPPKPA:GVYYATAYWMPAEKTVQVKN-VMDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQTLSNEIIMFVAGFLEGYLTAP
>PLBD1_ponAbe MTRGGPGGRPGLPPPPPLLLLLLLPPLLLVAAEPANSA:GVYYATAYWMPTEKTVQVKN-VMDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQALSNEIIMFVAGFLEGYLTAP
>PLBD1_rheMac MTRGGPGGCPGLPPPLPLLLRLLLPPLLLVTAESPNPA:GVYYATAYWMPAEMTVEVKN-IMDKNGDAYGFYNNSVETTGWGILEIRAGYGSQALSNEIIMFVAGFLEGYLTAP
>PLBD1_papHam MTRGGPGGCPGLPPQLPLLLRLLLPPLLLVTAESPNPA:GVYYATAYWMPAEMTVEVKN-IMDKNGDAYGFYNNSVETTGWGILEIRAGYGSQALSNEIIMFVAGFLEGYLTAP
>PLBD1_calJac MTRGGPGGRLGLPPPPLLLLLLLLLPPLPTTAEPPTPA:GISYATAYWMPAEKTVQVKN-VMDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQALSNEIIMFVAGFLEGYLTAL
>PLBD1_otoGar MANRTLDRRLGLPPPPLLLLLLLPPPPLLVTAARKNPP:GVYYATAYWKPAEKTVEVKK-VIDKNGDAYGFYNNSMNATGWGILEIRAGYGSQALSNEMTMFVAGVLEGYLTAP
>PLBD1_musMus MCHRSPGRSLRPPSPLLLLLPLLLQPP-WAAALPASPT:GVHCATAYWSPESKKVEIKT-VLDKNGDAYGYYNDSIKTTGWGILEIRAGYGSQVLSNEIIMFLAGYLEGYLTAL
>PLBD1_ratNor MCHRSHGRSLRPPSPLLLLLPLLLQSP-WAAAPLRSSA:GVHYATAYWLPDTKAVEIKM-VLDKKGDAYGFYNDSIQTTGWGVLEIKAGYGSQILSNEIIMFLAGYLEGYLTAL
>PLBD1_cavPor MALCGPGCSPGLPPSPLLLLPLLL----LAAAWSPSPP:GIHYATAYWIPDTKTVEVKD-ILDKDGDAYGYYNNSMEATGWGILEIKAGYGSQELTNEIIMFVAGFLEGYLTAL
>PLBD1_speTri MSRRSLGCGRW-PPPPLQLLPLLLLLLPLAAAQP----:EVYYATAYWIPSEKSIKVKH-VMDKSGDAYGYYNDSMETTGWSILEIRAGYGSQALSNEIIMFVAGFLEGYLTAP
>PLBD1_oryCun MALWLPPLLFPLL---------------LAAAEPPSPE:GVSYATAYWMDAEKKVQVRN-VLDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQALSNEIIMFVAGFLEGYLTAP
>PLBD1_turTru MSRRSPDGSLGLLSPPALLLLLL------AAVVPSGLA:GVYYATAYWMPTEKRIQVQN-VLDRNGDAYGFYNNSVKTTGWGILEIRAGYGSRSLSNEIVMFAAGFLEGYLTAP
>PLBD1_bosTau MSRHSQDERLGLPQPPALLPLLLLL----AVAVPLSQA:GVYYATAYWMPTEKTIQVKN-VLDRKGDAYGFYNNSVKTTGWGILEIKAGYGSQSLSNEIIMFAAGFLEGYLTAP
>PLBD1_oviAri MPRHRRDERLGLPPPPARLPLLLLLL---AAAVPLSQA:GVYYATAYWMPTEKRIQVKN-VLDRKGDAYGFYNNSVKTTGWGILEIKAGYGSQSLSNEIIMFAAGFLEGYLTAP
>PLBD1_susScr MSRRSRDGRLGLPAPPAPL-LLLLLL---AAAVPPSLA:GVYYATAYWMPTEKRMLVKN-VLDRNGDAYGFYNDSMKTTGWGILEIRAGYGSQSLSNNIIMFAAGYLEGYLTAP
>PLBD1_equCab MARHRPDGRLGLPAPPAPPLPPLLLLLLV-AAVSPSQA:VVYSATAYWMPAEKTVQVKN-VMDRNGDAYGFYNNSVKTTGWGILEIRAGYGSQTLSNDITMFVAGFLEGYLTAL
>PLBD1_felCat MARRSRDGRPGLSAPPTPPLLPLLLL---AAAVSPSLA:EVHYATVYWMPAEKTIQVKN-VLDRNGDAYGFYNDSVKTTGWGVLEIRAGYGSQALSNEIIMFVAGFLEGYLTAP
>PLBD1_canFam MPRRARDARLEPCPPLLPLLLLLL-----AAAVPQGRA:EVYYATAYWIPDEKTIQVKN-VLDRNGDAYGFYNDSVKTTGWGILEIRAGYGSQILSNEITMFVAGFLEGYLTAP
>PLBD1_pteVam MSRRSLDGRLGLPATSAPPLLLLLLL---AAAVPPSLA:evyYATAYWMPAEKTVNVKN-LLDKNGDAYGFYNNSMNTTGWGILEIKAGYGSQTLSNDIIMFVAGYLEGYLTAP
>PLBD1_eriEur MSRRSRDGRLGLLLSPPLLLLLLLL-----AAAPPSLQ:EIYYATAYWMPEEEEIQVKN-VLDKNGDAYGFYNDSMLTTGWGILEIKAGYGSHQLSNDVVMFVAGFLEGYLTAP
>PLBD1_sorAra MARGGGDGPPALLPLPLLSLLLALL----AAAVPPSLA:EVHYATAYWMPDEQRVEIKT-TLDKKGDAYGYYNDSVLTTGWGILEIRAGYGSQDLTDEITMFVAGALEGYLTAP
>PLBD1_loxAfr MSSRSRGRHHGPAPQLPQLLLLLLLLLLVAAAAPPSLA:EVHYATVYWMSSEKTMQVKD-VLDKKGDAYGYYNDSVLTTGWGVLEIKAGYGSQALSNDIIMFAAGYLEGYLTAL
>PLBD1_proCap MCSRSV--PCRLSPPLSPPLSLPLLLLLLAAAAPPSLA:EVHYATVYWMSSEKTMQVKD-TLDKNGDAYGFYNDSMQTTGWGVLEIKAGYGSQGLSNDVIMYAAGYLEGYLTAp
>PLBD1_echTel MSTHSRGGR--PAPPLSPSLSLTPLLLL-AALVAPSLA:EIHYATAYWMSSEKTIQIKD-VLDKSGDAYGFYNDSVNATGWGILEIRAGYGSQNLSNDIIMFAAGFLEGYLTAP
>PLBD1_choHof MSRSCQAERLGPVPRRRLLLLLL-----VASAAPPSVA:EVFYATAYWIPSEKKIVVKD-ILDQNGDAYGFYNDSMKTTGWGILEIKAGYGSHIPSNEIIMFTAGFLEGYLTAE
>PLBD1_triVul MSRRSRDGRLGLPAPPAPLLLLLLL----AAAVPPSLA:GVYYATAYWMPTEKRMLVKN-VLDRNGDAYGFYNDSMKTTGWGILEIRAGYGSQSLSNNIIMFAAGYLEGYLTAP
>PLBD1_monDom MTRFSCFGRLQLW--PLQVLLLLLL----TFGAPVTQA:GIHYATVYWNSSTSSAEVKD-SLDPDGDAYGFYNDTIQTTGWGILEIRAGYGANSLTDEIIMFVAGFLEGYLTAQ
>PLBD1_ornAna MSRTCRGGRSGPPQPAPTPAGLLLLLL--TVASPLLQS:HVRYATAYWESATQTVRVKD-VLDWDGDAYGFYNHTVQTTGWGTLEIRAGYGAQALSDEVVMFVAGFLEGYLTAP
>PLBD1_taeGut MARAGGGVCRCCCWALVLLWAAAGGRA-----------:ELRYATVYWNRAEKILQVKN-TLDRSGDAYGFYNNSLQTTGWGVLEIRAGYGSQTLSNEDIMYVAGFLEGYLTAP
>PLBD1_galGal MARLGGGALCCCWGLVLLWAVAGGRA------------:EMRYATLYWNKAQKILQVKN-ILDRSGDAYGFYNNTVQTTGWGVLEIKAGYGHQTLSNEDIMYAAGFLEGYLTAP
>PLBD1_melGal MARLGGGPLCCCWGLVLLWAVAGGRA------------:EMRYATLYWNKAQKILQVKN-ILDRSGDAYGFYNNTVQKTGWGVLEIKAGYGHQTLSNEDIMYAAGFLEGYLTAP
>PLBD1_sisCat MIRFGNPSSSDTRRQRCRSWYWGGLLLLWAVAETRA--:DIHYATVYWLEAEKSFQIKD-VLDKNGDAYGYYNDTIQSTGWGILEIKAGYGNQPISNEILMYAAGFLEGYLTAS
>PLBD1_ambMex MGGLRQLLPLCALLLLQPLGAR----------------:AIRYATVYWTD-RKTVLVKE-VLDKGGDAYGFYNDTIQSTGWGVLEIRAGYAPTSRTNEEIMFAAGYLEGYLTAL
>PLBD1_takRub MFLLTSTCAFVLLTLPATSSTADG--------------:GTAAATVYWDPQHKTVLLKEGVLEQEGDAYGYFNDTLSSTGWSVLEIRAGYGTTPETDEVIFFLAGYLEGFLTAQ
>PLBD1_danRer MPDFSFCVLFLIGFLFSSRSD-----------------:KLK-ATVYWDATHKSAVLKQGVLDPAGASYGYYDNVLLSTGWGVLEVRAGYGDTTQTDDITMFTAGYLEGFLTAP
>PLBD1_ictPun MTEFMVCVCMFLCAVIAVRTDS----------------:VHK-ATAYWDPDSKTVLLKDGVLEDTGDAYGFYNDSFSETGWGVMEVRAGYGQTPRADERTFFLAGYLEGFLTAR
>PLBD1_perFla MEKQSIKLCVLLSTLAASVQTY----------------:QLQEATVYWDGAQKSVILKEGVMETEGGAYGYFNDTLLLSGWGVLEICAGHGGITQEDETTFFLAGYLEGYLTAG
>PLBD1_gasAcu MFLEKTLYVLLLCSVSTTSSAD----------------:KMTAATVYWDPQHKVVLLKEGVLEKEGDAYGYLNDTLSSTGWSVLEIRAGYGETPETDEVTFFLAGYLEGFLTAQ
>PLBD1_oryLat MKLEVFLLLHVIATFASSQ-------------------:KLTAATVYWDAQHKLVLLKEGVLETEGDAYGYLNNTLSTSGWSILEIRAGYGKTPEDDEITFFLAGYLEGFLTAQ
>PLBD1_pimPro MDTNSICVLLLLCSVSTTSSAD----------------:KMTAATVYWDPQHKVVLLKEGVLEKEGDAYGYLNDTLSSTGWSVLEIRAGYGETPETDEVTFFLAGYLEGFLTAQ
>PLBD1_dicLab MPLVTRLYVFLLFTVVTSFASAD---------------:KMTAATVYWDPLHKLVKLKEGVLETEGDAYGYLNDTLSSSGWSILEIRAGYGKTPETDELTFFLAGYLEGYLTAQ
>PLBD1_salSal MKRVCLLFFFYVAASFASAD------------------:EMKAATVYWDATHKTVQLKEGVIEKEGDAYGYLNDTLSQTGWSVLEIRAGYGETLEHDEVTYFLAGYLEGFLTAP

Difference alignment of exon 1 from placental mammals:
PLBD2_homSap   MVGQMYCYPGSHLARALTRALALALVLALLVGPFLSGLAGAIPAPGGRWARDGQVPPASRSRSVLLDVSAGQLLMVDGRHPDAVAWANLTNAIRETG
PLBD2_panTro   .......S.............................................P...........................................
PLBD2_ponAbe   ......GSS.....----...................................P.T...........A......L......................
PLBD2_rheMac   .......SS..P....................L.................H..P.T..........HAAT....L....Q...........S.H...
PLBD2_papHam   .......SS..P....................L.................H..P.T...........AAT....L......................
PLBD2_calJac   ...K..SS.S.R..Q...............A.L.....S..............S..SG.G....V..AA.....L..................H...
PLBD2_otoGar   ...P..GS..GR..................I.L...C......P..SGR....LIT.....S.....ATTD..RL..............S...H...
PLBD2_musMus   .AAPVDGSS.GWA....R.....TSL..S.T.LL...P...L.TL.PG.Q.QNPD..V..T..L...AAS...RLE..F..................
PLBD2_ratNor   .AAP.DRTH.GRA....R.....----.S.A.LL.......L.TL.PG.R.QNPE.....T..L...AAS...RLEY.F..................
PLBD2_dipOrd   .AAPP.GSR.GRP.GS.S...V.----.V...LSP..P...V.S..D..G.HKPE..V.......V.AAS...RL...L..G...............
PLBD2_cavPor   .AAPT.VSLDGRPV..--......PA.C....LS.GR....V....P.G....P..A.--C......AAS...RL...LQ.G...........P...
PLBD2_oryCun   ..APRDGCA.GR.....A--------....T.LL.G.....A.....GEQ..PPS....CC..A...AAT...RL..................H...
PLBD2_ochPri   .AATRDSSA.CR...V.......---...PT.L....P.....VR.DGEE.GRPA.SG..C....V.AES...RL......A...........H...
PLBD2_turTru   ..DP..GC..GR....................L.....T....T.R.HRGPGRP......C......PET...RL......................
PLBD2_bosTau   ..AP..GS..GR....V...............L.....T....T.R.QRG.GMP......C..L...PET...RL......................
PLBD2_oviAri   ..AP..GS..GR....V...............L.....T....T.R.QRG.GMP......C..L...PET...SL......................
PLBD2_susScr   ..AP..GS..GR....................L.....TS...T.K.YRGSGRS.............TET...RL..................H.N.
PLBD2_ursAme   .AAP..GS..GR....................L.....T....IS.RQ.GPN.P...D.........AET...RL......E...............
PLBD2_myoLuc   ..APPSRS..GR.TP..S..P...PG....A.L....WT....T.RDP.GPN.P..........V..ART...QL....Q.............H...
PLBD2_pteVam   ..AP.DRS..GR..G....T.E.T....P.A.L....RTS..QT..S..GSE.P.S...........PQT...RL..................H...
PLBD2_eriEur   ..AP.CGS..GRP...........PA......L...S......P.EDN.G.N.SF..V..C......SET...RL..............S...H...
PLBD2_loxAfr   ..APV.GS..GR......Q...V.........L.....T...SLT.H..GP..PA............TAT...RL......................
PLBD2_echTel   ..ATE.GS..GR........P....M......L.....T...SPA...REPN.R.....S...A...PAT...RLA.....E...........H...
Consensus      MVAPMYGSPGGRLARALTRALALALVLALLVGLFLSGLAGAIPaPGGRWGRDGPVPPASRSRSVLLDAATGQLRLVDGRHPDAVAWANLTNAIRETG

Alignment of first two exons of PLBD1 from vertebrates showing onset of conservation:

PLBD1exon12.jpg

Understanding conserved residues in PLBD1 and PLBD2

Although the gene duplication creating these paralogs took place in early unicellular eukaryotes with PLBD1 quite diverged in primary sequence from PLBD2 today, it is nonetheless instructive to compare individual residues and residue patches that are still conserved, given the folds have diverged rather little. Here we wish to exploit the situation that more is known about the maturation and substrates of PLBD1 whereas excellent crystallographic structures exist for PLBD2 and certain of its ancient homologs. 

Localization of conserved residues within compared secondary structures: s = beta sheet, h = alpha helix

38    PTGVHCATAYWSPESKKVEIKTVLDKNGDAYGYYNDSIKTTGWGILEIRAGYGSQVLSNEIIMFLAGYLEGYLTALHMYDHFTNLYPQLIKN----PSIV PLBD1          
61    PPVSRTRSLLLDAASGQLRLEDGFHPDAVAWANLTNAIRETGWAYLDLST---NGRYNDSLQAYAAGVVEASVSEELIYMHWMNTVVNYCGPFEYEVGYC PLBD2          
        ssssssssss    sssssss     ssssssssss   ssssssssss       hhhhhhhhhhhhhh   hhhhhhhhh              hh           
        ssssssssss    sssssss     ssssssssss   sssssssss   s    hhhhhhhhhhhhhh   hhhhhhhhhh           hhhh           
  
134   KKVQDFMEKQEMWTRQNIKAQKDDPFWRHTGYVVTQLDGLYLGAQKRASEE-KIKPMTMFQIQFLNAVGDLLDLIPSLSPTKSSSMMKFKIWEMGHCSAL PLBD1          
158   EKLKNFLEANLEWMQREMELNPDSPYWHQVRLTLLQLKGLEDSYEGRLTFPTGRFTIKPLGFLLLQISGDLEDLEPALNKTN----------GSGSCSAL PLBD2          
      hhhhhhhhhhhhhhhhhhhh    hhhhhhhhhhhhhhhhhhhhh                       hhhhhhhhh                    sss           
      hhhhhhhhhhhhhhhhhhhh    hhhhhhhhhhhhhhhhhhhhh                       hhhhhhhhh                    sss           
  
233   IKVLPGFENIYFAHSSWYTYAAMLRIYKHWDFNIKD------KYTLSKRLSFSSYPGFLESLDDFYILSSGLILLQTTNSVYNKTLLKQVVPK-TLLAWQ PLBD1          
253   IKLLPGGHDLLVAHNTWNSYQNMLRIIKKYRLQFREGPQEEYPLVAGNNLVFSSYPGTIFSGDDFYILGSGLVTLETTIGNKNPALWKYVQPQGCVLEWI PLBD2          
      sssss  sssssssssssss    ssssssss               sssssss           ssss sssssssss     hhhh          hh           
      sssss  sssssssssssss    ssssssss sss      sss  sssssss           ssss sssssssss     hhhh          hh           
  
326   RVRVANMMAEGGKEWAQIFSKHNSGTYNNQYMVLDLKKVTINRSL-DKGTLYIVEQIPTYVEYSDQTNV-LRKGYWASYNIPFHKTIYNWSGYPLLVHKL PLBD1          
353   RNVVANRLALDGATWADVFKRFNSGTYNNQWMIVDYKAFLPNGPSPGSRVLTILEQIPGMVVVADKTAELYKTTYWASYNIPYFETVFNASGLQALVAQY PLBD2          
      hhhhhhhh   hhhhhhhh        sssssssss             ssssssss  sssssss hh    sssss      hhhhhh   hhhhhhh           
      hhhhhhhh   hhhhhhhh        sssssssss             ssssssss  ssssssshhhhhhhsssss      hhhhhh   hhhhhh            
  
424   GLDYSYDLAPRAKIFRRDQGNVTDMASMKYIMRYNNYKEDPYSKGDPC-------STICCREDLNGAS---------PSPGGCYDTKVADIFLASQYKAYAISGPTVQDGLPPFNWNRF--NETLHRGMPEVFDFNFVTMK -          
453   GDWFSYTKNPRAKIFQRDQSLVEDMDAMVRLMRYNDFLHDPLSLCEACNPKPNAENAISARSDLNPANGSYPFQALHQRAHGGIDVKVTSFTLAKYMSMLAASGPTW-DQCPPFQWSKSPFHSMLHMGQPDLWMFSPIRVPWD
               hhhhhhh        hhhhhhhhh                          sss                 ssssssssss hhhhh   ssssss         sss              sss    sss           
               hhhhhhhh       hhhhhhhhh         sss     sss      sss                 ssssssssss hhhhh   ssssss         sss              sss    sss

Shared conserved residues: defining specializations of phospholipases

Suppose a phylogenetically broad set of curated PLBD1 and PLBD2 are aligned together. After careful consideration of gap placement, a restricted number of residues will prove very deeply conserved in both proteins throughout eukaryotes. Of these, some are universal localizational, modificational, structural, or catalytic features basic to the entire NTN clan of 12 protein families and so not particular to phospholipases.

