IKGNVDLVFLFDGSMSLQQDEFEKIVDFMKDVMKKLSNSSYQFAAVQFSTDFKTEFTFLDYNRQ55556.com

BioMedCentralPage1of12(pagenumbernotforcitationpurposes)BMCVeterinaryResearchOpenAccessResearcharticleThewildboar(Susscrofa)Lymphocytefunction-associatedantigen-1(CD11a/CD18)receptor:cDNAsequencing,structureanalysisandcomparisonwithhomologuesPhilippeGACVandenBergh,LaurentLMZecchinon,ThomasFettandDanielJMDesmecht*Address:PathologyDepartment,FacultyofVeterinaryMedicine,UniversityofLiege,ColonsterBoulevard20B43,B-4000Liege,BelgiumEmail:PhilippeGACVandenBergh-pvdbergh@student.
ulg.
ac.
be;LaurentLMZecchinon-lzecchinon@ulg.
ac.
be;ThomasFett-thomas.
fett@ulg.
ac.
be;DanielJMDesmecht*-daniel.
desmecht@ulg.
ac.
be*CorrespondingauthorAbstractBackground:Themostpredominantbeta2-integrinlymphocytefunction-associatedantigen-1(LFA-1,CD11a/CD18,alphaLbeta2),expressedonallleukocytes,isessentialformanyadhesivefunctionsoftheimmunesystem.
Interestingly,RTXtoxin-producingbacteriaspecificallytargetthisleukocytebeta2-integrinwhichexacerbateslesionsanddiseasedevelopment.
Results:Thisstudyreportsthesequencingofthewildboarbeta2-integrinCD11aandCD18cDNAs.
PredictedCD11aandCD18subunitsshareallthemainstructuralcharacteristicsoftheirmammalianhomologues,withalargerinterspeciesconservationfortheCD18thantheCD11a.
Besidesthesestrongoverallsimilarities,wildboaranddomesticpigLFA-1differby2(CD18)and1or3(CD11a)substitutions,ofwhichoneislocatedinthecrucialI-domain(CD11a,E168D).
Conclusion:AsmostwildboarsareseropositivetotheRTXtoxin-producingbacteriumActinobacilluspleuropneumoniaeandbecausetheyhavesustainedcontinuousnaturalselection,futurestudiesaddressingthefunctionalimpactofthesepolymorphismscouldbringinterestingnewinformationonthephysiopathologyofActinobacilluspleuropneumoniae-associatedpneumoniaindomesticpigs.
BackgroundCelladhesionreceptorsplaycrucialrolesinmulticellularorganismsbymediatingthedirectcell/cellorcells/extra-cellularmatrixproteinsinteractions.
Thesemolecularinteractionsconditionthestructuralintegrityofcellsandtissuesandcontributetothesignallingtransductioninter-veninginthecellulardynamic[1].
Celladhesionrecep-torsaresubdividedinseveralmembrane-associatedproteinfamilies,includingintegrins,cadherins,immu-noglobulinsuperfamilycelladhesionmolecules,selectins,andsyndecans.
Integrinsareafamilyofcellsur-faceadhesionandsignallingglycoproteinsmadeupofnon-covalentlyassociated120–180kDaαand90-110kDaβsubunits[2].
Thereare19distinctαsubunitsand8βsubunitsthatarecombinedtoform25differentheterodimericreceptors[1].
Eachsubunitpossesses(i)alargeextracellularN-terminaldomainassociatingwiththatofthecompanionsubunittoformtheintegrinhead-piecewhichcontainstheligandbindingsite,(ii)asingletransmembranestretch,and(iii)ashortcytoplasmicC-Published:15October2007BMCVeterinaryResearch2007,3:27doi:10.
1186/1746-6148-3-27Received:3May2007Accepted:15October2007Thisarticleisavailablefrom:http://www.
biomedcentral.
com/1746-6148/3/272007Berghetal.
;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
BMCVeterinaryResearch2007,3:27http://www.
biomedcentral.
com/1746-6148/3/27Page2of12(pagenumbernotforcitationpurposes)terminaltailwhichmediatesinteractionswithcytoskele-tonandsignallingproteins[1,3,4].
