Phospholipases PLBD1 and PLBD2

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 metazoa) 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 mutation in the reference sequence individual (with the SNP representing wildtype) as proline is quite well conserved throughout mammals. 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.

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

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:

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.

(to be continued)

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). Bizarely, 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 orgenelle 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.

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 relevence 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.

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 occured 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.

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 occured 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 modelled by SwissModel using the mouse PLBD2 structure as template but in fact have not, though modelling 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+ submitted
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

(to be continued) --tom 14:35, 9 November 2010 (UTC)

Arthropods (drosophila)

(to be continued)

Nematodes (C. elegans)

(to be continued)

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.

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 arefacts 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_droMel Drosophila melanogaster (fruitfly) U57314 retinal lamina neuron ancestor (lama) PMID: 16077094,8892229
0 MERPEYDGTYCATALWTKQVGFQIENWKQQNDLVNIPTGVGRICYKDSVYENGW 0
0 AQIEVETQRTYPDWVQAYAAGMLEGSLTWRNIYNQWSN 2
1 TISSSCERDESTQKFCGWLRDLLTTNYHRLKRQTEKAENDHYWHQLHLFITQLEGLETGYKRGASRARSDLEEEIPFSD
FLLMNAAADIQDLKIYYENYELQNSTEHTEEPRTDQPKNFFLPSATMLTKIVQEEESPQVLQLLFGHSTAGSYSSMLRIQK
RYKFHYHFSSKLRSNTVPGVDITFTGYPGILGSTDDFYTIKGRHLHAIVGGVGIKNENLQLWKTVDPKKMVPLVARVMAANRI
SQNRQTWASAMSRHPFTGAKQWITVDLNKMKVQDNLYNVLEGDDKHDDAPVVLNEKDRTAIQQRHDQLRDMVWIAEQLPGMMTKK
DVTQGFLVPGNTSWLANGVPYFKNVLELSGVNYSEDQQLTVADEEELTSLASVDKYLRTHGFRGDLLGSQESIAYGNIDLKLFS
YNARLGISDFHAFAGPVFLRFQHTQPRTLEDEGQDGGVPPAASMGDERLSVSIEDADSLAEMELITERRSVRNDMRAIAMRKIGSGP
FKWSEMSPVEEGGGHEGHPDEWNFDKVSPKWAW* 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_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*

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 reliable sequence. Note too that exon boundaries differ between the two paralogs and so differentially delimit what can be collected in practise from genomic contigs using tblastn from a necessarily diverged query. 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.

(to be continued)