This class of residues must be found by structural alignment of crystallographic structures, as primary sequences are too diverged for these to be accurately located by ClustalW or similar tools. Since the fold of PLBD2 was originally recognized by the fold comparison (via Dali) to all of PDB, these are known already: the autocatalytic cysteine residue at the N-terminus of the 40 kDa fragment and the three active site residues noted above.

An additional 6%-14% of structurally equivalent amino acids (themselves only half of the chain) are identical as amino acids, with IMPC (inosine monophosphate cyclohydrolase) being the highest but PVA (penicillin acylase V) and CBAH (conjugated bile acid hydrolase) also significant. Dali provides a primary sequence multiple alignment and so the super-invariant amino acids (plus those with narrow reduced alphabets, while eliminating accidental matches) not specific to phospholipases PLBD1 and PLBD2. These largely lie within the beta strands of the core αββα sandwich as they are better conserved than alpha helices within NTN hydralases but it is fair to say that this fold class is not understood until an explanation can be given for each of the universally conserved residues. 

1oqz CA   cephalosporin acylase
3pva PVA  penicillin V acylase
1k5s PGA  penicillin G acylase
2bjf CBAH choloylglycine hydrolase
2ntm IMP  cyclohydrolase
1ryp ---  a chain among 28 of yeast proteasome

Dali report on 3fgr vs 1oqzB: AyaA is a candidate super-invariant region but only second A occurs in both PLBD1 and PLBD2

DSSP  leeeeeeeeelllleeeeeelllllLLEEEEEEEEhhhhleEEEEEEELllllhhhHHHHH
Query vsrtrsllldaasgqlrledgfhpdAVAWANLTNAiretgwAYLDLSTNgryndslQAYAA   61
ident                                                          A  A
Sbjct ---------------pqapiaaykpRSNEILWDGY------GVPHIYGV-------DAPSA   33
DSSP  ---------------llllllllllLLLEEEEELL------LLEEEELL-------LHHHH

DSSP  HHHHHHHHHHHHHHHHHhhLLLL--LLLL------LLLH-HHHH--HHHHHHHHHHHHH
Query GVVEASVSEELIYMHWMntVVNY--CGPF------EYEV-GYCE--KLKNFLEANLEWM  109
ident                                                            
Sbjct FYGYGWAQARSHGDNIL-rLYGEarGKGAeywgpdYEQTtVWLLtnGVPERAQQWYAQQ   91
DSSP  HHHHHHHHHHHHHHHHH-hHHHHhlLLHHhhhlhhHHHHhHHHHhlLHHHHHHHHHHLL

DSSP  hhhhhhllllhhhhhHHHHHHHHHHHHHHHHL---------llllllllllLLLLlLHHHHL-HHHH-HHHHhhlLLL
Query qremelnpdspywhqVRLTLLQLKGLEDSYEG---------rltfptgrftIKPLgFLLLQI-SGDL-EDLEpalNKT
ident                       L                                     
Sbjct ---------------SPDFRANLDAFAAGINAyaqqnpddispdvrqvlpvSGAD-VVAHAHrLMNFlYVAS---PGR
DSSP  ---------------LHHHHHHHHHHHHHHHHhhhhlhhhllhhhhlllllLHHH-HHHHAHrLMNFlYVAS---PGR

In effect, these residues must be 'subtracted off' the larger set of phylogenetically invariant residues between PLBD1 and PLBD2 to reach conserved residues defining what is specifically important to phospholipases. These sites developed subsequent to the divergence of phospholipases from generic NTN hydralases but prior to the gene duplication giving rise to PLBD1 and PLBD2 because convergent evolution later is implausible. They are evidently critical to structure/function, being retained in both paralogs in all surviving species up until the present day. Several active site residues fall into this category as described earlier.

Conserved residues determining mannosylation

PLBD1 and PLBD2 are among the 2500 human proteins carrying signal peptides targeting them initially to the endoplasmic reticulum and subsequently to the golgi, where they are sorted and packaged according to final destination (lysosome, plasma membrane, extracellular secretion). Many of these proteins are initially glycosylated via a limited repertoire but few of the soluble members are ultimately targeted to the lysozome (about 50 of 2500).

These receive an additional post-translational modification in the golgi of terminal mannose residues to mannose 6-phosphate making them recognizable to the two lysosomal mannose receptors (IGF2R and M6PR). Bizarrely, this simple phosphorylation is not accomplished by a kinase but rather requires 3 separate gene products, GNPTAB (UDP GlcNAc-1-phosphotransferase, internally cleaved into α,β catalytic and recognition subunits), GNPTG (γ regulatory subunit of the α2β2γ2 hexamer) and NAGPA (N-acetylglucosaminidase, or uncovering enzyme).

Overall, trafficking is very imperfect, with some protein molecules [eluding the uncovering enzyme yet http://www.ncbi.nlm.nih.gov/pubmed/20615935 still ending up in the lysosome] and while others even with M6P markers are improperly secreted.

An alternate pathway exists: β-glucocerebrosidase (of Gaucher disease) localizes to the lysozyme via the coiled coil of theLIMP2 protein encoded by SCARB2.) This presumably continues despite mutations (inclusion-cell disease) in GNPTAB, GNPTG, NAGPA, IGF2R and M6PR that disable the main pathway. Conversely, mutations in SCARB2 (causing action myoclonus renal failure syndrome) do not appear to affect proteins using the M6P lysosomal trafficking route. The SORT1 gene product sortilin may also let proteins sidestep the M6P pathway to the lysosome.

PLBD1 and PLBD2 likely use the conventional M6P pathway, nonetheless raising difficult bioinformatic issues. The 50 proteins using it have unrelated folds. They share the generic glycosylation NxT/S sequence motif but this is completely insufficient to differentiate them from proteins not targeted to the lysozome. There is no support for a larger motif enveloping NxT/S or a supplementary contiguous motif elsewhere.

Recognition must reside within individual GNPTAB/GNPTG/NAGPA subunits but their roles are complex, providing no clear bioinformatic handle, only two separated lysines forming a critical triad with M6P at 34 angstrom distance.

However in homologous lysosomal sulfatases ARSA and ARSB, the two identified lysines did not occupy homologous positions, perhaps because sulfatases duplicated and diverged prior to the evolutionary appearance of the lysosome.

This then illustrates convergent evolution rather than conservation of homology. This applies too to the many signal peptides and NxT/S motifs that must have arisen independently a great many times (as no such mobile elements could have provided them).

PLBD2 targeting is then fairly complex from a predictive standpoint as its 40 lysines and 6 M6P would yield a great many candidate pairs, depending on the softness of the 34 angstrom requirement. As PLBD1 and PLBD2 may have diverged later than sulfatases (ie, after recruitment to the lysosome), the lysine pairs might still be homologous. Looking at human gene alignment, this still gives an unwieldy set of 7 homologous pairs (some poor quality because of gap uncertainty).

However these lysines need to be invariant within phylogenetically dispersed PLBD2 and PLBD1 orthologs. Under that assumption, only 3 of the lysines are strong candidates (red), 1 is so-so (magenta) and the rest are implausible (blue). The lysine in CSALIK may be part of the autocleavage motif, leaving two candidates to be compared to the six M6P sites for the required 34 angstrom geometry. 

PLBD2:   189 LKGLEDSYEGRVSFPAGK-FTIKPLGFLLLQLSGDLEDLELALNKTK---IK--PSLGSG 242
             + GL    + R      K  T+  + FL     GDL DL  +L+ TK   +K       G
PLBD1:   169 IDGLYVGAKKRAILEGTKPMTLFQIQFL--NSVGDLLDLIPSLSPTKNGSLKVFKRWDMG 226

PLBD2:   243 SCSALIKLLPGQSDLLVAHNTWNNYQHMLRVIKKYWLQFREGPWGDYPLVPGNKLVFSSY 302
              CSALIK+LPG  ++L AH++W  Y  MLR+ K +W  F      D      ++L FSSY
PLBD1:   227 HCSALIKVLPGFENILFAHSSWYTYAAMLRIYK-HW-DFNVID-KD---TSSSRLSFSSY 280

PLBD2:   303 PGTIFSCDDFYILGSGLVTLETTIGNKNPALWKYVRPRGCVLEWVRNIVANRLASDGATW 362
             PG + S DDFYIL SGL+ L+TT    N  L K V P   +L W R  VAN +A  G  W
PLBD1:   281 PGFLESLDDFYILSSGLILLQTTNSVFNKTLLKQVIPE-TLLSWQRVRVANMMADSGKRW 339

PLBD2:   363 ADIFKRFNSGTYNNQWMIVDYKAFIPGGPSPGSRVLTILEQIPGMVVVADKTSELYQKTY 422
             ADIF ++NSGTYNNQ+M++D K  +    S     L I+EQIP  V  +++T  L +K Y
PLBD1:   340 ADIFSKYNSGTYNNQYMVLDLKK-VKLNHSLDKGTLYIVEQIPTYVEYSEQTDVL-RKGY 397

PLBD2:   483 DFLHDPLSLCKACNPQPNGENAISARSDLNPANGSYPFQALRQRSHGGIDVKVTSMSLAR 542
             ++  DP S    CN        I  R DLN  N S P         G  D KV  + LA 
PLBD1:   458 NYKKDPYSRGDPCN-------TICCREDLNSPNPS-P--------GGCYDTKVADIYLAS 501

The phylogenetic emergence of these features could time the onset of targeting to the lysozyme during the evolutionary history of phospholipases PLBD1 and PLBD2. Mutations localizing at M6P receptor targeting sites would lead to non-recognition, non-arrival, and perhaps lysosomal storage disease (as the proteins would then be secreted).

The evolution of the lysozyme itself, preposterously claimed a vertebrate innovation as late as 2006, instead uses components that readily trace back to slime mold. These surface as the eight loci responsible for flawed lysosome related organelle biogenesis (Hermansky-Pudlak syndrome).

Ragged carboxy terminus in PLBD1

The deuterostome orthologs of PLBD1 display an unusual pattern of extensions and contractions of the carboxy terminus. These do not correspond to subclades, implying numerous separate events affecting the stop codon in fairly recent times (which is supported by read-through past the stop codon, lower case). The precise location of the stop codon may not be at all important but conservation does continue on to the proline near the end (which is strongly invariant. Proline is a common helix terminating residue but here the structural model suggests it is terminating a beta strand.

PLBD2 is much more orderly at its carboxy terminus, though some extensions are seen in early deuterostomes. Again an almost-terminal proline is conserved. However past it, a tryptophan is also universally conserved. The xray structure fortunately includes both residues -- the goal for every protein is to provide an explanation for each conserved residue (for rational interpretation of possible disease SNPs) though here the focus initially is on activation and catalysis. 

PLBD1_homSap VADIYLASQYTSYAISGPTVQGGLPVFRWDRFNKTLHQGMPEVYNFDFITMKPILKLDIK*
PLBD1_panTro VADIYLASQYTSYAISGPTVQGGLPVFRWDRFNKTLHQGMPEVYNFDFITMKPILKLDIK*
PLBD1_ponAbe VADIYLASQYTSYAVSGPTVQGGLPVFRWDRFNKTLHQGMPEVYNFDFITMKPILKLDIK*
PLBD1_rheMac VADIYLASQYTSYAISGPTVQGGLPVFHWNRFNKTLHQGMPEVYNFDFITMKPILKRDMK*
PLBD1_papHam VADIYLASQYTSYAISGPTVQGGLPVFHWNRFNKTLHQGMPEVYNFDFITMKPILKRDMK*
PLBD1_calJac VSDIYLASQYTSYAISGPTVQGGLPVFRWNRFNKTLHQGMPEVYNFDFITTKPILK*hkmk
PLBD1_tarSyr VADIYLASQYTAYAISGPTVQDGLPVFHWNRFNKTLHQGMPEVYNFDFVTMKPILKLDIK*
PLBD1_micMur VsDIFPASQFTGHAINGPTVPSGlPVFYRPPFNKTPHQGIAEAYHFDFISKKPILKPDIK*
PLBD1_otoGar VADIYLASQYTAYAISGPTVQGGLPVFHWHRFNKTLHHGMPEAYNFDFITMKPVLKLDIK*
PLBD1_tupBel VADIYLASQYTAYAISGPTVQDGLPVFHWNRFNRTVHQGMPEAYNFDFITMKPVLKLDIK*
PLBD1_musMus VADIFLASQYKAYAISGPTVQDGLPPFNWNRFNDTLHRGMPEVFDFNFVTMKPILS*dkk*
PLBD1_ratNor VADIFLASQYKAYAISGPTVQNGLPPFNWNRFNDTLHQGMPDVFDFDFVTMKPILT*dkn*
PLBD1_perMan VADIFLAFQYTAYAISGPTVQDGLPAFDWKHFNKTLHEGMPDVFNFDFVTMKPILTEDIK*
PLBD1_dipOrd VSDIFLASKYIAYAISGPTVQDGLPAFSWRLFNKTLHQGMPEIYNFDFVLMKPFFND*qk
PLBD1_cavPor VADIHLASEYTAYAISGPTVQGGLPVFRWNRFNDTLHQGMPEVYNFDFITMKPILKPNVKRRRKMRE*
PLBD1_speTri VSDIYLASQYTAYAISGPTVQGGLPVFRWNRFNTTLHQGMPEAYNFDFITMKPVLKIDIK*
PLBD1_oryCun VSDIYLASRYTAYAISGPTVQGGLPVFHWNRFNKTLHQGMPEVYNFDFITTKPILKLDKR*
PLBD1_ochPri VSDVHLASQYTAYAISGPTVQGKLPVFHWSQFNKTLHQGMPDAYNFDFITMKPILKKMREDEAEGNRMK*
PLBD1_vicPac VADIYLASQSTAHAISGPTAEDGLPVFHWNRFNKTLHSGMPEVYNFDFITMKPIL*ldik*
PLBD1_turTru VADIHLASAYTAYAISGPTVQGGLPVFHWSRFNKTLHEGMPEAYNFDFITMKPIL*ldmk
PLBD1_bosTau VADIYLASKYKAYAISGPTVQGGLPVFHWSRFNKTLHEGMPEAYNFDFITMKPIL*ldik
PLBD1_oviAri VADIYLASKYKAYAISGPTVQGGLPVFHWSRFNKTLHEGLPEAYNFDFITMKPIL*ldik 
PLBD1_lamPac VADIYLASQSTAHAISGPTAEDGLPVFHWNRFNKTLHSGMPEVYNFDFITMKPIL*LDIK 
PLBD1_susScr VADIHLASTYTAYAISGPTVQDGLPVFHWNHFNKTLHEGMPEAYNFDFITMKPTL*LD
PLBD1_equCab VADIYLASKYTAYAISGPTVQGGLPVFHWNRFNKTLHEGMPEAYNFDFITMKPILKPYVKGRR*
PLBD1_ailMel VADIYLASEYTAYAISGPTVQGGLPIFHWNRFNKTLHKGMPETYDFDFITMKPILKRDKK
PLBD1_felCat VADIYLASAYTAHAISGPTVQDGLPVFHWNRFNKTLHQGMPETYNFDFIIMKPILKQDIK*
PLBD1_canFam VADIYLASEYTAYAISGPTTQGGLPVFHWNRFNKTLHKGMPEIYNFDFMTMKPILKHDRK*
PLBD1_myoLuc VADMYLALEYTAHAISGPTVQGGLPVFHWKRFNKTLHEGMPEAYNFDFITMKPILKPDIK*
PLBD1_pteVam VADIYLASQYTAHAISGPTVQGALPVFHWNQFNKTLHEGMPEAYNFDFVTMQPILKPDKK*
PLBD1_eriEur VADFYLTFKYTAYAISGPTVQDGLPAFHWNRFNKTLHKGMPEVYNFDFVTMKPVL*ldrk
PLBD1_sorAra VADIYLAAKFTAYAISGPTVQGGLPVFRWDPFNKTLHRGMPESFDFDFITVKPTL*qdkk
PLBD1_loxAfr VADMYLASEYTAYAISGPTVQNGLPVFHWNRFNKTLHHGMPEAYNFDFVTMRPILKPDRN*
PLBD1_proCap VSDMFLASEFIAYAISGPTVQNGLPVFHWNNFNKTLHQGMPEAYNFDFVTMQPILKLDRKL
PLBD1_echTel VADMWLASKYRAYAISGPTVQDGLPVFRWGSFNKTVHQGMPEAYNFDFTHMKPILT*gr*
PLBD1_dasNov VADIYLASQYTAYAISGPTVQGGLPVFHWNRFNKTLHEGMPEAYNFDFITMKPSLNSDIK*
PLBD1_choHof VADIYLASQYTAYAISGPTVQGGLPVFHWNRFNKTLHRGMPETYNFDFITMKPILT*ne*
PLBD1_monDom VADMFLASQFTAYAINGPTVDDGLPVFEWKKFNETIHKGLPEAYNFDFVTMKPLLEFCELHKEKKKRCGKQVRRWKRRN*
PLBD1_ornAna VSDMALAARLTAHAISGPTVQGGLPVFRWSRFNGTVHRGLPEAYDFDFVTMRPVLRPPWPREAGGR*
PLBD1_galGal VSDFRLASAFTATAINGPPVQGGLPVFTWRRFNNTRHQGLPESYNFKFVTMRPIL*
PLBD1_taeGut VSDFRLAAAFTASAINGPPVQGGLPAFSWRRFNRTRHQGLPESYNFDFVTMRPIL*
PLBD1_anoCar VADINMAMKFTSYAINGPPVEEGLPIFTWSRFNQTKHQGLPDSYNFDFITMKPVL*
PLBD1_botJar VADISMAAKFTAYAISGPTVEKGLPVFSWVHFNKTKHQGLPESYNFDFVTMKPVL
PLBD1_ambMex VSDFYLAATYTAHAINGPPVADGLPPFSWSPFHETIHEGLPEHYNFSFILTKPVL
PLBD1_tetNig VTDFLMAGKFRAEAINGPTTQSGLPPFVWDRFGSVSHQGLPQSYNFTFVPMQPLLF*
PLBD1_takRub VTDFFMAGKFRAEAINGPTTQNGLPPFAWDGFGNISHEGLPKYYNFTFVQMQPILFQP*
PLBD1_gasAcu VTDFHMAGDFRAEAVNGPTTQDGLPPFFWDKFSSMSHQGLPQFYNFTFIRMQPVLFEP*
PLBD1_oryLat VTDFFMAGDFTAEAVNGPTTQDGLPPFYWDKFSSISHQGLPRFYNFTFVTMKPLMFKP*
PLBD1_salSal VTDFHMAQEFRAEAVNGPTTQGDLPPFSWEDFNSTAHQGLPDHYDFPFISMQPALFMP*
PLBD1_danRer VADYRMAQMFTAEAVNGPTSQNGLPLFSWSRFNRTAHQGLPQTYNFTFITMQPLLFAFRDQAKTER...
PLBD1_ictPun VTDLNMAQQFVSEALNGPSTDGDLPPFTWDAFNRTTHQGLPRLYNYTFVTMHPVLFSP
PLBD1_petMar VADMRMAKKFMTSAVNGPTVEGKLPAFSWSPFDNIKHEGLPNTYKFPFVTMQPTLFTIP*
PLBD1_braFlo VSDYYLARNLTSFAINGPTLGTGLEPFSWSDKFKISHIGLPKVYNFSFVTMTPAEL*
PLBD1_strPur VTNLAMAAKQTSFVINGPTRGDGSLPPFKWVAPFTGWSHVGLPTVYDFNFVEMCPKEL*