Withintheintegrinfamily,theleukocyte-specificβ2-integrins(CD11/CD18)includefourmemberssharingincommontheβ2subunit(CD18)[5]:(i)themostpredom-inantisthelymphocytefunction-associatedantigen-1(LFA-1,CD11a/CD18,αLβ2)onallleukocytes[6];(ii)theCD11b/CD18(Mac-1,CR3,αMβ2)mainlyonactivatedgranulocytesandtissuesmacrophages[7];(iii)theCD11c/CD18(gp150/95,CR4,αXβ2,Leu-M5)alsoonactivatedgranulocytesandtissuesmacrophages[8],and(iv)theCD11d/CD18(αDβ2)abundantontheCD8+lymphocyteandonmacrophages[9-11].
Thesereceptorsareessentialforaneffectiveimmunesystemasobservedinrepeatedinfectionsassociatedwiththelymphocyteadhesiondeficiency(LAD)typeIsyndrome,adiseaseduetomutationsintheβ2(CD18)subunitgeneleadingtothelackoffunctionalβ2integrinsonthemembranesurfaceofleukocytes[12-14].
Theβ2-integrinLFA-1isessentialforthefollowingfunc-tionsoftheimmunesystem[15-20]:(i)interactionbetweenlymphocytes,(ii)interactionbetweenT-cellsandantigenpresentingcells,(iii)adhesionofnavelym-phocytestopost-capillaryhighendothelialvenulesofsec-ondarylymphoidtissues,(iv)adhesionofleukocytestoactivatedendotheliumatsitesofinflammationforextravasation,(v)controlofcelldifferentiationandprolif-eration,and(vi)antibody-dependentkillingbynaturalkillercellsandgranulocytes.
LeukocyteLFA-1-mediatedadhesionisengagedviathebindingoftheLFA-1inanactivatedconformationalstatetomembraneproteins,theso-calledintercellularadhesionmolecules(ICAM)-1to-5andthejunctionaladhesionmolecule(JAM)-A[21].
Interestingly,severalpathogenstargettheleukocytesβ2-integrinswhichleadstolesionsanddiseasedevelopment[22].
SeveralstudieshavehighlightedthecentralroleofLFA-1inthepathogenesisofdiseasescausedbyrepeats-in-toxin(RTX)-producingbacteria.
ThevirulenceofAggre-gatibacter(Actinobacillus)actinomycetemcomitans(localisedaggressiveperiodontitisinhumans),Mannheimiahaemo-lytica(pneumoniaincattle),andpathogenicstrainsofEscherichiacoli(extraintestinalinfections)hasbeenassoci-atedwithaligand/receptorinteractionbetweentheirrespectiveRTXtoxin(LtxA,LktA,andHlyA)andtheCD11a/CD18receptorresultinginleukocytesalterations[23-26].
Thisinteractiontriggerssynthesisandreleaseofawidearrayofcytokinesandchemoattractantsbytheleu-kocytesthatexacerbateinflammationandultimatelyresultsinamuchgreaterleukolysisworseningthelesions[25,27].
Actinobacilluspleuropneumoniae,acausativeagentofpleuropneumoniaindomesticpigs(Susscrofadomes-tica),responsibleforeconomiclossesandantibioticusageintheporkindustry,alsoproducesRTXtoxins(ApxIA,-IIA,-IIIA,and-IVA)[28,29].
WethereforehypothesizethatthepathogenesisofthisdiseasesimilarlyreliesonaninteractionwiththeSusscrofadomesticaLFA-1,whoseCD11a(αL)andCD18(β2)subunitshavebeenwellchar-acterised[30,31].
Onthebasisofthereportthatapproxi-mately50%ofwildboarsintheirnaturalenvironmentareserologicallypositiveforActinobacilluspleuropneumoniae[32]andbecausethesewildpigssustainlossesduetonat-uralselectionpressure,wehypothesizethatsomeLFA-1molecularpeculiaritiesconferringresistancetowildboarscouldhavebeenselected.
Inthiscontext,thepurposeofthisstudywastoreportthesequenceandanalysisofthecDNAsencodingwildboarLFA-1(WbCD11a/WbCD18)andtopointoutthewildboarLFA-1specificitiesthatmightconferresistancetoActinobacilluspleuropneumoniae-associatedpneumonia.
ResultsanddiscussionCharacterizationofWbCD11a-encodingcDNAanddeducedamino-acidsequenceTheWbCD11acDNAsequencecontainsanORFof3519bp[GenBank:EF585976]thatcodesfor1172aa(Fig.
