Phospholipases PLBD1 and PLBD2: Difference between revisions

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(to be continued shortly)
(to be continued shortly)


=== PLBD2 active site ===
=== Conservation at critical sites ===


[[Image:PLBD2activeSiteComp.png|left]]
[[Image:PLBD2activeSiteComp.png|left]]
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  (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  C249 H266 W269 T330 N432 R463 mouse numbering
  PLBD1 phospholipase B-like    ....  pred  C249 H266 W269 T330 N432 R463 [http://swissmodel.expasy.org/repository/?pid=smr03&uid=&token=&query_1_input=Q8VCI0&zid=async SwissModel]
  PLBD1 phospholipase B-like    ....  pred  C228 H245 W248 T303 N402 R433 [http://swissmodel.expasy.org/repository/?pid=smr03&uid=&token=&query_1_input=Q8VCI0&zid=async SwissModel]
  Cephalosporin acylase          pink  1OQZ  S170 .... H192 .... N413 R443
  Cephalosporin acylase          pink  1OQZ  S170 .... H192 .... N413 R443
  Conjugated bile acid hydrolase green  2BJF  C2  .... D21  .... N175 R228
  Conjugated bile acid hydrolase green  2BJF  C2  .... D21  .... N175 R228
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[[Image:PLBD1colored.png|left]]
[[Image:PLBD1colored.png|left]]
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<br clear ="all">
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 class of NTN hydrolases.
This is perhaps unsurprising since the active site was established a 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). Glycosylation is important in targeting of lysosomal proteins, more so than in generic endoplasmic reticulum proteins where motifs are often poorly conserved (as in sulfatases).
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 similarlly. 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:PLDB2consSites.png|left]]
[[Image:PLDB2consSites.png|left]]
<br clear ="all">
<br clear ="all">

Revision as of 12:52, 26 October 2010

Introduction

(to be continued shortly)

Conservation at critical sites

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  C249 H266 W269 T330 N432 R463 mouse numbering
PLBD1 phospholipase B-like     ....   pred  C228 H245 W248 T303 N402 R433 SwissModel
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 substitions:
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


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 class of NTN hydrolases.

This is perhaps unsurprising since the active site was established a 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). Glycosylation is important in targeting of lysosomal proteins, more so than in generic endoplasmic reticulum proteins where motifs are often poorly conserved (as in sulfatases).

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 similarlly. 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.

PLDB2consSites.png


PLDB1consSites.png


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_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

PLBD2 reference sequences

>PLBD2_homSap Homo sapiens (human) PMID: 19706171,19237744,17007843
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_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
MERPEYDGTYCATALWTKQVGFQIENWKQQNDLVNIPTGVGRICYKDSVYENGWAQIEVETQRTYPDWVQAYAAGMLEGSLTWRNIYNQWSNTISSSCERDESTQKFCGWLRDLLTTNYHRLKRQT
EKAENDHYWHQLHLFITQLEGLETGYKRGASRARSDLEEEIPFSDFLLMNAAADIQDLKIYYENYELQNSTEHTEEPRTDQPKNFFLPSATMLTKIVQEEESPQVLQLLFGHSTAGSYSSMLRIQK
RYKFHYHFSSKLRSNTVPGVDITFTGYPGILGSTDDFYTIKGRHLHAIVGGVGIKNENLQLWKTVDPKKMVPLVARVMAANRISQNRQTWASAMSRHPFTGAKQWITVDLNKMKVQDNLYNVLEGD
DKHDDAPVVLNEKDRTAIQQRHDQLRDMVWIAEQLPGMMTKKDVTQGFLVPGNTSWLANGVPYFKNVLELSGVNYSEDQQLTVADEEELTSLASVDKYLRTHGFRGDLLGSQESIAYGNIDLKLFS
YNARLGISDFHAFAGPVFLRFQHTQPRTLEDEGQDGGVPPAASMGDERLSVSIEDADSLAEMELITERRSVRNDMRAIAMRKIGSGPFKWSEMSPVEEGGGHEGHPDEWNFDKVSPKWAW*

>PLBD2_acyPis Acyrthosiphon pisum (aphid) XM_001948827
MLSIRCILLSLLFVWALQCSATQKNQTLLAVKTDNNRITIQPKHYSVKDKEIIIGKGKFIDRINSTGWAYLEIRTSQKAKDEDQAYGAGYLEGTLTADLIYSYWFNTAKGYCTDRPNVCQQLK
DYMTTNKNWIKSKLNESDPYWYQVGLYYKQLDGLYDGYMRGKSPSTPDLTWDDLYWLNALDDLGDLSIALYPSDISNRVLGSGSCSALIKLMPDNKDILVSHATWSGYETMLRIQKRYSLRFRKS
KKSNKLIRGFDMSFSSFPGGIQSGDDFYLISSGLTTMETTIENYNDSLWSNVKPVGQVLEFVRAMVANRLADNPTDWANLFKLHNSGTYNNQWMILNYAAFQPGSPLPPRDVLHVLEQIPGHVMHD
DFTGHLINRTYWASYNVPYFPFIFNVSGNYEMEQIYGSWFSYSETPRARIFARDHVKIHCDKCMLHLMRSNNYTRD
PESRCDCSPPYSAENAISSRNDLNPANGTYPIRALGHRSHGATDVKVTSSQLFQQLQFKAIAGPTQGSNNSLGPFCWSKSDFNDKVSHLGHPDCFNFKPVLHQWSL*

>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

>PLBD2_dicDis Dictyostelium discoideum (slime_mold) AAFI02000019 AF411829 introns both novel
0 MRVIRSLLLLTIAIIGSVLSQSSIDDGYTVFYSQPDNYYVKPGTFSNGVAQAIFSNEMMTTGWSFMSISSSEGLYPNDIIAAGAGYLEGYISQEMIYQNWMNMYNNEYHNVIGSD
VENWIQENLQYLQTMIDSAPSNDLYWQNVETVLTQITYMQRGYNQSVIDNGVDASQSLGITEFFLMNMDGDMIDLGPALNLTNGKQVTSPATATSPKQAFKEFMRRTGHCSALIKMTDDLSDLFSGHTTW 2
1 SSYYEMVRMFKVYNLKYLFNGQPPASKVTMFSGYPGTLSSIDDFYLLDTKIVVIETTNGLMNNNLYHLITSESVLSWIRVIVANRLATGGESWCQTFSLYNSGTYNNQ 0
0 WIIVDYNKFIKGYGALDGTLYILEQVPDYVEYGDQTAILRTGYWPSFNIPFYENIYGLTGFNETYAQFGNWFSYQASPRSMIFKRDANNIHSLTQFQAMLRYNNWQNDPFSQGNAGN
QISSRFDLVTADDPNNQYLDPDAFGGIDSKVVSADMVAALLVNAQSGPSHDNETPFTWNSQWNQKYTYAGQPTTWNFDWMTMSLQSMKPASPSSDSSSDSTTFN* 0
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