PLBD2_homSap VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD*
PLBD2_gorGor VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD*
PLBD2_ponAbe VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD*
PLBD2_nomLeu VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD*
PLBD2_papHam VTSMSLARILGLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD*
PLBD2_calJac VTSMSLAKILSLLAASGPTWDQVPPFQWSTSPFSTLLHMGQPDLwKFCAPKVSWD*
PLBD2_micMur VTSMSLAKALSLLAVSGPTWDQVPPFQWSASPFSSLLHMGQPDLWKFSPIRVWWQ*
PLBD2_otoGar VTSMSLAKALSLLAVSGPTWDQVPPFQWSTSPFSSLLHMGHPDLWKFLPIEVWWD*
PLBD2_tupBel VTSMSLAKTLSLVAASGPTWDQVPPFQWSTSAFSHLLHMGHPDLWRFSPIQVSWD*
PLBD2_musMus VTSFTLAKYMSMLAASGPTWDQCPPFQWSKSPFHSMLHMGQPDLWMFSPIRVPWD*
PLBD2_ratNor VTSVALAKYMSMLAASGPTWDQLPPFQWSKSPFHNMLHMGQPDLWMFSPVKVPWD*
PLBD2_dipOrd VTSMSLAKALGLLAVSGPTWDQVPPFQWSSSPFPDVLHMGQPDLWKFLPVEVLWGL
PLBD2_cavPor VTSMSMAKTLSLQAVSGPTWDQVPAFQWSTSPFRDMLHMGHPDLWKFAPVEVSWG*
PLBD2_oryCun VTSKSLAKAMSLLAASGPTWDQVPPFQWSTSPFRDQLHMGHPDLWKFLPIRVLWD*
PLBD2_ochPri VTSMSLAKALSLLADSGSTWDQVPPFQWSASPFRDKLHMGHPDLWKFLPFKVLWD*
PLBD2_vicPac VTNMALAKALRLLAASGPTWDQLPPFQWSTSPFSRLLHMGQPDLWKFSPIDVWWD*
PLBD2_turTru VTSTALAKALRLLAVSGPTWDQLPPFQWSSSPFSSLLHMGQPDLWKFSPIEVWWD*
PLBD2_bosTau VTSTALAKALRLLAVSGPTWDQLPPFQWSTSPFSGMLHMGQPDLWKFSPIEVSWD*
PLBD2_oviAri VTSTALAKALRLLAVSGPTWDQLPPFQWSTSPFSGMLHMGQPDLWKFSPIEVSWD*
PLBD2_lamPac VTNMALAKALRLLAASGPTWDQLPPFQWSTSPFSRLLHMGQPDLWKFSPIDVWWD*
PLBD2_susScr VTSMALARVFGLLAASGPTWDQLPPFQWSTSPFSHLLHMGQPDLWKFSPIEVSWD*
PLBD2_felCat VTSMALAKAFQLVAASGPTWDQLPPFQWSASPFSGLLHMGQPDLWKFSPIEVRWD*
PLBD2_canFam MTSMALAKAFHIIAVSGPTWDQVPPFQWSSSPFSGLLHMGQPDVWKFLPIETWWD*
PLBD2_ailMel VTSMALARAFHIIAVSGPTWDQLPPFQWSSSPFSSLLHMGQPDLWKFSPIEVWWD*  
PLBD2_myoLuc VTSMALAKALRLVAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFSPVKVSWD* 
PLBD2_pteVam VTSAALAKALRFLAASGPTWDQLPPFQWSTSPFSGLLHMGQPDLWKFSPIEVWWD*
PLBD2_eriEur VTNVSLVRALGLLAVSGPTWDQLPPFQWSTSPFSGLLHMGQPDLWKFspievwwd*
PLBD2_loxAfr VTSLAMAKALRLLAVSGPTWDQLPPFQWSTSPFRSLLHMGQPDLWKFLPIEVWWD*
PLBD2_proCap VTNLAMAKALRLRAVSGPTWDQLPPFQWSTSPFQSLLHMGQPDLWKFLPIEVWWN*
PLBD2_echTel VTSSGLAKSLRLWAVSGPTWDQLPPFQWSSSPFHNLLHMGQPDLWKFSPVEFGWD*
PLBD2_macEug VTSYELSKDLRLIAVSGPTWDQLPPFQWSSSPFDKLLHMGHPDLWKFFPIKVSWE*
PLBD2_ornAna VTSSQLAKDFRFVAASGPTWDQVPAFRWSSSPFKGLVHMGHPDLWRFSPVHVRWD*
PLBD2_galGal VTSFGMARTFGLVAASGPTWDDVPPFRWSTSPCSHLLRMGHPDLWRFPPVKVRWD*
PLBD2_taeGut VTSSAMVPTFGLVAVSGPAWDDVPPFRWSASPCSSLLHMGHPDLWTFPPVKVHWD*
PLBD2_anoCar VTSFEMAKLYSFVATSGPTWDDLPAFEWSSSPYRNLLHMGHPDLWRFSPIQVHWG*
PLBD2_xenTro LTSYEMAKKYEMVVVNGPTWDQVPPFQWSTSPFSSLMHMGHPDLWKFSPITIRWH*
PLBD2_ranCat VTSYEFAKEYMMFAVNGPTWDQVPPFQWSTSPFSNLMHMGHPDLWKFDPILIRWK*
PLBD2_cynPyr VTSFNMARVYGMVAVSGPTWDDLPPFQWSTSPFSVQLHMGHPDLWQFDPVEVLWWQ*
PLBD2_tetNig LTSYQMFRDYAMIAVSGPTWDQVPPFQWSTSPYKDLLHMGHPGTWTFKPVKVTWKP*
PLBD2_takRub LTSYKMFRDYGLIAVSGPTWDQVPPFQWSTSPYKDLLHMGHPDTWTFKPITVIWTP*
PLBD2_gasAcu LTSFEMFRDYAMLAVSGPTWDQVPPFQWSTSPYSDLMHMGHPDSWAFKPVKVSWNP*
PLBD2_oryLat MTSFGMFKEYGMIAVSGPTWDQLPPFQWSTSPYKDLVHMGHPDVWNFKPIKVTWTP*
PLBD2_salSal VTSYGLWREFGFLAASGPTWDQVPAFQWSSSPYSDLMHMGHPDTWAFTPIHVTWST*
PLBD2_danRer MTSSSMFRQWELLAASGPSCEQTPVFQWSRSPYSSLMHMGQPDRWDFPTVHVRWAT*
PLBD2_ictPun MTSYGLFKQYELLAVSGPTWDQVPAFEWSTSPYSSLTHIGHPDRWDFPTVHIRWSE
PLBD2_squAca VTSFELHSTYQMIAVNGPTWDEVPAFQWSKSPFSSLMHMGHPDLWRFLPVMVQWK*
PLBD2_calMil VTSSEMYKTFEMIAISGPAWDQVPPFQWSKSSYSGLIHMGHPDLWKFPPVMVRWS*
PLBD2_petMar ITSKAMVPRLEMVAQSGPTWDQQPPFQWSKSPFSSLSHVGQPDLWSFLPEHISWCKHSGQ*
PLBD2_braFlo MTSYSMHESHQMMAVSGPTHDQQQPFQWSTSDYDKQFYHLGHPDLFNFDPIHVIWFDQSDN*
PLBD2_cioInt VVGYSMMKNFEILAECGPTHDQQPPFVWSKSPFSHVSHKGMPDKYDFKPTLIIWDKFSPLKMLDKIHKSVNL*
PLBD2_halRor VTSYTLHKTLQMVAEAGPTHDQQPVFQWSTSPYASKSHEGHPDRFDFLPVLIKWDGEILPK* 
PLBD2_strPur VTTSSMVKSLSMVAVCGPTTDQQPPFQWSKSDFNQTLHLGHPDLFNFKPINVQWYD*
PLBD2_sacKow LTNYVMHKDLSFVAISGPTQDQQPVFQWSTSPYLDVLHLGHPDKFDFGPVQVNWKND*
PLBD2_aplCal LISYDLFKSLSFLAISSPTYDDLPPFQWSKSDYNYMSHLGHPDVWKFPRILFKGTDPLA*
PLBD2_creFor LTSAEMVKDLTFIAVGGPSWDQQPPFQWSKSDFKSTSHIGQPDLWKFPPVLFNISLIF*
PLBD2_eupSco LTNSSLARDLQFVAIAGPTYDQVTPFQWSKSDFKNTVSHIGHPDIFKFEPYIFGDDVMKFE*
PLBD2_craGig LTNSAMFKAMQFVAISGPTYDQFAPFQWSKSDFKDNTPHMGHPDTFKFDPVVFDGTTDFKPFQR*
PLBD2_helRob ITSYSMFKNFQFLAVSGPTRDQVAPFQWSKSDLKDTIRHAGHPDLWVFDPVQF*
PLBD2_capCap ITSYSMFKNFQFLAVSGPTRDQVAPFQWSKSDLKDTIRHAGHPDLWVFDPVQF*
PLBD2_limPol LTTFGLSQKFEFVAIGGPTHDPLPPFQWSKSDFSKDLPHYGHPDLWVFKPVTHHWK*
PLBD2_ixoSca VTNFALFERQVFYAVSGPTSDDQRPFRWSTSGFDNVSHAGHPDLWDFDPILAQWRY*
PLBD2_derVar LTTYQQFKEQQFFAISGPTWSQQPVFQWSTSGFNDSHVGHPDRWEFGPVLNYWGSCR*
PLBD2_dapPul VTSHQLMTSLDFIAVGGPTFDSLPAFRWSESDFVNMSHIGHPDLWKFEPVQTEWTL*
PLBD2_calCle VTSFDLLLRGSFIAGGGPTYDSVEPFQWSKADFEKDTPHFGHPDKWDFKPMRVEWDNVL*
PLBD2_lepSal ITSFDLFMKGSFIAGGGPTYDSVEPFQWSKTDFGKTTPHFGHPDKWAFKPIKVDWENAFNRDEDVV*
PLBD2_hydMag ATSFQLSKLMSQFIVGGPTYDQQPPFQWSKTEWNRPLGHPDIFKFNPELLDWRKEEWIYSKLNI*
PLBD2_nemVec ITSFELFQKFQCIAVSGPTHDQQPAFQWSTSEWEKPLGHPDKFDFEPVKVSWDNKD*
PLBD2_merSen ITSSELFKKFQCQAVSGPTHDQQPVFQWSKSDWKRPLGLPDKFDFSPVMVSWENEE*
PLBD2_acrPal ITNSEMVKSLECVAVSGPTHDQQPVFKWSASGWDTPLGHPDAWDFEPIVVKWQEN*
PLBD2_porAst ATSHQLVQQLSTIATCGPTHAQQPVFKWSESGETKPLGHPDAFDFPTVQIKWNKQ*
PLBD2_monFav VTNSELIKELQCMAVSGPTHDQQPVFKWSTSGWKRPTGHPDAWDFEPIKVTWE*
PLBD2_ampQue LTNSEMVKSLSCLATSGPTHSQQPVFKWSTSGFQDTPPLGHPDEFDFAPIVIKWGEIN*
PLBDa_dicDis VVSADMVAALLVNAQSGPSHDNETPFTWNSQWNQKYTYAGQPTTWNFDWMTMSLQSMKPASPSSDSSSDSTTFN*

Signature conservation in the final exon

Using the above collection of sequences for the final exon of PLBD1 and PLBD2, it is possible to compare the same 45 vertebrate species for both genes. When these are aligned retaining phylogenetic order and allowing 0,1,2,...,10 departures from absolute invariance at each site, the two tables below result.

From them, residues conserved in PLBD1 but not PLBD2, conserved in PLBD2 but not PLBD1 or conserved in both can be extracted. The former serve quite well to classify phospholipases in the very earliest diverging eukaryotes which otherwise might have poor overall Blast scores to their mammalian ortholog. That classification is usually supported by an indel location: PLBD2 has lost one residue relative to PLBD1 just before the conserved SGPT motif and PLBD1 has lost two residues relative to PLBD2 further down.

The two proteins have been conserved equally well over the 500 million years of vertebrate evolution, quantitated simply by a dot count (.) in the two tables relative to the total number of residues (45 spp x 55 sites = 2475). Oddly, this conservation often takes place at non-homologous (different) sites in the two proteins despite the overwhelming similarity in their folds. At two sites near the carboxy terminus, a residue is conserved but it differs for the two protein (F/P and P/W). Otherwise 11 residues are conserved in both proteins -- this conservation may date back to the original gene duplicated (and perhaps earlier in NTN hydratases). 

Vertebrate conservation of last exon of PLBD1 with increasing tolerance for exceptions:
 0 V.D..........A..GP.....LP.F....--F....H.G........F....P..
 1 V.D...A......A..GP.....LP.F.W..--F....H.G.P....F.F....P..
 2 V.D...A......A..GP.....LP.F.W..--F....H.G.P..Y.F.F....P.L
 3 V.D...A......A..GP.....LP.F.W..--F....H.G.P..Y.F.F....P.L
 4 V.D...A......A..GP....GLP.F.W..--F..T.H.G.P..Y.F.F..M.P.L
 5 V.D...A......A..GPT...GLP.F.W..--FN.T.H.G.P..Y.F.F..M.P.L
 6 V.D...A......AI.GPT...GLP.F.W..--FN.T.H.G.P..YNF.F..M.P.L
 7 V.D...A......AI.GPT.Q.GLP.F.W..--FN.T.H.G.P..YNF.F..M.P.L
 8 V.D...A....a.AI.GPT.Q.GLP.F.W..--FN.T.H.G.P..YNF.F.TM.P.L
 9 V.D..LA....a.AI.GPTVQ.GLP.F.W..--FN.T.H.G.PE.YNFDF.TM.P.L
10 V.D..LA....a.AI.GPTVQ.GLP.F.W..--FN.T.H.G.PE.YNFDF.TM.P.L

   V.D...A...............gL.......--Fn.t............F..m.P.L conserved PLBD1 not PLBD2
   .............A..GPt.....P.F.W.........H.G.P..w.F........  conserved PLBD1 and PLBD2
   .Ts............S...-WDq......S.SP......m...D.....P..v.W.. conserved PLBD2 not PLBD1

Vertebrate conservation of last exon of PLBD2 with increasing tolerance for exceptions:
 0 .T..............G..-....P.F.WS.S........G.P..W.F......W..
 1 .T..............G..-....P.F.WS.S........G.P..W.F......W..
 2 .T...........A.SGP.-WD..P.F.WS.SP.....H.G.PD.W.F......W..
 3 .T...........A.SGPT-WD..P.F.WS.SP.....HMG.PD.W.F.P....W..
 4 .TS..........A.SGPT-WDQ.P.F.WS.SP.....HMG.PD.W.F.P....W..
 5 .TS..........A.SGPT-WDQ.P.FQWS.SP.....HMG.PD.W.F.P..V.W..
 6 .TS..........A.SGPT-WDQ.P.FQWS.SP.....HMG.PD.W.F.P..V.WD.
 7 .TS..........A.SGPT-WDQ.PPFQWS.SP.....HMG.PD.W.F.P..V.WD.
 8 .TS..........A.SGPT-WDQ.PPFQWS.SP....LHMG.PDLW.F.P..V.WD.
 9 VTS..........A.SGPT-WDQ.PPFQWS.SP...LLHMG.PDLW.F.P..V.WD.
10 VTS..........A.SGPT-WDQ.PPFQWS.SP...LLHMG.PDLW.F.P..V.WD.

Lysosomal storage disease: allele prediction

Plbd2CpG.png

Experience has shown that it is fairly simple to predict about half of the common mutations that will eventually surface as disease alleles. These are comprised of (1) rare polymorphisms in the population that occur at phylogenetically conserved sites and and (2) CpG hotspot non-synonymous substitutions at similarly conserved residues. Here there will also be replication slippage errors in the regions of anomalous GC compositions such as the signal peptide; their outcome is difficult to predict but would lead to frameshifts 2/3 of the time (if not 3n).

The reason for this is both PLBD1 and PLBD2 are autosomal, meaning that both alleles must be affected. This occurs in the consanguineous case mainly for adverse polymorphisms that have managed to attain a reasonable frequency in the population. These can otherwise combine with the dominant de novo mutations, namely CpG mutations resolving to TpG or CpA. Indeed the Q54P discussed earlier for the reference genome arose as a transversional CpG mutation CCG (pro) -> CAG (gln), as can be seen from chimpanzee sequence.

The significance of a given substitution can then be read off the 46-way vertebrate alignment -- a departure from the evolutionarily tested reduced alphabet is rarely an adaptive innovation but rather almost always an unacceptable change reducing fitness (what else could account for observed conservation?).

As a practical matter, the 1000 Human Genome Project provides rare alleles to the 1% level for every gene (though currently these are difficult to extract for a particular gene). These are supplemented with previously observed SNPs. Consequences of CpG mutations are obtained by in silico substitution, followed by Blastx against wildtype to flag differences (55 for CpA, 50 more for less frequent TpG), followed by evaluation of the change, using mammals from the 46-way with MultAlin set to maintain input order.