1).
StartingfromtheN-terminalend,the1172aamatureWbCD11acontainsa23-residueputativeleaderpeptide(M1-S23),anextracellulardomainof1064residues(Y24-D1086),asinglehydrophobictransmembraneregionof24residues(M1087-Y1110)andashortcytoplasmictailof62residues(K1111-A1172)(Fig.
1).
SixN-linkedputativeglyc-osylationsites(Asn-Xaa-Ser/Thr)arefoundintheextra-cellulardomain(Fig.
1).
TheWbCD11apossesses22cysteineresidues,amongwhichoneislocatedinthecyto-plasmictail(Fig.
1).
Asubsetofintegrinαchains(α1,α2,α10,α11,αD,αE,αL,αMandαX),includingCD11a,con-tainsaI-domain(forInserteddomain,alsocalledαLI-domainorαLA-domain)thatishomologoustothefamilyofvonWillebrandFactor(vWF)A-typedomainsandtocartilagematrixprotein[33,34].
TheI-domainhasbeenassociatedwithligandbinding.
Itsthree-dimensionalstructureconsistsofafive-strandedparallelβ-sheetcoresurroundedonbothfacesbysevenα-helices(Fig.
1).
Ashortantiparallelstrandoccursononeedgeofthissheet[35].
TheI-domain(I149-D331)containsametalion-dependentadhesionsite(MIDAS)(residuesD160-S164,T229,D262)[35,36](Fig.
1).
TheI-domaincrystallisationhasdemonstratedthata"closed"(lowaffinity)andan"open"(highaffinity)formsexistandthatthemajorcon-formationalchangesduringtransitionfromtheclosedtoopenstatesincludearearrangementofthecation-coordi-natingresiduesintheMIDASsite,accompaniedbyasmallinwardmovementoftheα1helixandalargedown-wardshiftofthemobileC-terminalα7helix[37].
TheextracellulardomainofWbCD11acontainsseveninternalrepeats(FG-GAP)(G40-T89,S90-E147,S348-R398,A399-Q453,G455-D511,G513-F573,I576-P628)thatsurroundtheI-BMCVeterinaryResearch2007,3:27http://www.
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com/1746-6148/3/27Page3of12(pagenumbernotforcitationpurposes)domain(Fig.
1)[38,39].
ThedegreeofidentityishighestamongthethreeCOOH-terminalrepeats(18–31%)andtheircentralregion(D466-E474,D528-D536andD588-D596)issimilartotheEFhanddivalentcation-bindingmotifs(DCBM)oftroponinC,parvalbuminandgalactosebind-ingprotein[38](Fig.
1).
AllthecysteineresiduesandallbutoneN-glycosylationsitesarefoundoutsidetheI-regionanddivalentcationbindingmotifs(Fig.
1),con-sistentwiththehypothesisthattheseregionsmayundergoconformationalchangesimportantinligandbinding[38,40].
BetweentheFG-GAP7andthetrans-membranedomainstandsthethighdomain(M629-K766),thegenu(N767-C774),andtheCALFdomains(E775-L920,N921-D1086)[41].
ThecytoplasmicportionofWbCD11acontainsfourpotentialphosphorylationsitesandalsoaconserved"G1113FFKR"basicsequencenearthetrans-membraneregion(Fig.
1).
Theintegrinsbecomeconstitu-tivelyactivewhenthissequenceisdeleted.
The"G1113FFKR"motifthusnormallyfixestheintegrinsinaninactivestate[11,42].
Besidethecomplexmechanismsofaffinity/avidityregula-tionoftheintegrins,theexistenceofseveralisoformsissuedfromalternativesplicingcomplicatesthebiologicalunderstandingoftheseglycoproteins[43].
Previously,wehavecharacterisedtwodifferentformsofPoCD11aduetothepresenceofasupplementary"cag"codonthatcodesforaglutamine(Q)inposition744[Gen-Bank:DQ013284,GenBank:DQ013285][31].
Theaddi-tionofaGlnatthesamepositionwasalsoobservedinthehuman(Q746)[GenBank:NM_002209,Gen-Bank:AY892236],thesimian(Q746)[44],ovine(Q743)[45]andcaprine(Q743)[GenBank:AY773018,Gen-Bank:AY773019]CD11acDNAs(Fig.