Neanderthal has 4 substitutions (three arising from CpG) relative to human, none of which are predicted to affect protein function: 

human genome location      #reads change  N   H  comment
chr12:112280948-112280949  1C>0A(CCG>CAG) P 54Q  reference genome has a mutation at an ultra-conserved site
chr12:112294980-112294981  0G>3C(GCT>CCT) A182P  human has a phylo-acceptable change
chr12:112297191-112297192  0C>2T(CGG>TGG) R286W  human has a change at an unselected residue
chr12:112309977-112309978  0G>3A(GAC>AAC) D496N  human has a change at an unselected residue

To these should be added fatal CpG disruptions of splice donors, which PLBD2 has in 3 consecutive exons, all of phase 2 which give rise in the CpG hotspot effect either to a phylogenetically non-conservative change in 2nd letter of codon -- shown at left: T373M, P405L, P429L for CpG mutating to TpG -- and/or loss of GT-AG splice donor soon leading to a premature stop codon for for CpG transitioning to CpA: 

agGTGACACTGGAGACCACCATTGGCAACAAGAACCCAGCCCTGTGGAAGTATGTGCGGCCCAGGGGCTGTGTGCTGGAGT
GGGTACGCAACATCGTGGCCAACCGCCTGGCCTCGGATGGGGCCACCTGGGCAGACATCTTCAAGAGGTTCAACAGCGGCACgt 2 T373M

agGTATAACAACCAGTGGATGATCGTGGACTACAAGGCGTTCATCCCGGGTGGGCCCAGCCCCGGGAGCCGGGTGCTTACCATCCTGGAGCAGATCCCgt 2 P405L

agCGGCATGGTGGTGGTGGCTGACAAGACCTCGGAGCTCTACCAGAAGACCTACTGGGCCAGCTACAACATACCgt 2 P429L

No stop codons result from CpG mutations in PLBD2 but numerous non-conservative changes occur at invariant sites. A sampler of these is shown flanking the GSGSCSALIK cleavage motif below: E153K, R183Q, E197K, R199H, R272H, R282Q, G294S are all predicted to be disruptive. Dashes indicate lack of data rather than deletions -- input sequences could be greatly improved by curation as is done below -- and at much greater phylogenetic depth -- for the terminal exon. 

       160       170       180       190       200       210       220       230       240       250       260       270       280       290       300
        |         |         |         |         |         |         |         |         |         |         |         |         |         |         |
CpG2CpA YCKRLKSFLEANLEWMQEEMESNPDSPYWHQVQLTLLQLKGLEDSYKGHVSFPAGKFTIKPLGFLLLQLSGDLEDLELALNKTKIKPSLGSGSCSALIKLLPGQSDLLVAHNTWNNYQHMLHVIKKYWLQFQEGPWGDYPLVPSNKLVFS
homSap  ..E.............................R.............E.R........................................................................R.........R...........G......
panTro  ..E.....................Y.A.....R.............E.R........................................................................R.........R...R.......G......
gorGor  ..E.......................A.....R.............E.R.....................................................................Y..R.........R...RSFETVFNASG.QAL
ponAbe  ..E.......................A.....R.............E.R........................................................................R.........R...R.......GS.....
rheMac  ..E.......................A.....R.............E.R........................................................................R.........R...Q..S....G......
papHam  ..E.......................A.....R.............E.R........................................................................R.........R...Q..S....G......
calJac  ..E.................Q.....A.....----------------------------------------------------------------------------------------------------------------------
micMur  ..E.......V.......QVAL.K..V.....R.............E.R.N..TE.............I........P......V.AA..A.......................HS.....R......F..R...RD.D....G..V...
tupBel  ..E..................R.Q..T....KTKV.-------------------------------------------------------..GCS....---.RH.VATR----W.....R....CL.-IHQ..QE.S...........
musMus  ..EK..N..........R...L..........R.............E.RLT..T.R............I........P.....NT..................GH..........S..N..RI....R...R...QEE....AG.N....
ratNor  ..EK.............R...LS.........R.............E.RLT..T.R.N..........I........P.....NT...V..............SH..........S..N..RI....R...R...QEE...IAG.N.I..
dipOrd  ..EK.........A...A...QQVN.T.....R.............E.R.T..TE.......X--............P....SNS..V....A....V.....HR..........A..S..R.........R...---------------
cavPor  ..E.......T......QQ..LH..C......R.............E..L...T.R.S..........I.............S.TRAT.........L......R..........A..Y..R.L...Q...RV..RE.F..A.G..V...
speTri  ..EK..H..........R..QWDQ..G...K.R.............E.R....T..............I........P.....RTRAV............M...R.....L.--S.S.N..-I....TS.I--..QK.AL...G.QM...
oryCun  ..E...R..............R.K..T.....R.............E.R.T..T.R............I........P...R..TQAA...............HR..........S.....R......F..R...QE......G.RVI..
ochPri  ..E...RY.........A.....RG.M.C...R.............E.R....M.R............I........P......T.AV................Q..........S.....RI.....F..RKD.Q..S....G.RVI..
turTru  ..E...K..........K...L.NG.A.....R.............E.S.T..T..............I...............T.HAT.........................HS..Y..RIM....F..R...QEQSTRA.G.RVI..
bosTau  ..E...N..........K...L.NG.A.....R.............E.S.A..T....V.........I........V......TNHAM...............R.........HS..Y..RIM....F..R...QAESTRA.G..VI..
equCab  ..E......DT............K..A.....R.............E.S.A..T.R............I....G...P.....EARRT..A.A....V.....HR..........S..S..RI.....--------S.A.PA.G.R....
felCat  ..EK..N...T.......Q.QR.KG.A----.R.............ESS.T..T.R.............A...G...P......S.HVM...................I.....SS..N..RI.....F..R.D.QVNS..A.G......
canFam  ..E..MR...T..D..L.LIKL.K.FA..--.R.............E.SMT..T.R.................G...P......T.HIM...................I.....SS..N..RI.....F..R.D.QENS..A.G......
myoLuc  ..E...G..........KQV.L.K..A.....R.............E.S.A..T..............I...............T.HT...............H...........S..N..RI.....F......QE.S..A.G......
pteVam  ..EK.................L.K..A.....R.......-.....E.S.T..T..............I...............TRHT..A....V.......HG..........S..N..R.....SF..RQ..QE.S..A.G......
loxAfr  ..EK.................L.R..A.....R.............G.RMT..T.......................P....S.T.RA................R..........S.....R.V...SFH.H...RENSL.A.G.SM...
proCap  --------------...K...L.R..A.....R......-......G.RMT..T.N.....................P......T.RA................R..........S.....R.L...SFH.R...QE.SV.A.G.T....
echTel  ..EK......T..........R.K..T.....R.A...........G...T..T.R.....................P......T.R.M...............R..........S.....RIV...SF..H...QD.SL.I.G...I..
dasNov  ..E...R..........R..ALS.HDA.....R.............E.R.T.......F.........V---------------------------------------------------------------------------------
macEug  ..EK..NYI.L..A...KQIA.SK.DE.....E.A...........Q.RIA..SKN...T.F....F..........P.....EQRRVM.............NN.E...S.D...S.....RI....DFR.RTL.K---..I.G..Q...
monDom  ..EK...YI.M..A...KQ.A.GK.AE.....E.A...........Q.RIA..SKN...T.F....F..........P.....AHRRVM.............NRKE...S.D..SS.....RI....KFH.RTL.Q.EA..I.G.EQ...

A serious CpG mutation -- G551S -- could occur via G -> A in the terminal exon whereas other substitutions are nearly neutral:
                               +                  -            -
CpG2CpA        VTSMSLARILSLLAASSPTWDQVPPFQWSTSPFS-SLLHMGQPDLWKFTPVKVSWD
PLBD2_homSap   ................G..................G............A.......
PLBD2_gorGor   ................G..................G............A.......
PLBD2_ponAbe   ................G..................G............A.......
PLBD2_nomLeu   ................G..................G............A.......
PLBD2_papHam   ..........G.....G..................G............A.......
PLBD2_calJac   .......K........G..................T............CAP.....
PLBD2_micMur   .......KA.....V.G............A..................S.IR.W.Q
PLBD2_otoGar   .......KA.....V.G........................H......L.IE.W..
PLBD2_tupBel   .......KT...V...G..............A...H.....H....R.S.IQ....
PLBD2_musMus   ...FT..KYM.M....G.....C......K...H..M.........M.S.IR.P..
PLBD2_ratNor   ...VA..KYM.M....G.....L......K...H.NM.........M.S....P..
PLBD2_dipOrd   .......KA.G...V.G............S...P.DV...........L..E.L.G
PLBD2_cavPor   .....M.KT...Q.V.G.......A........R.DM....H......A..E...G
PLBD2_oryCun   ...K...KAM......G................R.DQ....H......L.IR.L..
PLBD2_ochPri   .......KA.....D.GS...........A...R.DK....H......L.F..L..
PLBD2_vicPac   ..N.A..KA.R.....G.....L............R............S.ID.W..
PLBD2_turTru   ...TA..KA.R...V.G.....L......S..................S.IE.W..
PLBD2_bosTau   ...TA..KA.R...V.G.....L............GM...........S.IE....
PLBD2_oviAri   ...TA..KA.R...V.G.....L............GM...........S.IE....
PLBD2_lamPac   ..N.A..KA.R.....G.....L............R............S.ID.W..
PLBD2_susScr   ....A...VFG.....G.....L............H............S.IE....
PLBD2_felCat   ....A..KAFQ.V...G.....L......A.....G............S.IE.R..
PLBD2_canFam   M...A..KAFHII.V.G............S.....G........V...L.IETW..
PLBD2_ailMel   ....A...AFHII.V.G.....L......S..................S.IE.W..
PLBD2_myoLuc   ....A..KA.R.V...G..................G............S.......
PLBD2_pteVam   ...AA..KA.RF....G.....L............G............S.IE.W..
PLBD2_eriEur   ..NV..V.A.G...V.G.....L............G............S.IE.W..
PLBD2_loxAfr   ...LAM.KA.R...V.G.....L..........R..............L.IE.W..
PLBD2_proCap   ..NLAM.KA.R.R.V.G.....L..........Q..............L.IE.W.N
PLBD2_echTel   ...SG..KS.R.W.V.G.....L......S...H.N............S..EFG..
PLBD2_macEug   ...YE.SKD.R.I.V.G.....L......S...D.K.....H......F.I....E
PLBD2_ornAna   ...SQ..KDFRFV...G.......A.R..S...K.G.V...H....R.S..H.R..
PLBD2_galGal   ...FGM..TFG.V...G....D....R.....C..H..R..H....R.P....R..
PLBD2_taeGut   ...SAMVPTFG.V.V.G.A..D....R..A..C........H....T.P....H..
PLBD2_anoCar   ...FEM.KLY.FV.T.G....DL.A.E..S..YR.N.....H....R.S.IQ.H.G
PLBD2_xenTro   L..YEM.KKYEMVVVNG....................M...H......S.ITIR.H
PLBD2_ranCat   ...YEF.KEYMMF.VNG..................N.M...H......D.ILIR.K
PLBD2_cynPyr   ...FNM..VYGMV.V.G....DL............VQ....H....Q.D..E.L.W
PLBD2_tetNig   L..YQMF.DYAMI.V.G...............YK.D.....H.GT.T.K....T.K
PLBD2_takRub   L..YKMF.DYG.I.V.G...............YK.D.....H..T.T.K.IT.I.T
PLBD2_gasAcu   L..FEMF.DYAM..V.G...............Y..D.M...H..S.A.K......N
PLBD2_oryLat   M..FGMFKEYGMI.V.G.....L.........YK.D.V...H..V.N.K.I..T.T
PLBD2_salSal   ...YG.W.EFGF....G.......A....S..Y..D.M...H..T.A...IH.T.S
PLBD2_danRer   M..S.MF.QWE.....G.SCE.T.V....R..Y....M......R.D.PT.H.R.A
PLBD2_ictPun   M..YG.FKQYE...V.G.......A.E.....Y....T.I.H..R.D.PT.HIR.S
PLBD2_squAca   ...FE.HSTYQMI.VNG....E..A....K.......M...H....R.L..M.Q.K
PLBD2_calMil   ...SEMYKTFEMI.I.G.A..........K.SY..G.I...H......P..M.R.S
PLBD2_petMar   I..KAMVPR.EMV.Q.G.....Q......K.......S.V......S.L.EHI..C
PLBD2_braFlo   M..Y.MHESHQMM.V.G..H..QQ.......DYDKQFY.L.H...FN.D.IH.I.F
PLBD2_cioInt   .VGY.MMKNFEI..ECG..H..Q...V..K.....HVS.K.M..KYD.K.TLII..
PLBD2_halRor   ...YT.HKT.QMV.EAG..H..Q.V.......YA..KS.E.H..RFD.L..LIK..
PLBD2_strPur   ..TS.MVKS..MV.VCG..T..Q......K.D.N.QT..L.H...FN.K.IN.Q.Y
PLBD2_sacKow   L.NYVMHKD..FV.I.G..Q..Q.V.......YL.DV..L.H..KFD.G..Q.N.K
PLBD2_aplCal   LI.YD.FKS..F..I....Y.DL......K.DYN.YMS.L.H..V...PRILFKGT
PLBD2_creFor   L..AEMVKD.TFI.VGG.S...Q......K.D.K..TS.I........P..LFNIS
PLBD2_eupSco   L.NS....D.QFV.IAG..Y...T.....K.D.KNTVS.I.H..IF..E.YIFGD-
PLBD2_craGig   L.NSAMFKAMQFV.I.G..Y..FA.....K.D.KDNTP...H..TF..D..VFDGT
PLBD2_helRob   I..Y.MFKNFQF..V.G..R...A.....K.DLKDTIR.A.H....V.D..QF---
PLBD2_capCap   I..Y.MFKNFQF..V.G..R...A.....K.DLKDTIR.A.H....V.D..QF---
PLBD2_limPol   L.TFG.SQKFEFV.IGG..H.PL......K.D..KD.P.Y.H....V.K..THH.K
PLBD2_ixoSca   ..NFA.FERQVFY.V.G..S.DQR..R....G.D.NVS.A.H....D.D.ILAQ.R
PLBD2_derVar   L.TYQQFKEQQFF.I.G...S.Q.V......G.-.NDS.V.H..R.E.G..LNY.G
PLBD2_dapPul   ...HQ.MTS.DFI.VGG..F.SL.A.R..E.D.V.NMS.I.H......E..QTE.T
PLBD2_calCle   ...FD.LLRG.FI.GGG..Y.S.E.....KAD.EKDTP.F.H..K.D.K.MR.E..
PLBD2_lepSal   I..FD.FMKG.FI.GGG..Y.S.E.....KTD.GKTTP.F.H..K.A.K.I..D.E
PLBD2_hydMag   A..FQ.SKLM.QFIVGG..Y..Q......KTEWN.--RPL.H..IF..N.ELLD.R
PLBD2_nemVec   I..FE.FQKFQCI.V.G..H..Q.A......EWE.--KPL.H..KFD.E.......

Four serious mutations could arise from C -> T mutations at CpG sites: A548V, T553M, P559L, and S565L
                            +    +     +     +                 -
CpG2TpG        VTSMSLARILSLLVASGPMWDQVPLFQWSTLPFS-GLLHMGQPDLWKFVPVKVSWD
PLBD2_homSap   .............A....T.....P.....S.................A.......
PLBD2_gorGor   .............A....T.....P.....S.................A.......
PLBD2_ponAbe   .............A....T.....P.....S.................A.......
PLBD2_nomLeu   .............A....T.....P.....S.................A.......
PLBD2_papHam   ..........G..A....T.....P.....S.................A.......
PLBD2_calJac   .......K.....A....T.....P.....S....T............CAP.....
PLBD2_micMur   .......KA....AV...T.....P....AS....S............S.IR.W.Q
PLBD2_otoGar   .......KA....AV...T.....P.....S....S.....H......L.IE.W..
PLBD2_tupBel   .......KT...VA....T.....P.....SA...H.....H....R.S.IQ....
PLBD2_musMus   ...FT..KYM.M.A....T...C.P....KS..H.SM.........M.S.IR.P..
PLBD2_ratNor   ...VA..KYM.M.A....T...L.P....KS..H.NM.........M.S....P..
PLBD2_dipOrd   .......KA.G..AV...T.....P....SS..P.DV...........L..E.L.G
PLBD2_cavPor   .....M.KT...QAV...T.....A.....S..R.DM....H......A..E...G
PLBD2_oryCun   ...K...KAM...A....T.....P.....S..R.DQ....H......L.IR.L..
PLBD2_ochPri   .......KA....AD..ST.....P....AS..R.DK....H......L.F..L..
PLBD2_vicPac   ..N.A..KA.R..A....T...L.P.....S....R............S.ID.W..
PLBD2_turTru   ...TA..KA.R..AV...T...L.P....SS....S............S.IE.W..
PLBD2_bosTau   ...TA..KA.R..AV...T...L.P.....S.....M...........S.IE....
PLBD2_oviAri   ...TA..KA.R..AV...T...L.P.....S.....M...........S.IE....
PLBD2_lamPac   ..N.A..KA.R..A....T...L.P.....S....R............S.ID.W..
PLBD2_susScr   ....A...VFG..A....T...L.P.....S....H............S.IE....
PLBD2_felCat   ....A..KAFQ.VA....T...L.P....AS.................S.IE.R..
PLBD2_canFam   M...A..KAFHIIAV...T.....P....SS.............V...L.IETW..
PLBD2_ailMel   ....A...AFHIIAV...T...L.P....SS....S............S.IE.W..
PLBD2_myoLuc   ....A..KA.R.VA....T.....P.....S.................S.......
PLBD2_pteVam   ...AA..KA.RF.A....T...L.P.....S.................S.IE.W..
PLBD2_eriEur   ..NV..V.A.G..AV...T...L.P.....S.................S.IE.W..
PLBD2_loxAfr   ...LAM.KA.R..AV...T...L.P.....S..R.S............L.IE.W..
PLBD2_proCap   ..NLAM.KA.R.RAV...T...L.P.....S..Q.S............L.IE.W.N
PLBD2_echTel   ...SG..KS.R.WAV...T...L.P....SS..H.N............S..EFG..
PLBD2_macEug   ...YE.SKD.R.IAV...T...L.P....SS..D.K.....H......F.I....E
PLBD2_ornAna   ...SQ..KDFRFVA....T.....A.R..SS..K...V...H....R.S..H.R..
PLBD2_galGal   ...FGM..TFG.VA....T..D..P.R...S.C..H..R..H....R.P....R..
PLBD2_taeGut   ...SAMVPTFG.VAV...A..D..P.R..AS.C..S.....H....T.P....H..
PLBD2_anoCar   ...FEM.KLY.FVAT...T..DL.A.E..SS.YR.N.....H....R.S.IQ.H.G
PLBD2_xenTro   L..YEM.KKYEMV.VN..T.....P.....S....S.M...H......S.ITIR.H
PLBD2_ranCat   ...YEF.KEYMMFAVN..T.....P.....S....N.M...H......D.ILIR.K
PLBD2_cynPyr   ...FNM..VYGMVAV...T..DL.P.....S....VQ....H....Q.D..E.L.W
PLBD2_tetNig   L..YQMF.DYAMIAV...T.....P.....S.YK.D.....H.GT.T.K....T.K
PLBD2_takRub   L..YKMF.DYG.IAV...T.....P.....S.YK.D.....H..T.T.K.IT.I.T
PLBD2_gasAcu   L..FEMF.DYAM.AV...T.....P.....S.Y..D.M...H..S.A.K......N
PLBD2_oryLat   M..FGMFKEYGMIAV...T...L.P.....S.YK.D.V...H..V.N.K.I..T.T
PLBD2_salSal   ...YG.W.EFGF.A....T.....A....SS.Y..D.M...H..T.A.T.IH.T.S
PLBD2_danRer   M..S.MF.QWE..A....SCE.T.V....RS.Y..S.M......R.D.PT.H.R.A
PLBD2_ictPun   M..YG.FKQYE..AV...T.....A.E...S.Y..S.T.I.H..R.D.PT.HIR.S
PLBD2_squAca   ...FE.HSTYQMIAVN..T..E..A....KS....S.M...H....R.L..M.Q.K
PLBD2_calMil   ...SEMYKTFEMIAI...A.....P....KSSY....I...H......P..M.R.S
PLBD2_petMar   I..KAMVPR.EMVAQ...T...Q.P....KS....S.S.V......S.L.EHI..C
PLBD2_braFlo   M..Y.MHESHQMMAV...TH..QQP.....SDYDKQFY.L.H...FN.D.IH.I.F
PLBD2_cioInt   .VGY.MMKNFEI.AEC..TH..Q.P.V..KS....HVS.K.M..KYD.K.TLII..
PLBD2_halRor   ...YT.HKT.QMVAEA..TH..Q.V.....S.YA.SKS.E.H..RFD.L..LIK..
PLBD2_strPur   ..TS.MVKS..MVAVC..TT..Q.P....KSD.N.QT..L.H...FN.K.IN.Q.Y
PLBD2_sacKow   L.NYVMHKD..FVAI...TQ..Q.V.....S.YL.DV..L.H..KFD.G..Q.N.K
PLBD2_aplCal   LI.YD.FKS..F.AI.S.TY.DL.P....KSDYN.YMS.L.H..V...PRILFKGT
PLBD2_creFor   L..AEMVKD.TFIAVG..S...Q.P....KSD.K.STS.I........P..LFNIS
PLBD2_eupSco   L.NS....D.QFVAIA..TY...TP....KSD.KNTVS.I.H..IF..E.YIFGD-
PLBD2_craGig   L.NSAMFKAMQFVAI...TY..FAP....KSD.KDNTP...H..TF..D..VFDGT
PLBD2_helRob   I..Y.MFKNFQF.AV...TR...AP....KSDLKDTIR.A.H....V.D..QF---
PLBD2_capCap   I..Y.MFKNFQF.AV...TR...AP....KSDLKDTIR.A.H....V.D..QF---
PLBD2_limPol   L.TFG.SQKFEFVAIG..TH.PL.P....KSD..KD.P.Y.H....V.K..THH.K
PLBD2_ixoSca   ..NFA.FERQVFYAV...TS.DQRP.R...SG.D.NVS.A.H....D.D.ILAQ.R
PLBD2_derVar   L.TYQQFKEQQFFAI...T.S.Q.V.....SG.-.NDS.V.H..R.E.G..LNY.G
PLBD2_dapPul   ...HQ.MTS.DFIAVG..TF.SL.A.R..ESD.V.NMS.I.H......E..QTE.T
PLBD2_calCle   ...FD.LLRG.FIAGG..TY.S.EP....KAD.EKDTP.F.H..K.D.K.MR.E..
PLBD2_lepSal   I..FD.FMKG.FIAGG..TY.S.EP....KTD.GKTTP.F.H..K.A.K.I..D.E
PLBD2_hydMag   A..FQ.SKLM.QFIVG..TY..Q.P....KTEWN.--RPL.H..IF..N.ELLD.R
PLBD2_nemVec   I..FE.FQKFQCIAV...TH..Q.A.....SEWE.--KPL.H..KFD.E.......

>CpG2CpA 55 changes for PLBD2
MVGQMYCYPSSHLAQALTQALALALVLALLVRPFLSSLAGAIPAPGGHWAHNGQVPPASHSHSVLLDISAGQLLMVDRHHPDTMAWANLTNTIHKTGWAFLELGTSGQYNDSLQAYAASV
VEAAVSEELIYMHWMNTVVNYCSPFKYEVSYCKRLKSFLEANLEWMQEEMESNPDSPYWHQVQLTLLQLKGLEDSYKGHVSFPAGKFTIKPLGFLLLQLSGDLEDLELALNKTKIKPSLG
SGSCSALIKLLPGQSDLLVAHNTWNNYQHMLHVIKKYWLQFQEGPWGDYPLVPSNKLVFSSYPSTIFSCNNFYILGSGLVTLETTIGNKNPALWKYVQPRGCVLEWVHNIMANHLASDGA
TWADIFKRFNSSTYNNQWMIMDYKAFIPGGPSPRSQVLTILEQIPSMVVVADKTSELYQKTYWASYNIPSFKTVFNASGLQALVAQYGDWFSYDRSPQAQIFQQNQSLVQDMDSMVRLMR
YNDFLHDPLSLCKACNPQPNGENAISTHSNLNPANGSYPFQALHQHSHGGINVKVTSMSLARILSLLAASSPTWDQVPPFQWSTSPFSSLLHMGQPDLWKFTPVKVSWD*

>CpG2TpG 50 changes for PLBD2
MVGQMYCYPGSHLAWVLMWVLVLALVLALLVGLFLSGLVGVIPVLGGCWVCDGQVPPASCSCLVLLDVLVGQLLMVDGCHPDAVAWANLTNAICETGWAFLELGTSGQYNDSLQAYAAGV
VEAAVLEELIYMHWMNMVVNYCGPFEYEVGYCERLKSFLEANLEWMQEEMESNPDSPYWHQVWLTLLQLKGLEDSYEGCVSFPAGKFTIKPLGFLLLQLSGDLEDLELALNKTKIKPSLG
SGSCSALIKLLPGQSDLLVAHNTWNNYQHMLCVIKKYWLQFWEGPWGDYLLVPGNKLVFSSYPGTIFSCDDFYILGSGLVTLETTIGNKNPALWKYVWPRGCVLEWVCNIVANCLALDGA
TWADIFKRFNSGMYNNQWMIVDYKVFILGGPSPGSWVLTILEQIPGMVVVADKTLELYQKTYWASYNILSFETVFNASGLQALVAQYGDWFSYDGSPWAQIFWWNQSLVQDMDSMVRLMR
YNDFLHDPLSLCKACNPQPNGENAISACSDLNLANGSYPFQALCQCSHGGIDVKVTSMSLARILSLLVASGPMWDQVPLFQWSTLPFSGLLHMGQPDLWKFVPVKVSWD*

Homologs in model organisms

In this section, species are discussed that have an existing (or potential) phospholipase experimental literature for either PLBD1 or PLBD2. Their relevance to the human situation varies greatly depending on divergence and other complications. Drosophila LAMA -- despite two early papers will prove completely irrelevant. Nematodes have a reasonably comparable protein and could be quickly studied by genetic techniques. Oddly slime mold may be the most useful of the three because it has been used for decades as a model system for lysosomal targeting.

Slime molds (Dictyostelium)

Although slime molds seem an improbable model system for mammals given the immense time since divergence from the last common ancestor, Dictyostelium works fairly well for the lysosomal sorting process via the M6P pathway, establishing its great antiquity, with 220 on-topic papers dating back decades. A phospholipase B, presumably from lysosomes, was characterized in 2004. Called PLBDa here, it is only one of seven found in the newly sequenced Dictyostelium genome.

It is not immediately clear how this gene family expansion relates to mammalian PLBD1 and PLBD2. In one scenario, a single gene in the ancestral stem had expanded to the two PLBD1 and PLBD2 clades at the time of the last common ancestor. No further expansion occurred in the lineage leading to human but each clade expanded further in slime molds. This would cause the seven Dictyostelium genes to classify clearly with one branch or another of the gene tree. The data below do not support this.

PLBDa dicDisPDB.png

A second scenario envisions a single gene at the time of species divergence. This duplicated subsequently in the lineage leading to human and expanded independently in Dictyostelium. This would give rise to the gene tree ((PLBD1, PLBD2),(dicDis subtree)). The data fit this better; it predicts a slightly later diverging species can be found in which the PLBD1/PLBD2 duplication had already occurred and the Dictyostelium-type genes are missing. The choanoflagellate Monosiga fits this description. It also predicts earlier diverging species than Dictyostelium will only have ambiguously classifying PLBDx (assuming ancestral features do not predominate in PLBD1 or PLBD2, respectively).

Note all seven genes are perfectly conserved for the six active site residues, meaning this had been established earlier. Thus any or all of them could be used as a generic structural model for mammalian PLBD1 or PLBD2, an important consideration in view of the similar processing, activation, and known substrate.

It can be seen from Blastp against SwissProt that all of the Dictyostelium proteins have been assigned accession numbers. This means they might have already been modeled by SwissModel using the mouse PLBD2 structure as template but in fact have not, though modeling requests can be submitted using the 3FGW template. Note PLBDd and PLBDg are the most favorable for PLBD1; whereas PLBDb, PLBDc, and PLBDa have better matches to PLBD2. Only PLBDa has its substrates determined. 

Q550U9   PLBLA_DICDI   PLBDa_dicDis  SwissModel+ interactive
Q55BJ6   PLBLB_DICDI   PLBDb_dicDis  SwissModel-
Q54M94   PLBLC_DICDI   PLBDc_dicDis  SwissModel-
Q54PS7   PLBLD_DICDI   PLBDd_dicDis  SwissModel-
Q54ZI6   PLBLE_DICDI   PLBDe_dicDis  SwissModel-
Q554H5   PLBLF_DICDI   PLBDf_dicDis  SwissModel-
Q55FN1   PLBLG_DICDI   PLBDg_dicDis  SwissModel-

PLBD1_homSap Homo sapiens (human) FLJ22662             2968  1.2e-313 100% blastp query PLBD1
PLBD1_musMus Mus musculus (mouse)                      2321  4.5e-245  79%
PLBD1_braFlo Branchiostoma floridae (lancelet)         1369  3.4e-144  50%
PLBD1_nemVec Nematostella vectensis (anemone)          1349  4.5e-142  51%
PLBD1_strPur Strongylocentrotus purpuratus (urchin)    1122  5.1e-118  47%
PLBD1_monBre Monosiga brevicollis (choanoflagellate)   1089  1.6e-114  51%
PLBDd_dicDis Dictyostelium discoideum (slime_mold)      822  3.1e-86   37% d
PLBDg_dicDis Dictyostelium discoideum (slime_mold)      746  1.2e-84   36% g
PLBD2_triAdh Trichoplax adhaerens (trichoplax)          721  1.2e-82   38%
PLBD2_braFlo Branchiostoma floridae (lancelet)          722  2.0e-80   36%
PLBDb_dicDis Dictyostelium discoideum (slime_mold)      762  7.2e-80   34% b
PLBDc_dicDis Dictyostelium discoideum (slime_mold)      733  8.5e-77   35% c
PLBDa_dicDis Dictyostelium discoideum (slime_mold)      717  4.2e-75   33% a
PLBDe_dicDis Dictyostelium discoideum (slime_mold)      717  4.2e-75   35% e
PLBD2_acyPis Acyrthosiphon pisum (aphid)                667  4.7e-75   37%
PLBD2_homSap Homo sapiens (human) LOC196463             639  2.0e-72   36%
PLBD2_musMus Mus musculus (mouse)                       631  6.0e-71   37%
PLBDf_dicDis Dictyostelium discoideum (slime_mold)      640  6.1e-67   32% f
PLBD2_monBre Monosiga brevicollis (choanoflagellate)    547  3.3e-66   32%
PLBDx_droMel Drosophila melanogaster (fruitfly)         142  5.5e-19   32%

PLBD2_homSap Homo sapiens (human) LOC196463            3142  0.0      100%  blastp query PLBD2
PLBD2_musMus Mus musculus (mouse)                      2571  1.4e-271  81%
PLBD2_braFlo Branchiostoma floridae (lancelet)         1528  4.8e-161  54%
PLBD2_triAdh Trichoplax adhaerens (trichoplax)         1293  3.9e-136  50%
PLBD2_acyPis Acyrthosiphon pisum (aphid)               1129  9.2e-119  47%
PLBDb_dicDis Dictyostelium discoideum (slime_mold)      873  1.2e-91   39% b
PLBD2_monBre Monosiga brevicollis (choanoflagellate)    835  1.3e-87   38%
PLBDc_dicDis Dictyostelium discoideum (slime_mold)      819  6.5e-86   39% c
PLBDa_dicDis Dictyostelium discoideum (slime_mold)      805  2.0e-84   38% a
PLBD1_nemVec Nematostella vectensis (anemone)           702  1.6e-78   38%
PLBD1_musMus Mus musculus (mouse)                       688  5.9e-77   38%
PLBDd_dicDis Dictyostelium discoideum (slime_mold)      731  1.4e-76   37% d
PLBD1_homSap Homo sapiens (human) FLJ22662              639  2.0e-72   36%
PLBDg_dicDis Dictyostelium discoideum (slime_mold)      685  1.0e-71   36% g
PLBDe_dicDis Dictyostelium discoideum (slime_mold)      661  3.6e-69   32% e
PLBD1_braFlo Branchiostoma floridae (lancelet)          652  3.2e-68   36%
PLBD1_monBre Monosiga brevicollis (choanoflagellate)    646  1.4e-67   38%
PLBDf_dicDis Dictyostelium discoideum (slime_mold)      640  6.1e-67   35% f
PLBD1_strPur Strongylocentrotus purpuratus (urchin)     534  7.7e-61   35%
PLBDx_droMel Drosophila melanogaster (fruitfly)         218  2.9e-38   30%

Alignment of PLBDa_dicDis with conserved residues of PLBD1 or PLBD2 shows no clear classification:

VTDLNMAQQFVSEALNGPSTDGDLPPFTWDA--FNRTTHQGLPRLYNYTFVTM best PLBD1 match
V   +M    +  A +GPS D + P FTW++  +N+ T+ G P  +N+ ++TM 
VVSADMVAALLVNAQSGPSHDNETP-FTWNSQWNQKYTYAGQPTTWNFDWMTM PLBDa_dicDis
+ S  MV  L + AQSGP+ D + P F W+       ++ GQP  W+F
ITSKAMVPRLEMVAQSGPTWDQQPP FQWSKSPFSSLSHVGQPDLWSFLPEHI best PLBD2 match

V.D...A...............gL.......--Fn.t............F..m.P.L conserved PLBD1 not PLBD2
.............A..GPt.....P.F.W.........H.G.P..w.F........  conserved PLBD1 and PLBD2
.Ts............S...-WDq......S.SP......m...D.....P..v.W.. conserved PLBD2 not PLBD1

Arthropods (Drosophila)

The standard fruitfly has a very odd PLBD2-type gene product LAMA (lamina ancestor) that has been the subject of two publications. Note first that a poor choice of name has lead to widespread confusion: this protein has nothing whatsoever to do with the important vertebrate protein laminin (whose homolog in fly is called LamA). Instead it has to do with an obscure neuroanatomical structure in insects called the lamina.

The gene, call PLBD2x here, is so diverged from conventional phospholipases that a number of hypotheses have to be considered:

  • Fraud: It is not unusual for annotational hoaxes to be perpetrated in the scientific literature, complete with bogus submissions to GenBank. Here however simple tBlastn searches show that the most recent assembly of the drosophila genome does contain a letter-perfect match to the LAMA entry and further that this protein has a more or less a full length match to conventional vertebrate PLBD2. This is beyond anyone's ability to manipulate.
  • Pseudogene: This is a large protein of almost 600 amino acids. Should it have lost its function, rapid divergence would result (as seen), with accrual of frameshifts and stop codons (not seen). The divergence would occur equally rapidly at ultra-conserved residues as at ones not under selection (not seen), either in D. melanogaster or one of the other 11 drosophilids with sequenced genome (not seen). However recent pseudogenization in certain lineages cannot be ruled out as comparative genomics becomes insufficient.
  • Horizontal transfer: The LAMA protein might fail to nest within the gene tree as expected from the fruitfly position in the species tree. This happens rarely in metazoan from gene transfer from other species, notably parasites, commensals, and intra-cellular symbionts. In this case, tBlastn would locate the appropriate clade, even if the exact species had no data because genomic coverage is so pervasive today. However the LAMA gene has no outside affinities.
  • Role change: If this gene lost its original function and acquired another unrelated one, then the selection profile could change radically across the gene. For example, if the catalytic function were redirected, then the six active site residues might experience relaxed constraint. The fold itself might be conserved to assure structural stability but otherwise rapid evolution to perfect the new role might alter many otherwise conserved residues. Here the change is so rapid and so drastic that a simple change to a new substrate can be ruled out -- indeed, the two experimental papers provide evidence for a regulatory role.
  • Role loss: Along the lines of Piatigorsky, a single gene product may have multiple roles, sometimes quite distinct (eg aldehyde dehydrogenase ALDH3A1 also serving as a lens crystallin). Should the gene subsequently be duplicated, these pre-existing roles can be split up in the two copies (rather than one copy continue with the parental role and the other copy scramble to acquire a new adaptive function). Selection initially acts on all the functions in proportion to their importance.
DroMelPLDB2X.png

In this last scenario, phospolipase activity was lost, leaving selection to act solely on the residual regulatory function. However within the 12 drosophilid genomes, the PLBD2 are quite diverged, with percent identity quickly ranging from 75% down to 45%. This suggests the protein is not yet well adapted to what was initially a secondary function or that few of its features are needed.

This raises the question whether PLBD2 also serves a cryptic non-catalytic function in other species (eg mammals). It is thus important to date the onset of loss of phospholipase activity (coupled with retention of within the PLBD2 locus) within arthropods because this also provides some dating of the secondary function origin. This can be dated to the last tens of millions of years because only a few other insects (eg tsetse Glossina morsitans, EZ422576) outside drosophilids carry a comparable gene.

Within insects, some species such as Acyrthosiphon carry both a normal and derived PLBD2. However the normal form has been lost in other insects with derived PLBD2. Although no chelicerate has the derived PLBD2, Ixodes has multiple copies of normal PLBD2, some closely related and others fairly diverged but still clearly classifying as PLBD2. Molluscs, annelids, and trematodes have PLBD2 but not PLBD1 or PLBD2x.

The loss of active site residues characteristic of NTN hydratases and more specifically those shared by both PLBD1 and PLBD2 phospholipases is taken here as a proxy for loss of phospholipase (and indeed all) catalytic activity. 

Active site residues are lost in derived PLBD2 though the first disulfide is retained:

               <---- human active site ----> <-- human disulfides --> <-- glycosylation-->
PLBD2_homSap   C244 H261 W264 T325 N427 R458   C142-C152 C492-C495         N436S N515S
PLBD2x_droMel  A216 H238 A241 G305 G438  ?      C98-C108 
PLBD2x_glomor  A210 H231 A234 G298 G424  ?      C94-C104
PLBD2x_acrPis  A233 Q250 S253 V315 S413 V436   C132-C139
PLBD2_acrPis   C208 H225 W228 T288 N390 R421   C112-C119 C434-C437         N399S N477T

Unless there is some counterpart to the secondary function, the tempo and mode of evolution of the LAMA protein makes it largely irrelevant to its human ortholog and lysosomal storage diseases. Indeed, fruitflies have in effect lost both PLBD1 and PLBD2, proving they are non-essential to lysosome function. 

Drosophila PLDB2x at SwissModel: little of the original fold remains:

PLDB2x    45         CATAL WTKQV-GFQI ENWKQQNDLV NIPTGVGRIC YKDSVYENGW
3fgrA     63    vsr--trsll ldaasgqlrl ed-----g-- fhpdavawan ltnairetgw
                      ssss ss    ssss s               sssss sssss   ss
                sss  sssss ss    ssss ss     s        sssss sssss   ss

PLDB2x    89    AQIEVETQRT YPDWVQAYAA GMLEGSLTWR NIYNQWSNTI SSSCERDEST
3fgrA     104   ayldlstngr yndslqayaa gvveasvsee liymhwmntv vnycgpfeye
                ssssssss     hhhhhhhh hhhhhh   h hhhhhhhh             
               ssssssss     hhhhhhhh hhhhh   hh hhhhhhhh             

PLDB2x    139   QKFCGWLRDL LTTNYHRLKR QTEKAENDHY WHQLHLFITQ LEGLETGYKR
3fgrA     154   vgyceklknf leanlewmqr emelnpdspy whqvrltllq lkgledsyeg
                hhhhhhhhhh hhhhhhhhhh hhhh    hh hhhhhhhhhh hhhhhhhhh 
                hhhhhhhhhh hhhhhhhhhh hhhh    hh hhhhhhhhhh hhhhhhhhh 

PLDB2x    189   GASRARSDLE EEIPFSDFLL MNAAADIQDL KIYYENY -- -         
3fgrA     204   rltfptgr-- ftikplgfll lqisgdledl epalnktgsg s         
                                   hh h   hhhhhh hhh                  
                                   hh h   hhhhhh hhh

Nematodes (C. elegans)

Potentially a better model organism than fruitfly, C. elegans has 3 phospholipase B genes, with the best match to human PLBD2 having 44% identity. These appear to be a lineage-specific expansion of PLBD2, whereas PLBD1 has been lost from this and other nematodes. (The C. elegans genome is assuredly complete and the detection methods are sufficiently sensitive.) 

>PLBD2a_caeEle Caenorhabditis elegans (nematode) NP_499668 Y37D8A
MNWIFIFLAAAVAIGCEARQERTYTVCQKPEGDLHYFKEGRKTDEELCAKRLATAYFHDEVNQTGWAFLEVDVISPKIPHYLQGYAAGFAEGRATRHLID
LHIINTVNGYCDGAKHFCDELGEFMVDNLKWMEQEIRENPEDEYWQQVNLTVNQLFGLIHGYENQLGAEIDFKQIAVHPIFMIQIAGDLEDLAMKFKKPE
NPKKVFSGPGHCSALVKLLPKNEDILFSHVTWSSYGTMLRINKKYSFKTGDPGQIYSFSSYPASITSTDDFVLTSAKLAILETTIGNYNEKSLDLITPNT
VLTWIRAEIAHRTASSGLQWAEAFGRHNSGTYNNEWVVVDYKQFHRGKEVQPETGIIHVVEQMPGHIVHSDKTAHLFRETYWPGYNQPYYKQIIRFSDTD
KMVEKFGDWYSYDKTPRALIFKRDHNTVTDMSSMIALMRSNNYTKDPLSKCDCNPPYSAENAIACRSDLNPLNGTYPFKSLGFRDHGAIDVKVTNSKLIN
SLQFTAVSGPPGGVTKDVPIFDWKTSPLREKVPHFGQPDRWNFAPVTYKWRKDAHRHYHLYQKLHKELSSL*

>PLBD2b_caeEle Caenorhabditis elegans (nematode) NP_510509 F09B12
MTRLIRSKKQFLIRSLHSVFYYLGSLLHSTFEMNVFIGLLLATVVASQSSEGRDESYTYKQLCIVDDKPQVLDGFDCRNQVAVARWQNAVNTTGWTFLEV
ETKENYCPQLQAYSAGYLEGLLSKTVLTYHLKNAQEDYCKNFTGYCSRLSDFLTENQKWIQSSLETVAPDDLYWGAVNRTYHQVSGLIDAYEGREFKPRI
TYELHPILYLNLNGDFYDLEKKLNKTRDPAFEQTGGKCSGLIKVAPGNADLFISQVTMSGFQNMLRVIKLYKFGYDRQFYPGYASSFSSYPGLLYSSDDF
ALQTSGLAVIETTISVFNTSLFENTKPVGQLPTWIRAIVSNQLARDAREWCKLYSLYNSGTYNNQWAVLDYKKFKPNQPLPKNGLFYVLEQMPGKIVYSD
LTWFVEKYSYFPSYNIPFFKEITEISGFIGQAAKMGDWFKWGASPRAKIFERDHGNVHDLDSLTALMRYNDYKNDEFSKCKCNPPYSAEAGISARGDLNP
ANGTYEFPGQGHVNHGALDYKGTNVELMKKLQFVAQGGPTWGKVPSFKWSEFDFKDKVNHVGHPDEWKFNTLVHKWETEINA*

>PLBD2c_caeEle Caenorhabditis elegans (nematode) NP_497570 Y54F10AM
MKLLFFLFGLIFAVEQEKPYLDNNRVPVEQILNDHSSAKFDYTYVSVCVNSTDETLLDIVYAKECKNAASRVALGKYSNQVNTTGWGILEIETFASHSYD
VQAYGAGVAEGELTRLQIYYHYRNTIETMCNNHTLFCKRLYIYLQQNLDWMRSQVQANPPTDPFWRQVNLTFAQLTGIYDAYSKRNLTPEIGFDLHPIYM
MQLAGDMFDLNKLLNKTADPMEYPEGGRCSGFVKLAPGNKDMFMAHVSMSSLSWMQRVLKIYKFGYDVNEVPGHIVTFSGYPGVLISTDDYTITSAGLTS
IETTIAIFNQTLYTDKFMKPEGQVHCWIRSMISNLLSRTGKQWVDMFGRYNSGTYNNQWTVLDWKQFTPEKELPDKDVLWISEQTPGYYETRDMTWYLKK
YTYFASYNIPFLPKVSEISGFDNKARQFAWFDWGGSPRARIFDRDHSKVTDIDSLTKLMRYNDYTHEEFARCKCTPNPYTGEGGISARGDLNTPGGTYEV
ESMGFRDHAGLDFKGTNYEMFKKMRFRAWGGPPYDPLPVFDWNHTNLTNVRHFGQPDVWNFTYVDLEWQLAAQVQLTPYDN*

Reference sequences

These two genes are well represented at GenBank though some predicted gene models are erroneous. A genomic alignment of 46 species of vertebrates at UCSC (under protein fasta section of the gene details page) allows this large set of orthologs to be collected as needed. These sequences -- which are no better than the underlying assemblies -- also contain extensional errors at the termini. Insertions relative to human also are not shown. However these sequences provide a strong starting point for correction at the wgs contig division of NCBI blast.

Below, only a few key sequences are shown -- ones with an experimental literature and some from deeply diverged pre-bilaterans. These have been intronated by blast of protein against genome assemblies.

A separate section holds anomalous PLBD2x insect sequences. These resulted from a duplication of PLBD2, followed bu severe specialization of one copy and often loss of the original. These nonetheless will retain most features of the fold, though use of SwissModel here would require considerable intervention after the fact. Despite an experimental literature, these are largely irrelevant to human considerations.

In the last section, sequences have been trimmed to the region covered by the xray structural determination. Species with one-off insertions that confuse gap placement have been adjusted. This helps avoid artifacts in alignment.

PLBD1 reference sequences

>PLBD1_homSap Homo sapiens (human) FLJ22662 PMID: 19019078,20093120
0 MTRGGPGGRPGLPQPPPLLLLLLLLPLLLVTAEPPKPA 1
2 GVYYATAYWMPAEKTVQVKNVMDKNGDAYGFYNNSVKTTGWGILEIRAGYGSQTLSNEIIMFVAGFLEGYLTAP 2
1 HMNDHYTNLYPQLITKPSIMDKVQDFME 2
1 KQDKWTRKNIKEYKTDSFWRHTGYVMAQIDGLYVGAKKRAILEGTK 0
0 PMTLFQIQFLNSVGDLLDLIPSLSPTKNGSLKVFKRWDMGHCSALIK 0
0 VLPGFENILFAHSSWYTYAAMLRIYKHWDFNVIDKDTSSSRLSFSSYP 1
2 GFLESLDDFYILSSGLILLQTTNSVFNKTLLKQVIPETLLSWQRVRVANMMADSGKRWADIFSKYNS 1
2 GTYNNQYMVLDLKKVKLNHSLDKGTLYIVEQIPTYVEYSEQTDVLRK 1
2 GYWPSYNVPFHEKIYNWSGYPLLVQKLGLDYSYDLAPRAKIFRRDQGKVTDTASMKYIMRYN 1
2 NYKKDPYSRGDPCNTICCREDLNSPNPSPGGCYDTK 0
0 VADIYLASQYTSYAISGPTVQGGLPVFRWDRFNKTLHQGMPEVYNFDFITMKPILKLDIK* 0

>PLBD1_musMus Mus musculus (mouse) NM_025806 note earlier stop codon
0 MCHRSPGRSLRPPSPLLLLLPLLLQPPWAAGAASQSDPT 1
2 GVHCATAYWSPESKKVEIKTVLDKNGDAYGYYNDSIKTTGWGILEIRAGYGSQVLSNEIIMFLAGYLEGYLTAL 2
1 HMYDHFTNLYPQLFKNPSIVKKVQDFME 2
1 KQEMWTRQNIKAQKDDPFWRHTGYVVTQLDGLYLGAQKRASEEKIK 0
0 PMTMFQIQFLNAVGDLLDLIPSLSPTKSSSMMKFKIWEMGHCSALIK 0
0 VLPGFENIYFAHSSWYTYAAMLRIYKHWDFNIKDKYTLSKRLSFSSYP 1
2 GFLESLDDFYILSSGLILLQTTNSVYNKTLLKQVVPKTLLAWQRVRVANMMAEGGKEWAQIFSKHNS 1
2 GTYNNQYMVLDLKKVTINRSLDKGTLYIVEQIPTYVEYSDQTNVLRK 1
2 GYWASYNIPFHKTIYNWSGYPLLVHKLGLDYSYDLAPRAKIFRRDQGNVTDMASMKYIMRYN 1
2 NYKEDPYSKGDPCSTICCREDLNGASPSPGGCYDTK 0
0 VADIFLASQYKAYAISGPTVQDGLPPFNWNRFNDTLHRGMPEVFDFNFVTMKPILS* 0

>PLBD1_braFlo Branchiostoma floridae (lancelet) XM_002595538
0 MEGRACRSCRLHHLSAVFLLFLVTIAA 1
2 GAEIQATAYLQAQGKVQVKLGVLDKQNGDAVATYDDR 2
1 LTENGWGVLNVVSGFGPKKLSDNDIMYLAGYLEGVLTQE 2
1 RIYQHYLNLYGIFFMGKSEDLVGK 0
0 VKKFYTAQDTWVRAQVKQSTDPVMKHLSYILSQYDGLVKGYNDN 0
0 LFPHVSFFQKLDIFAFQLLNGNGDTFDIIPAVNPSSRPDFSNMSRVEIDDWVSAHSHCSALVK 0
0 VLGAYENVYMSHSSWFNYAATMRIYKHYNFNIANPATATRKMSFSSYP 1
2 GYLESLDDFYLMDSGLVMLQTTNNVFNGTLYDLVKPESILAWQRVRTANMLARNGDQWGAIMNVHNS 1
2 GTYNNQYMIIDLNLIELGKTIHDGALYVVEQIPGLVMSADQTDILRA 1
2 GYWPSYNIPFYEKVYNLSGYPEFAKSQGLDYTYQLAPRAKIFRRDAGKVKDMESMKAIMRYN 1
2 DYLHDPYSKGNPCSAICCRKDLAKVGAKPDGCYDTK 0
0 VSDYYLARNLTSFAINGPTLGTGLEPFSWSDKFKISHIGLPKVYNFSFVTMTPAEL* 0

>PLBD1_strPur Strongylocentrotus purpuratus (urchin) XM_001192029
0 MANKFRMFKILTAFLVLVLVNLST 1
2 GELLQGTVYKQEDGTFTVSSGIIDKQGVAYGSYNNTLFQTGWGELHLFAGYSTADNVALSDADRMYAAGILEGALTAK 2
1 QISQTLRNINVTFFSAESDPEIWRRVADFFETQDAWMKGMIIERADEDPFWEGVGLVLAQFEGLIKGYEMSQFSNAST 0
0 SNGFLAMQVLNSCGDLLDLKSAVMPSLIPDWDKLTKKEFLKFIRTSGHCSALVK 0
0 ICAALVKVGRFAPPFQSLLYSIS 0
0 SYFKSQAILKLNSPSCQLFGIE 1
2 GFLESLDDFYIMSSGLSMLQTTNNIFNKTLYKYVKPQSLLAWQRVRVANMMARSGKDWARIVARYNS 1
2 GTYNNQYMVIDRTKIKPNVAILDDALWVVEQVPTLVASGDQTNILRA 1
2 GYWPSYNVPFYEEIYNISGYPEYAYKGGADISYQLAPRAKIFRRDQGNVVDMESFKKIMRFN 1
2 DYKNDPYSEGDPSKSICMRGDLMTSPMPNGCYDTK0
0 VTNLAMAAKQTSFVINGPTRGDGSLPPFKWVAPFTGWSHVGLPTVYDFNFVEMCPKEL* 0

>PLBD1_nemVec Nematostella vectensis (anemone) XM_001638165
0 MTLIRNSVMITVTFVLILFVFGCHGSQKSATVYYNRGQG 2
1 YSLKFGVVDKLMGVAYGTFEDSLNTTG 2
1 WYELNIVSGTGIEPYNDDVIMHAAGYLEGALTAS 2
1 QINDNYANLYGVFFKSEDDPMVAKVEKFFIEQ 0
0 DIWMRKMIALKSSNSSFWRQMGNIIAQFD 1
2 GLVEGYQKYPATDK 0
0 ALGVFAFQMLNGVGDLLDLTKALMPERMADWDHMTEKEILEK 0
0 VAMDGHCSALIKVLPAYENVFASHVS 2
1 WFTYSAMLRVYKHYHLNLKDETT 1
2 AAQRMSFSSYPGFLESLDDFYIMDS 2
1 KLVMLQTTNNVFNKSLYEQVVPESLFSWQRVRLANLVASSGRQWADIVGQYNS 1
2 GTYNNQYMVLDLKLIQLNNTIQDNALWVVEQIPT 2
1 LVASGDQTAILRAGYWPSYNVPFYEL 0
0 VYNLSGYPDFVARHGVQFSHELAPRAKIFRRDQSM 0
0 VHDLDSMKHIMRYNDFQHDPYSQGNPMNAICSRGDLIADGPRASGCYDGK 0
0 VTDFTMAQSLISHAINGPTHE 0
0 QQVPFHWSQYQFKNKHEGQ 0
0 PDLFNFDFVEMKPKF* 0

>PLBD1_monBre Monosiga brevicollis (choanoflagellate) XM_001745398
MSSLNNGIPEPLLKFLAAQFNWTRSQVAANQDDVFWQQVGLIMA
QYDGLRAGYGANVYDKHVLPEFAFQLLNGNGDFFDIIPKAVDVTKMSSREFHDWRMRN
GRCSALIKLTGDFSDLFMSHSAWYIYQAMNRIYKHCASYNFQATITHAKKISFSSYPG
YLESLDDFYLMSSGLVMLQTTNNVFNTDLQQYIQPESLQSWIRIRTATALAQTSEDWA
ELAGRHNSGTYNNQYMVMDLNKFTPGQPLLDGTLYVAEQIPGTWEYADVTKMLSLGYW
PSYNVPFFEKIYNLSGYPAVVKQHGTDDSYELAPRAKIFRRDQTTVVDLDSFKAIMRY
NDYKNDPYAKGDPYNAICSRGDLESDSPSPGGCYDTKVTTYSMALKLQSQVINGPTTS
HGLPPFSWSQFPNASHLGMPEVFNFTFETMDAGW*

PLBD2 reference sequences

>PLBD2_homSap Homo sapiens (human) LOC196463 PMID: 19706171,19237744,17007843,17105447
0 MVGQMYCYPGSHLARALTRALALALVLALLVGPFLSGLAGAIPAPGGRWARDGQVPPASRSRSVLLDVSAGQLLMVDGRHPDAVAWANLTNAIRETG 2
1 WAFLELGTSGQYNDSLQAYAAGVVEAAVSEE 0
0 LIYMHWMNTVVNYCGPFEYEVGYCERLKSFLEANLEWMQEEMESNPDSPYWHQ 0
0 VRLTLLQLKGLEDSYEGRVSFPAGKFTIKPLGFL 2
1 LLQLSGDLEDLELALNKTKIKPSLGSGSCSALIKLLPGQSDLLVAHNTWNNYQHMLRVIKKYWLQFREGPW 1
2 GDYPLVPGNKLVFSSYPGTIFSCDDFYILGSGL 0
0 VTLETTIGNKNPALWKYVRPRGCVLEWVRNIVANRLASDGATWADIFKRFNSGT 2
1 YNNQWMIVDYKAFIPGGPSPGSRVLTILEQIP 2
1 GMVVVADKTSELYQKTYWASYNIP 2
1 SFETVFNASGLQALVAQYGDWFSYDGSPRAQIFRRNQSLVQDMDSMVRLMR 2
1 YNDFLHDPLSLCKACNPQPNGENAISARSDLNPANGSYPFQALRQRSHGGIDVK 0
0 VTSMSLARILSLLAASGPTWDQVPPFQWSTSPFSGLLHMGQPDLWKFAPVKVSWD* 0

>PLBD2_musMus Mus musculus (mouse) NM_023625
0 MAAPVDGSSGGWAARALRRALALTSLTTLALLASLTGLLLSGPAGALPTLGPGWQRQNPDPPVSRTRSLLLDAASGQLRLEDGFHPDAVAWANLTNAIRETG 2
1 WAYLDLSTNGRYNDSLQAYAAGVVEASVSEE 0
0 LIYMHWMNTVVNYCGPFEYEVGYCEKLKNFLEANLEWMQREMELNPDSPYWHQ 0
0 VRLTLLQLKGLEDSYEGRLTFPTGRFTIKPLGFL 2
1 LLQISGDLEDLEPALNKTNTKPSLGSGSCSALIKLLPGGHDLLVAHNTWNSYQNMLRIIKKYRLQFREGPQ 1
2 EEYPLVAGNNLVFSSYPGTIFSGDDFYILGSGL 0
0 VTLETTIGNKNPALWKYVQPQGCVLEWIRNVVANRLALDGATWADVFKRFNSGT 2
1 YNNQWMIVDYKAFLPNGPSPGSRVLTILEQIP 2
1 GMVVVADKTAELYKTTYWASYNIP 2
1 YFETVFNASGLQALVAQYGDWFSYTKNPRAKIFQRDQSLVEDMDAMVR 0
0 LMRYNDFLHDPLSLCEACNPKPNAENAISARSDLNPANGSYPFQALHQRAHGGIDVK 0
0 VTSFTLAKYMSMLAASGPTWDQCPPFQWSKSPFHSMLHMGQPDLWMFSPIRVPWD* 0

>PLBD2_braFlo Branchiostoma floridae (lancelet) XM_002612057
0 MAACRNIFCGRMLSCLLLFSFVFSAVSDGSKLASVRYDEAAKTYQITDKLDPSAAAWANFTDRISSTG 2
1 WSFLTVTTNEKYDDSVQAYAAGLVEGYLTRD LMYNHWLNTVGAAFCSSRSAFCKNLESFLKTNLAWMQEQIQASGDTDDYWHQ 0
0 VKLTLQQLSGLDDGYNDDPRQPSLDINPFGFL 2
1 IFQIGGDMEDLQEALKDKDSHRVLGSGSCSALVKLLPGNADLLVAHDTWDTFQSMLRIIKKYQFPFKLGGKK 1
2 GEDKIPGHTVSFSSYPGVIYSGDDFYITSASL 0
0 VAQETTIGNSNPALWKYVQPQGQVLEWLRNIVANRLANKAMDWATIFKKYNSGT 2
1 YNNQWMIVDYKTFTPNKDLPEKGLLVVLEQLP 2
1 GMVMMDDVTSVLAKQAYWPSYNSP 2
1 YFEKIFNTSGLPAMVEKYGDWFSYEHTPRANIFRRDHGKVTDISSMIKLMR 2
1 YNDFQNDPLSKCDCTPPYSAENAISARSDLNPANGTYPFSALQHRCHGGTDMK 0
0 MTSYSMHESHQMMAVSGPTHDQQQPFQWSTSDYDKQFYHLGHPDLFNFDPIHVIWFDQSDN* 0

>PLBD2_acyPis Acyrthosiphon pisum (aphid) XM_001948827
0 MLSIRCILLSLLFVWALQCSATQKNQTLLAVKTDNNRITIQPKHYSVKDKEIIIGKGKFIDRINSTG 2
1 WAYLEIRTSQKAKDEDQAYGAGYLEGTLTADLIYSYWFNTAKGYCTDRPNVCQQLKDYMTTNKNWIKSKLNESDPYWYQ 0
0 VGLYYKQLDGLYDGYMRGKSPSTPDLTWDDLY 2
1 WLNALDDLGDLSIALYPSDISNRVLGSGSCSALIKLMPDNKDILVSHATWSG 2
1 YETMLRIQKRYSLRFRKSKKSNKLIRGFDMSFSSFPGGIQSGDDFYLISSGLTTMETTIENYNDSLWSNVKPVGQ 0
0 VLEFVRAMVANRLADNPTDWANLFKLHNSGTYNNQWMILNYAAFQPGSPLPPRDVLHVLEQIPGHVMHDDFTGHLINRTYWASYNVPYFPFIFNVSGNYEMEQIYGSW 2
1 FSYSETPRARIFARDHVKIHCDKCMLHLMRSNNYTRDPESRCDCSPPYSAENAISSR 2
1 NDLNPANGTYPIRALGHRSHGATDVKVTSSQLFQQLQFKAIAGPTQGSNNSLGPFCWSKSDFNDKVSHLGHPDCFNFKPVLHQWSL* 0

>PLBD2_lepSal Lepeophtheirus salmonis (crustacean)
MEILKGSIFPIFLLISTRALGTSSLKHSLSYDGKDFTLLPGHVSGSNILLHGSFVNLINQTGWSILKINTMSHFPDNIQAYGAGYIEAYMTKDLIYNHWR
NTIVGYCDGKEKICEGIQGFLSKNLNYIRSMTTNSKREKSSYWHHVALFYDQIRGITEGYNKYAPPGRSISAEDIFFMNVMGDVEDLEQVFSHRLNISLP
DKVLGSGSCSALIRLLPGNRDLYAAHDTWNSYQSMLRIIKKYKFGLHWVLQKFPEKDDKHTIPGRSMSFSGYPGTVFSMDDFNVIPSSGLVSIETTIGNG
NASLWQYVQPIGTVLEGIRATVANRLARSGGHWVSVFSKRNSGTYNNQWMVVDYKRFKPGEKIIRPGLLWVLEQIPGRIHYTDVTGVLKKIGYWPSYNSP
YFKDIFNMSGGPEAVAKFGNWFSYEKTPRALIFKRDAPKVHNMGSMMKIMRYNDFRHDPISRCNCTPPYSAENAISARCDLNPKNGTYPFGALGHRSHGG
TDMKITSFDLFMKGSFIAGGGPTYDSVEPFQWSKADFGKTTPHFGHPDKWAFKPIKVDWENAFNRGEDVV*

>PLBD2a_ixoSca Ixodes scapularis (chelicerate) XM_002413831
MKNSYAPRFPSHVLGGSGSEFFCSKLNVYCSSTHRNMFVTLAVSALLALQAAAETPGDASCQYAWASLGPSGGDLHIHDGKPGQGAVAWGTFQNDIRFSG
WAFLDLQSNASYADDVQAYASGAVEAHLTRDLIEKHYNNMYSRYCDGQSEYCERLSKFLQENIKYSNDQEHLYYSTDPYWHMVHLQMKQLAGLSDHLENK
ALNTSNEYLNVTRALYLNLDGDLMDLEAYLRRVGDENSIFQTNACSVLIKVLPDNEDILFAHNTWFLYRSMLRIEKKYTFPWHVTSESSEIIPGHTISMP
SYPGKLLSLDDFYIASTGLAITETSLENNNDSLRNLIKPENAPLTWVRCMVATRLAVTGSQWVEYFGKLNSGTLNNQWMVLDYKLFTPGAAIGKDTFWIL
EQMPNTTKTKDVSEYLQNQKYWASYNIPFLPEIFNLSGQPQLVKKYGNYYSHDMCPRAQLFRREQGKVEDVDSMTALMRYNDYTHDPVSGCNCTPPYNAI
YAISSRYDLLDPEGSYDMPNMARRAVGATDMKLTNYGMSRRLEFIAINGPTYTNDSSVPPFQWSTSGFQDLHEGHPDKWTFGPTHHRWDSCPNF*

>PLBD2b_ixoSca Ixodes scapularis (chelicerate) XM_002405205 frag
98 TGWSSIVVHTNEFYLDDQQAFAAGLVEGRLTRTLIQEQFTNEFNKYCEEDPQFCAELFKYLHANIESMVANVAEYSSTDPYWHQVRKTAFYDKPTEQRNK
ACFGHSNQPVNEFFYTKRLLNLHGDLSDLETKFNRTNSRKINPTGSGSCSAIVKLAPGSRDVYFAHATWTHYNSMNRILKKYRLNYHTAPMSKDLIPGSS
IVFSGYPARIFSGDDFYLINTGLAVMETTIGNENSDLWKKVTPIDTVPTSIRNMVANRLSLTGQHWTNTFARFNSGTYNNEWMVLDYKRFESTSQKLNRG
FLWILEQIPGLVMSKDVTPILEEQGYWASYNSPYFTEIFNESGLPALVEKYGDWYTYDKSPRALMFKRDQEKAVDLRSVMKLMRYNDFTNDPLSRCTGCD
PPYSAENAISARSDLNPANGTYPFKALGRRAHGGTDAKVTNFALFERQEFYAVSGPTSDDQRPFRWSTSGFDNVSHAGHPDLWDFDPILAQWRY*

>PLBD2_plaOce Placobranchus ocellatus (mollusc) HP171048 
MAIIMNSRLCVQLFVAISIFSLCNAAVTKLWVTYNTESSIFEVSDQQATDYVAVASFDNTVNQTGWAKLDVITQAGPKRKYNDSVQAYAAGFVEGHITKS
LMTMHWANTGAWVCPEPLTSQCIQIKKFLESNLKWVLENIKTFSTTSPFWHHVRLFLEQTAGLQDGFAGMKGQLNLDIDVMSVYMWQVGGDMETLLNVFT
GSEDNQYKTPDPLAMGMGHCSALLRLLPDYSDLYVAQDTWSDFSSMLRVLKRYSFEMLKSPQYPDIPAPGNTMTFSSYPGVLFSGDDFYIMSSGLVSQET
TIGYANSSLNQYIKPTSVLEGVRTMVANRLAASGREWAQIFSLINSGTYNNEWMIVDYKLFRPGQVIPVPGLFTVLDQIPGTIVYDDLTLHLYKYKYFGS
YNTPFFPEVFNKSGQPALVAKFGDWFTHDKTPRALIFKRDAPKVNDLNSMIKLMRYNDFKHDPLSRCNCTPPYSAENAIAARCDLNPANGTYPFGALGHR
PHTATDMKVTTFEMAKSLSFRAVSSPPwDDLPVFQWSTSSF*

>PLBD2_aplCal Aplysia californica (mollusc) est frag
YPGPLMSVDDFYAISSGLVSQETTIGFSNAELVKYITSEAVMEGFRSMVANRLASSGGEGSKLFAQYNSGTYNNQWMIVDYKRFVPSRETGVPGTLYLIE
QIPGTIEYADLTDYLYEKSYFGSYNVPYFPDIFNKSGLPPLVKKLGDWFTYDKNPRALIYKRECSKVTDLDSMTKLMRYNYFKNDPLSRCNCTPPYSAEN
AISCRDDLNPANATYPFGALGH

>PLBD2_helRob Helobdella robusta (annelid) EY564899 frag
FAYLEIQTNEKVSDIEQAYTAGLAEGLLTSDLIKLHWYNTFQDYCKAPLNEFCAKLQNAMEANFNWMANQIFLEANLNPFWHQVELILVQLHGLIDGYKF
EQRPRDTLTKFVFKPDVTGLLMLFLSGDISDLESVLGGNKFNVYKPFDHLSGPNTCSALIKLLPNNKDLLISQTTRSNYGTMLRILKKYDFGYHTVTGGQ
LVPAKVISFSSYPGVQFSVDDFYLLSSGLVAQETTIGNSNADRWKLVKPESLLEVIRNMVANRLATTGEEWADLFVRYNSGTYNNQWMVLNYKLFTPGAK
EIKDGLLYVVEQIPGLTVGQDVTFVLRNQTYWPSYNSPYFKEIFNASGCLENVKKYGDWFTYEKTPRALIFKRDHSKVVDMNSMMRLMRYNDFTHDPLSA
CNCTPPYSAENAIAARCDLNPKDGKYPFDALGHRGHGATDVKITSYSMFKNFQFLAVSGPTRDQVAPFQWSKSDLKDTIRHAGHPDLWVFDPVQF

>PLBD2_alvPom Alvinella pompejana (annelid) frag
YCRIKVWILGLFSFFISISTAVDVYVKWDTVSNNFQISEKRLSDPVAWATFGDEIAKTGWSYLEVYTNGAYDDSKQAYAAGLAEGYITQDLITKHWYNTY
KGFCTKPLNANCKKLQAFIDKNLKWMASKIATEAHKDSYWHQVQLFLEQQAGLLDGYSGQPQFPSNYSMDVTGIALLQLSGDIKDLQTALGIASANKLWFGTKFGIRGHK*

>PLBD2_schJap Schistosoma japonicum (trematode) FN318739
MYFTVVFTIVISFIEIHTQHPSKALILANSHKPYLYFDYVSDDNILQNPHIIAMAVFKEEINTTGWSSLTVSTSSDFPDYLQAYWAGFLETNLTFSLTAS
QWANTVRDMCPLPLSKDCQALRKYLSENMAYMLNEAYKNDKHSSFWYHVALQLWQLKGMSDAFDKRFIKRADLLNRNYLDELVDNVMGIYLLQLNGDLGD
LVSALSLPTLKEGCNQNGHPFIASSSCSALIKVVDSNVYLSHVTWSPYSIMLRVLKHYNFPWKIVNNTGSQKIPGFAITFSSYPTYTSSVDDFYITSANL
TITETTNNVYNKTLWEIVRNGSKNAVLTFMRGMVASRLAKTGEEWITYFKYNNSGTYNNQWMIFDAKQWPKNKGSLLIAEQLPGIVSSLDVTKILKINGY
WASYNLPFIGDIYTLSGTEEMAKMFGDWYVHNKTARAKIFRRDHHKVVDFPSMLSLMRYNDFMNDPLSTCPCKPPYTSNSAISARDELNDPKGQYPIPSW
SYRLHGGTDAKIVDLSMINQLNMIAISGPTYDDLPPFRWSLIKDVKKPLMHPDKWQFPPVITDFIDYPKANSNISARFLSFESLF*

>PLBD2_triAdh Trichoplax adhaerens (trichoplax) XM_002107718 introns largely conserved
0 MAQCGKFLIYFSIFIITLATLCSCQSGSVIYKDGLYTFSKGINKRAASYGTFTDKIASSG 2
1 WTYLDVHTNPQDDDFITAYAAGYVEGILTAKY IYMHWKNTVGDYCKQKSIYCQKLKSFIMKNNQWMATQIKHRPHSIYWYH 0
0 INLTLIQQKGLRDGYHKAMPHKPIDEFSFL 2
1 LIELSGDLESLETALKDEDTHHVLGSGSCSAFIKVLPDNRDLYFAHDTWTGYQTMLRIYKYYELNFSMLPKTN 1
2 VTVPGTRISFSSYPGTILSGDDYYLIGSGL 0
0 ATMETTNGNSNEKLWKYVTPSSVLEWIRTIIANRLTSSGNDWVKIFSKYNSGT 2
1 YNNQ 00 WMILDYKLFAPKRPLNPNTLWVLEQIP 2
1 GKIESADVTNVLKKQGYWASYNVP 2
1 YFSSIFNMSGNQEQAKKYGNWFTHDKCPRALIFKRDQHKVNSMESLMKLMR 2
1 YNDFKHDPLSRCNCTPPYSAENAISARSDLNPADGKYNIGALGHRCHGGTDSK STNYTMFHSGLKSYAIAGPTHEQQPPFRWSTAKFNMTKPLGHPDLFNFTRQLVSWD* 0

>PLBD2_monBre Monosiga brevicollis (choanoflagellate) introns all novel
0 MWSCGAAAAAVVAVVVLASPATATVARFVEQTDVQTTYASVFYVESDDSYVVKTENHPWDGDFEKDE 0
0 AVRIKYTPGYLVAGWDQLHVKSNSAMDDATVAYAAGYGEAQLTAEMIYNYAYNNGYDTFTPNDKLADYLAKNQAFMAASIASNRSDANGYWYHVDLILRQLQGVCDGYNSSD
FAKSFPLPCESMLAINLMGDMEDLSDALASSDEWYTEDRFFRATHCSALVKLVGGASSPSDIYISQDTWSSLNSMTRIMKRYDLNFLQ 2
1 AKGADDRIAGSSIVFSSYPGSLYSGDDFYLTSAGMAVIETTIGNSNPELYQYIVPDTVLEWIRNIMANRLASNSQTWYEVYRQFNSGT 1
2 YNNMNMILDYKQFKPQEALQDELLTIVEQIP GTVTKTDVTGYLRNMTYWGS 1
2 YNVAFDQNIRELSGANQAEQLYGPW 2
1 FSYWNTSRALIFAREQKNVSSLEDLKRLMRLNQFKTDPL 2
1 YRGWTNCTPAYTAENVIATRGDLNDP 0
0 NGIYSLSSFGLRNHVATDSKISTFSTYDSNNLNVWAIS 2
1 GPTNGPPPNQPVFNWSTSYYKDTRHRGMPEAFDFDWVNFNWPF* 0

>PLBDa_dicDis Dictyostelium discoideum (slime_mold) AAFI02000019 AF411829 introns both novel
0 MRVIRSLLLLTIAIIGSVLSQSSIDDGYTVFYSQPDNYYVKPGTFSNGVAQAIFSNEMMTTGWSFMSISSSEGLYPNDIIAAGAGYLEGYISQEMIYQNWMNMYNNEYHNVIGSD
VENWIQENLQYLQTMIDSAPSNDLYWQNVETVLTQITYMQRGYNQSVIDNGVDASQSLGITEFFLMNMDGDMIDLGPALNLTNGKQVTSPATATSPKQAFKEFMRRTGHCSALIKMTDDLSDLFSGHTTW 2
1 SSYYEMVRMFKVYNLKYLFNGQPPASKVTMFSGYPGTLSSIDDFYLLDTKIVVIETTNGLMNNNLYHLITSESVLSWIRVIVANRLATGGESWCQTFSLYNSGTYNNQ 0
0 WIIVDYNKFIKGYGALDGTLYILEQVPDYVEYGDQTAILRTGYWPSFNIPFYENIYGLTGFNETYAQFGNWFSYQASPRSMIFKRDANNIHSLTQFQAMLRYNNWQNDPFSQGNAGN
QISSRFDLVTADDPNNQYLDPDAFGGIDSKVVSADMVAALLVNAQSGPSHDNETPFTWNSQWNQKYTYAGQPTTWNFDWMTMSLQSMKPASPSSDSSSDSTTFN* 0

>PLBDb_dicDis Dictyostelium discoideum (slime_mold) XM_640726 2 exons
0 MNKLKSNFILNIVILFTILIFNINFINCENQKQQQHQQQQQQQQQQSSSTTTTLPIYSIKFSSETGFTIYSGNDSTSIAQSGFSNEMMTMGW
AYLTITTNSQFEDSLQAEAAGYIEGYLTFEMIWQCWYNILVNEYQNQTIPNQVLNWANENIAYMKQQVATNENDPYWINIGLVLTQLSGMVD
GYNAANQDPSRQLSFLDFILINMDADLGDISSTFNQSTSFSEISNFKKSMDHIKKTDHCSGLIKLTDDLTELYSA 2
1 HTSWSSYINMLRIFKSYNFKFSSITNIKSKLTLFSGYPATIASLDDFYLLDTKLVVLETTNGLNNNDLYYLIKPESVLTWMRVIIANRLANGGQSWCETFERENSGTYNNQWMI
VDYNKFVPGVKVRDGTLFVLEQVPGYIEFADVTNVLRTGYWPSYNIPYFETIFNMSGFNDELTDSSDYEAYEEDARSQIFRRDANKVYSLTDFQAIMRYNNFQNDPLSHGDAANQI
SSRFDLNSPDSQDYDAFGGVDSKVTSFSLVNQLLVIAQSGPTHDQEPPFQWSSANWSNIYPSIGMPNLYDFGWVNFTDISYNY*

>PLBDc_dicDis Dictyostelium discoideum (slime_mold) XM_632848
MNKIIILISLFLNFLFGYVVCLNENNQKSQNLIIPTYSIKLKSDGEYLVYSGNDTLSIAQASYTNEMMSIGWGYISITTNPKYNDSLQIEAAGYLEGYLSYE
MIWQNWNNMMVNQNANNSFGNDIISWAKENILYMNQQIQLNQNDPYWINVNLVLQQLNGLTNGYSDANQNPDRQLSLMDFILLNMNVEIYDIMNSLKNNSSSFYQQPNNNSFDNNQ
HCSALIKLTDDLTELYTGHTTWSDYYQMVRMIKSYNFRFSKLVAAKSNTTMFSGYPGVLMSVDDFYMLDSKLVVLETTNGIKDNDSELFKLIKPQSVLTWIRIIVTNRIAHSGKSW
CEIFEKENSGTYNNQWMIVDYNKFIKGVRVQDGTLYVFEQLPGYVEYADVTNILRTGYWPSFNVPYFETISNMSGFNYQSSSSDSSSSSGSIAYEQYPRSQIFRRDSNKVYSISDF
QAFMRYNDFQNDPLAYGDPGNQISSRFDLITPQNNASAAGGIDSKVTSLELINQFLMIAQSGPTHDQEPPFSWSSENWKNKYPTIGQPDTFDFEWVTFSTTSFGSFPSASNEKNY*

>PLBDd_dicDis Dictyostelium discoideum (slime_mold) XM_633485
MIIFKNLLKLLIILLTIKLYFCIEIKREEHLTILNELNENSDVIQYSILPGNNEEYEIVKGIQEDAIVYGYYMSNVEVNGWAYLSLVSNDKYNDSTQSRAFG
YLEGYLTKDLIWNSKVNYYKNAFNSSEIPNKLDDWLTENIESIHTFIVNNRKSRYWNQITLVMDQINGMLDGYNEANTNSSETLSLHDFFVLNMFGDLFDLMPALNLDKEYKYFQK
DLNDIQDWFKRSQHCSALIKVSSDYSELYSGHTTWSGYYTMLRIFKSYNQQFSSDVSGTLSKRNIFSSYPGALISVDDFYLLGDTRMVVIETTNSLVTNDLYHLIRPTTVLSWMRV
IVSNRMSTNGKEWCENFQRYNSGTYNNQWMIVSYNLFVPYNELKDGALYVLEQIPGYIEFSDQTQALRQGWWNSYNIPFYETIYDASGYNNYTANNYSDSTIYYMSYQTCPRAEIF
RNFAGYVESLEDFQSLLRYNDFEYDPLSHKLPFYAIASRYDLSKKNPSPFGATDTKVTCNSMIDQNTIVAISGPTTSNGQPIFEWNSKIDFMESTSHLGCPEKYNFPWVSFSDTTFRNL*

>PLBDe_dicDis Dictyostelium discoideum (slime_mold) 
MKLFILLIVIVFLISNSYSLSSSDSSSDSGSDVQYYSLTSQFQVVQGKQIPGSIAWGYFKDEMNKDGWGKLSIETVSTVSDNIAFKAAGYLEGYLTWEYIYK
FSGNYFNSFFNTSNIKEIPTETLTFVSDNWEYMMERVNSSSTTDPYWIQIRNAMSQQIGLYEGYNAAAGEDYQKTFIEIYMINLYGDMGDIVTLTTTPNNEFIPMDRKEVEQLMAT
TGHCTSIIKLTNNCSDLMSAHTSWADFSVMIRIYKRINIPVASTPYGSETLFSSYPGLLVSIDDFYQIRPSKLHLTETLNTILNQTLYQQINAQSFMYWVRNLVANRLANNGFQWV
SIFVENNSGTNNIQFVVLDYKLFTPYSTELQSDLLWIVEQYPGGYQAADVTLTLWEQGYWPSYNRPYFEEVFDILGYPYYVEKFGDLFTYEYNPRANIFRRDHSKLETLQDMMNII
DYNQYKTDPFSMGYPGNSINARFDIKGGSLPSGNPIYSWFYHGTHGGIDGKAINYDMVNSFTAVARNGPTVTSDCPPFNWNDWSLISHQYMPQIYNFTWISINI

>PLBDf_dicDis Dictyostelium discoideum (slime_mold) XM_633485
MKIINSFVFIFVLLFVFNTNAIKLSEDEKYSIEEKPEWYSIDSNFAVSPGKISDALGWGYYKNEILKDGWGKLYVEMNKFMTPENSSIYYEATGYLEGYLTW
STTWNYSQAYFQNYMNGTNESDIPTPLVEFLKINYDWMTSTFSNRDESVYDTQVSNVIYQFEGFARGYQQAADSDKQLTTLQLLLLQYAGDLEDVAGYLEYEMAQNKTEYINRVKS
LKEIETLFAVKGRCSGLVRITPDYGELFISHTTWGSYFTAGYRIFKRIIIPDPTVPGNEILFASYAGVLTSDDDFFMIPSTEMVIIETTNDILNTSLYQYVTPNSLLYFVRSIIAN
RLSNTAQEWTNNFIQYNSGTYSNQWMIVDYKLFTPYQPLQPNTFWIIEQLPGGFMSADMTEVLALGNWPSFNRPFFPEIYDAMGYSYYEKLYGDIISYDLNPRSKMFRRDVPNVMS
LDGMQQIMTQNNYKSDPFSGGFPGNAIAARYDLGGGPAEPLSWSFIGLHGAIDSKITSYSLLQQNQAIAINGMTVTPDCPPFTWNSNWSTVSAHYGSPETFDFDWISITITDK

>PLBDg_dicDis Dictyostelium discoideum (slime_mold)
MIKSYYLFFIILIFLIFINNFILCENNNNNKYNNNNNYPFINVDKIIYTNSSNSIYLVWSNEQFNLILQSSYNEEFDDIVMIGEYFDKIETTGWGELNITFN
SNSATIQISDNEQAYFTGFIESVLTGERINQMYNNFAASEFTNSDHTPSPKLIDFLNTQMEFVRDQVFENNGSSQYWYSTGLIMSQFDGLVNGYQQSPFPQLSEIQLYILTSAGDL
ETLVTLFPSSSSSSSSTTNENKNKNIKISSIKPNFKDELTDCSGFIRILPDYSDVYFGHTTWRYYYALLRIYKFINLQFNFQDTPMEYKVSFSSSPGFISSKDDFYITGNKLAIME
TTNNIYNESLYQYTIPQSVLVWQRAMIANMIATNSSDWVKIFSEFNSGTYQNQWMVFDYKLFIPNKQQSSSSLPPNTFWIAEQIPGQVKTADLTNILNEQGYWKSYNIPYFESIYN
ISGYSEKTDLPPSYQYSYEKCPRSLIFSRNASDVLNFEDMKSLMQFNNYKTDPLSYSSPLNSISSRGDLLTIENGNRSAVAFGGVDSKITSFNQVLTLSCTAISGPSTNGGTLPPF
TWSQSPLFQNITHIGVPETFNFDWQEMGPYPN*

Derived PLBD2 sequences

>PLBD2x_droMel Drosophila melanogaster (fruitfly) Q9VRK8 retinal lamina neuron ancestor (lama) PMID: 16077094,8892229
0 MERPEYDGTYCATALWTKQVGFQIENWKQQNDLVNIPTGVGRICYKDSVYENGW 0
0 AQIEVETQRTYPDWVQAYAAGMLEGSLTWRNIYNQWSN 2
1 TISSSCERDESTQKFCGWLRDLLTTNYHRLKRQTEKAENDHYWHQLHLFITQLEGLETGYKRGASRARSDLEEEIPFSD
FLLMNAAADIQDLKIYYENYELQNSTEHTEEPRTDQPKNFFLPSATMLTKIVQEEESPQVLQLLFGHSTAGSYSSMLRIQK
RYKFHYHFSSKLRSNTVPGVDITFTGYPGILGSTDDFYTIKGRHLHAIVGGVGIKNENLQLWKTVDPKKMVPLVARVMAANRI
SQNRQTWASAMSRHPFTGAKQWITVDLNKMKVQDNLYNVLEGDDKHDDAPVVLNEKDRTAIQQRHDQLRDMVWIAEQLPGMMTKK
DVTQGFLVPGNTSWLANGVPYFKNVLELSGVNYSEDQQLTVADEEELTSLASVDKYLRTHGFRGDLLGSQESIAYGNIDLKLFS
YNARLGISDFHAFAGPVFLRFQHTQPRTLEDEGQDGGVPPAASMGDERLSVSIEDADSLAEMELITERRSVRNDMRAIAMRKIGSGP
FKWSEMSPVEEGGGHEGHPDEWNFDKVSPKWAW* 0

>PLBD2x_gloMor Glossina morsitans (tsetse) EZ422576 59% PLBD2_droMel V for iMet, C confirmed by ests
VYCATAFWTKQFGFLVRLSIEYWKQQNDLLNIPKGAARICYKDSIYENGWAQIEIETQRTSPDWVQAYGAGVIEGSLTWNQIYNQWLNTI
MSSCDRDENAQHFCTWLRELLENNFNEIKETAASKSEHDHYWHQINLYFIQLKGLETGYRQGAKRARSDLEGAIPWSDFLLMNSAADIHDLKIYYENYIPNG
INRTTDETEPKNFFLPSATMITKIYPTGNASTWQLLFGHSSAGSYTSMLRIQKRYKFHYHIASNQNSNTVPGMDIAFTGYPGILASTDDFYIIKGRQVHAIVAGVGIKNENLELWQ
TVNVQKAMPLAARVMAANRLAQSRRSWTKALSRHPFTGSKQWITIDLNKLSPLESLYELDENNLELDEEAASIERQNHLKGLVWIAEQLPGRMHMRDVSALFLETVDNSTTWLASG
IPYYKEILEASCVNNEAYITNLTAAESRELNNLEAIDKFLRTRGFRGDLLDDEQSVAYGNIDIKLFAYNARLGMSDYHAFAGPVFIRLQHVQTRSLPAAMGYRGDLAPASLIRDDR
LSVVVDDAAELAELQVITERRIVRDDMRAVAMSQISSSPFKWSSVAEKMATLQHAGHPDEWNFGKVSPKWAW*

>PLBD2x_aedAeg Aedes aegypti (mosquito) XM_001658072
MDRPNYSGTYCATAYWARNSGFRVEFWGQREELGEVPPGAVRACFKHSLMESGWSQLELESQPEYPDSIQAFAAGMLEGTLTWNNIYLHWSNTIESECNR
DDQSEEFCHWLRRIISTNVETVKKMADMKGKNDHYWYQIGLFFDQLDGLEFGFRKGVRRSRMDYEIPMEDFLLMNSAVDIRDLKAYYMNFLDSESGMHVE
PNKGMMLLKLVESVTGKVNILLGHTSDGSYASMLRMMKKYTLNYHFAEESIVERVVPNTNIVFTGYPAVLASLDDFYMLSGKHHRMVVGGIKIKNDNLNL
WTKIDLVRSVSLAPRVMACNRLAHSGRVWSKYFARSPSTGAKQWLLVDLTRLNRENELLDDSAEVEEMLLSHAEYDDPKAMRKAQSTDYVEVDFEGIVRK
MTSIGKISDGGLFWVVDQLPGRLHAEDMTEKIVRDGYWLGTGIPTFEELADIGQVKTNGTDSQRHSIQEKILNNITDLEGLAKFIRQSAYRGDLDQQHPT
AFGNIDMKLFVETNASNGTGIFQAYSGPLYDPLTDGQAVKRSVENDGEVEAEAIPVAAAKKKRVKPFDWDSAKKLDVPHQGQPTLWNFRRQSPKWAWI*

>PLBD2x_anoGam Anopheles gambiaei (mosquito) XM_316013
MLKVVGASWHKTRIGSYILIGALMLAVAALFLADMERPSYNGTYCATTYWARNSGYRLEFWGQRNDLDQVPKGAVRACFRDSILTNGWSQLELESQSTYT
DTVQAYAAGIMEGALTWHNIYMHWSNTIDAVCSKDEESEEFCDWLRGLITTNVDTVKKMADMKGKHDYYWYQIALFYDQLDGLEIGFRKGVKRSRMDYEI
PKQDFLLMNAAVDIRDLKIYYTNFLMENSGLKPEPAKGIMLLKLLQQPNGQSKVLLGHTSDGSYSSMLRMVKKYTLRYHLSAETARTVVSAQAAEDEDRA
VVPGTNIVFTGYPGVLASLDDFYVISGRRQHRLVAAGVKMENEHVDLWRKIDLVRSVSLAPRVMAANRLGHSGRSWARYFARNPSTGVKQWLVIDMSRFV
GSNETEVELLATSPASVAVDQSVEDNDIAAEGGQDVVADGEHQRIVTKRMEDYVDDDFKEHLSAIRQVRMVTIPKAPEPVAGEELVVEPSVGHTFGSGMF
WVVDQLPGRLHAEDITDKIVSDGYWLTNGVPYFKEALDIGQATANATNTTAPSSTMEKILQNITDLETLARFFRQSAYRGDLDQDEPAAFGNIDLKMYTE
TADGTVHFRAYSGPLYDPARSTPGQAAKRSIVKSEQLHQSTATDQKSAPASHGDSATVASHPFDWSRILPFEVRHQGHPDVWDFEHITPEWAWI*

>PLBD2x_apiMel Apis mellifera (bee) HP560493
MLKVVGASWLQTRISTYILVAVALLGIGAIILGEFGHVEQDGIYSATVLWNRKGGYRIDFWGQGNDLTAVPLHAARAYYKTGIFEYGWSYIEIETSSKYP
DTVQAYAAGLLEGSLTWQLIHHHWYNTIRLECEAKPIECRKLMRYLRDNTAVNRKRAELKESTDSFWHMVRLFYAQLDGLEAGWKFAVRRSRVSASLESE
DFLWLALASDLCGFQQIFNISDSLVSSMIYFKSLPRENSGPLIAISHHTSASYTQMLRLLKKYTFGYHILPVTKTAALIPSRSIVMSSYPGALSSRDEFY
LINGENRELVVTGTSLLMTNRTEWSFLYPDDYVMLTVRLMAANRLATNSQSWFNTLSYQNDGASALQWIIFEPRSMTLLLVEQLPSITVPINYTEEFKKI
GFISYIGISNFKSTNNIVQPAKKNLNLWKTRLTRLQKNITTFEQFRNMMRGCSQEGCSAVNQKTDYLQELMFRGDLEDVPIPYGIIDTKILTVDIDGFKG
FEAISGPSQSRTPFKWSQSFPNISHVGHPDTFNFESVTPKWVWI*

>PLBD2x_pedHum Pediculus humanus (louse) XM_002424573
MLKVVGASWVKIRMSTWIIGIVLVFGIIVVILGQMGESEEDGNYSATAYWNEETGFEIKFWGQGLEKDKIPKGVARVYFQPHIDTTGWAIIDVETQSEYP
DWVQAYAAGMLEGSLSWQLIYWHWKNTVQSVCQNQTLFCKYIKKYLEENFKSVKELYENTDDPYWHQAYLFYIQLEGMQQGFEHGVKRKGLIIEEIEFID
FLWMNAASDIIDLEYKFYDSKPYSTTTTNSSSFSANNNNNSNLLLKKLIQGRSDNVESMEKLSFVGCGVTLGSALLNSFQNMRNYFSPIIPLDQTKLLVA
GTPITVFNKNLWNLVSPLKQLLSGVRVVVANRLSNNASDWGSYLSKYNSGTGNKQWLIIDVDKFKRKINDSSVGERENFFWVAEQLPGHFKIDDLTENLI
NSSYWASYGLPFYKDMYEMSGTNYMKENFGDAFSYENSPLAKIYRRDHNKSTSIKSILNLMRSNSFKRDPLSLGNPRCAIGARGDIPNKVSNSFPIGVID
SKIISLEKKLENVTSNDTTTNNNNNIIPFNNNVSNDELIIFKAVAGPAHSLHKNSENLKGNDEIRKEKNETVEVEGIGVGGGDDDTLKPFNWQNSVFNEF
PHNGHPNNWDFDAISLSP*

>PLBD2x_acyPis Acyrthosiphon pisum (aphid) XM_001948052
MLKVVGASWAQTQLSWCLIIAITLLGLLAFYFGGTIRNEYDGKYAATAFWSKEFGMRIDFCGQNNDPLTVRKGVARAYYRPDLSENGWAVLEVETQAEYP
DYVQAKAAGYLEGSLTWRMIYWHWKNTVENTCIGRRNFCERIRKYLEENAEEIKRMAKRRGESDPFWHQVNMFYTQLKALEDGWRFGVKRSRQDIDIPSV
DFLWMNIMPDLKNFEQKWNASKDFNPDKPPLSATLVKIISTNPIDFVLAQSASGYYGSMLRIQKRYNFGFHETQSDDSALVNGKIVEFTSYPGSIYSQDD
FYKVIKKGSKTETTVVGTELQNNNRQLWEKIMKTDQVLLGARIMAANRLASNSKKWYEVFSRNNSGTGNKQWLVISTNSTSIAFGVIEQMPGIVSYEELS
KTLLSTGYWVSNSNPCLKETVYMSGADGRDAADHPVANILRSGQLNVTDIGTLVDLMRGTEMSLIGRTDLLGVKTNAMFRRFANQSFVEQLTAANRLQSA
DATAPLKDDRTTIVTNRLPSSAASDDKLDVDKCFTGTVDLKVTSANMNGYYAASGPPFTMDRSEVEPFRWSESPIRHLPHYGHPDVWDFEVEGVVWVWK*

Trimmed alignable sequences

It is useful for various purposes to trim protein sequences to their conserved core and matured length. Here, since compilations of signal peptides have been previously considered, they can be discarded, greatly simplifying acquisition of reliably alignable sequence.

Note too that exon boundaries mostly differ between the two paralogs and so limit the matched pairs from a fixed species that be collected from genomic contigs using tblastn from a necessarily diverged query. However the final exons do match and are already provided above from a very large and phylogenetically disperse species set. In some cases, there is a benefit to removing single-species insertions that introduce a gap in the rest of the alignment because then other gapping can be done more accurately.

Finally, as the xray structural determination did not extend over the whole protein, flanking sequence needs be included only to the extent it is strongly conserved. Thus the boundaries of the protein are established, allowing comparative genomics on the alignable core.

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