2).
ThisadditionlocatedinthethighdomainoftheextracellularpartofCD11a,justabovethegenu,increasesthelengthofanα-helixinthePoCD11aaccordingtotheGORIVbioinfor-maticprogram.
Untilnow,itwasnotclearwhetherthisadditionrepresentedtwoallelesorwasgeneratedbyanalternativesplicing.
WehaverecentlyclonedandsequencedathirdPoCD11aformcharacterisedbyanThenucleotideanddeducedaminoacidsequencesofwildboarCD11acDNAFigure1ThenucleotideanddeducedaminoacidsequencesofwildboarCD11acDNA.
¤1:MNSCIIVMRLLLSGPFLFAAAWSYNLDVRHVQHFSFPLAGRHFGYRVLQVGNGVFF:551:ATGAACTCCTGCATCATCGTGATGAGGCTGCTGCTGTCCGGGCCTTTTCTCTTTGCCGCCGCCTGGAGCTACAACCTGGACGTGCGGCACGTACAACACTTCTCCTTCCCACTCGCCGGCAGGCACTTTGGGTACCGTGTCCTGCAGGTTGGAAATGGGGTTTTC:165¤¤#¤56:VGAPGEGNSMGSLYQCQPDNGQCMQVTPSGSNYTSKYLGMTLATDPTTNSLLACDD:110165:GTGGGAGCTCCAGGTGAGGGGAACAGCATGGGAAGCCTCTATCAGTGTCAGCCAGACAATGGACAATGCATGCAAGTCACACCAAGTGGTTCCAACTATACCTCCAAGTACTTGGGAATGACCTTGGCGACAGATCCCACAACGAACAGTCTTTTGGCCTGTGAC:330¤¤¤ββββ1MIDAS111:PGLSRTCDQNIYLSGLCYLFHQGLGGPVLQGRPGYQECIKGNVDLVFLFDGSNSLL:165331:CCTGGGCTGTCTCGGACATGTGATCAGAACATCTATCTAAGTGGCCTGTGTTACCTCTTCCACCAGGGCCTGGGGGGTCCTGTGCTGCAAGGGCGCCCTGGTTATCAGGAATGTATAAAGGGCAACGTGGACTTGGTATTTCTGTTTGATGGATCAAACAGCTTG:495(D)αααα1#ββββ2αααα2αααα3166:SPEEFRKIVDFMKDVMKKLSNTSYQFAAVQFSTTYRTEFTFLDYAKQKNPDVLLNN:220496:AGTCCCGAAGAATTTCGGAAAATCGTGGACTTCATGAAGGATGTGATGAAGAAACTCAGCAACACTTCCTACCAGTTTGCTGCTGTTCAATTTTCCACAACCTACCGTACAGAATTTACTTTCTTGGATTATGCTAAACAGAAGAACCCGGATGTTCTGCTGAAT:660MIDASαααα4ββββ3MIDASαααα5221:NVKHMRELTNTFGAIDYVASHVFQQKLGARPDATKVLIIITDGEATDGKDISSAKK:275661:AATGTCAAACACATGCGCGAGTTGACCAACACCTTTGGTGCCATCGACTATGTTGCGAGTCACGTGTTCCAGCAAAAGCTGGGGGCCCGTCCAGATGCCACAAAAGTGCTTATCATCATCACGGACGGGGAAGCCACCGACGGAAAGGACATCAGCTCCGCCAAG:825ββββ4αααα6ββββ5αααα7276:DIVRYIIGVGKHFKTTKSQETLHQFASKPVEEFVKILDTFEKLKDLFTELQKKIYY:330826:GACATCGTCCGCTACATCATTGGGGTCGGAAAGCATTTTAAGACCACGAAAAGTCAAGAAACACTTCACCAGTTTGCCTCAAAACCCGTGGAGGAGTTTGTAAAGATTCTGGACACGTTTGAGAAGCTGAAAGATCTATTCACCGAGCTTCAAAAAAAGATCTAT:990331:DIEGTSKQDLTSFDMELSSSGISADLSKGHGIVGAVGAEDWAGGFLDLKADLQGD:385991:GACATTGAGGGCACAAGCAAACAGGACCTGACGTCCTTCGACATGGAGCTGTCCTCCAGCGGGATCAGCGCGGACCTCAGCAAGGGCCACGGCATCGTAGGGGCTGTCGGAGCCGAGGACTGGGCTGGGGGCTTCCTCGACCTGAAGGCAGACCTGCAGGGGGAT:1155386:TFVGNVPLTPEVRAGYLGYTVTWLPSRGTMSLLAAGAPRYQHVGRVLLFQKPEDK:4401156:ACATTTGTTGGGAATGTACCACTGACACCAGAAGTGAGGGCGGGATATTTGGGTTACACAGTGACCTGGCTGCCCTCCCGAGGAACCATGTCACTGCTGGCAGCTGGAGCCCCCCGGTACCAGCATGTGGGGCGGGTGCTGCTGTTCCAAAAACCAGAGGACAAA:1320¤DCBM1441:GPWRQIQKIDGTQIGSYFGGELCGVDLNQDGETELLLIGVPLFYGEQRGGRVFVY:4951321:GGACCCTGGAGGCAGATCCAGAAAATAGATGGGACCCAGATTGGCTCTTATTTTGGAGGCGAGCTGTGTGGTGTCGACCTGAACCAAGATGGGGAGACAGAGCTGCTACTGATTGGAGTCCCCCTGTTCTATGGGGAGCAGAGAGGAGGCCGGGTATTCGTTTAC:1485DCBM2496:QRKQLEFQMISELHGDPGYLLGRFGAAITALMDLNGDGLTDVAVGAPLEEQGAVY:5501486:CAGAGAAAACAGCTGGAGTTCCAAATGATCTCAGAGCTACATGGGGATCCCGGCTACCTCCTTGGGCGATTTGGAGCAGCCATCACTGCCCTGATGGACCTCAATGGGGACGGGCTGACGGACGTGGCTGTGGGAGCCCCTCTGGAGGAGCAGGGGGCTGTGTAC:1650DCBM3551:IFNGKPGGLSAWPSQRIAGAQMFSEIRWFGRSIHGVKDLGGDGLADVAVGAEGQV:6051651:ATCTTCAATGGGAAGCCCGGGGGGCTGAGCGCCTGGCCCAGTCAGCGGATAGCGGGCGCCCAGATGTTCTCAGAAATTCGATGGTTTGGACGCTCCATCCACGGGGTGAAGGACCTTGGAGGGGATGGCCTGGCAGATGTGGCGGTGGGGGCGGAGGGCCAGGTG:1815(E)¤¤606:IVLSSRPVVDINTTIDFSPAEIPMHEVECSYSTSNQKKEGVNLIVCFQVKSLISQ:6601816:ATCGTGCTGAGCTCCCGGCCTGTGGTGGACATCAACACGACCATCGACTTCTCCCCGGCCGAGATCCCAATGCACGAAGTCGAGTGCTCCTATTCAACCAGTAACCAGAAGAAGGAAGGCGTTAACCTCATAGTCTGCTTCCAGGTCAAGTCTCTCATCTCACAG:1980#¤¤661:FQGPLVANLTYTLQLDGHRTRSRGLFPGGTHELSGNTAVTSDQSCIRFKFHIPVC:7151981:TTCCAAGGGCCCCTGGTTGCCAACCTCACTTACACTCTGCAGCTGGATGGGCATCGGACCAGAAGCCGGGGCTTGTTCCCAGGAGGGACCCATGAACTCAGCGGGAACACAGCTGTCACCTCAGACCAGTCCTGCATCAGATTCAAGTTCCACATCCCGGTATGC:2145##¤716:VQDLISPINVSLNYSLWEEEATSRDQRADKDIQPILRPSAHSETREIPFEKNCGE:7702146:GTTCAAGACCTCATCTCGCCGATCAACGTCTCCCTAAATTACTCTCTCTGGGAGGAAGAAGCGACATCGAGGGACCAAAGGGCGGACAAGGACATCCAGCCCATCCTGAGACCCTCAGCACACTCAGAAACCAGGGAGATCCCTTTCGAGAAGAACTGTGGAGAG:2310¤771:DKKCEADLRVAFSPESSKVLRLTPSTSLAVRLTLQNVKEDAYWVHLSLSFPWGLS:8252311:GACAAGAAATGTGAGGCAGACCTGAGGGTGGCCTTCTCCCCTGAAAGCTCCAAAGTCCTGCGTCTGACCCCGTCCACCAGCCTCGCAGTGCGGCTGACACTGCAAAACGTGAAGGAAGACGCGTACTGGGTCCACCTCAGCCTGAGCTTCCCCTGGGGCCTCTCC:2475¤¤#826:FRKVEVLKPHSQMPVSCEELLEETSLQSRAVSCNVSSPIFKANSSVDIQVMFDTL:8802476:TTCCGCAAAGTGGAGGTGCTCAAGCCTCACAGCCAGATGCCTGTGAGCTGCGAGGAGCTTCTGGAGGAGACCAGTCTTCAGAGCAGAGCCGTCTCCTGCAACGTGAGCTCTCCCATCTTCAAAGCAAACAGCTCGGTTGATATCCAGGTGATGTTTGATACGCTA:2640¤881:SNSSWEDLVELKADVRCNNEDTGHLIDNWAATSIPVLYPLNILTKDQENSTLYIS:9352641:TCCAACAGCTCCTGGGAGGACCTTGTCGAGCTGAAGGCTGATGTGCGCTGCAACAACGAGGACACAGGCCACCTGATTGACAACTGGGCTGCCACCAGCATCCCGGTCCTGTACCCCCTCAACATCCTCACCAAGGACCAGGAAAACTCCACGCTGTATATCAGT:2805936:FTPKGPKTHHVKHSYQVKIQPSVYDHNMPALEALVGVPQPHPKGPITHKWSVQME:9902806:TTCACCCCCAAAGGTCCCAAGACCCACCATGTCAAGCACAGCTACCAGGTGAAGATCCAGCCTTCTGTCTATGACCACAACATGCCTGCCCTGGAGGCCTTGGTTGGGGTACCACAGCCCCACCCCAAGGGGCCCATCACACACAAGTGGAGCGTGCAGATGGAG:2970¤¤¤991:PPVTCHQEEVESLPSVAEPLCQLGAQFRCPVVFKQEILVQVIGTVELVGDIEASS:10452971:CCTCCTGTCACCTGCCACCAGGAGGAGGTGGAGAGTCTGCCCAGTGTGGCTGAGCCGCTGTGTCAGCTTGGAGCCCAGTTCCGCTGCCCAGTTGTCTTCAAGCAGGAGATCCTCGTCCAAGTGATCGGGACAGTGGAGCTGGTGGGGGACATCGAGGCCTCCTCC:3135¤1046:VFSLCSSLAISFNSSKHFHLYGSNASLAQVNMKVDIIYEKDMLYLYVLSSIGGLLL:11003136:GTGTTCAGCCTCTGCAGCTCCCTCGCCATCTCCTTCAACAGCAGCAAGCACTTCCACCTCTATGGCAGCAACGCCTCCCTCGCCCAGGTCAACATGAAGGTGGACATTATATACGAGAAGGACATGCTCTATCTCTACGTGCTGAGCAGCATCGGGGGGCTGTTG::33001101:LLLLIFLVLYKVGFFKRNLKEQMEATEEASNDTPEEDSGRPASGEDARDPGCLEP:11553301:CTGCTGCTGCTGATTTTCTTAGTTCTGTACAAGGTTGGCTTCTTTAAACGGAACCTGAAGGAGCAGATGGAAGCGACTGAAGAGGCTTCAAATGACACCCCGGAAGAAGACTCTGGGAGGCCAGCATCTGGGGAAGACGCCAGGGATCCGGGCTGCCTGGAGCCT:34651156:LHNDNTQDAVGATEAQA*:11723466:CTCCACAACGACAACACCCAGGACGCAGTGGGAGCGACTGAAGCCCAGGCCTGA:3519Boxesframingthesequencesrepresentthededuceddifferentconstitutivepartsoftheprotein:theputativeleaderpeptide(),thesevenrepeatsthatsurroundtheαI-domain()andthecentraldivalentcation-bindingmotifsofthethreeCOOH-terminalrepeats(),theαI-domain()withitsMIDAS(),thethighdomain(),thegenu(),theCALF-1()andCALF-2()domains,thetransmembraneregion()andthehighlyconserved"GFFKR"motif().
Theimportant"I149KGN"motif,P215,E333,K1120areframedinblackboxes.
Cysteineresidues(¤),potentialN-glycosylationsites(#),potentialcytoplasmic-tailphosphorylationsites(+)andallelesE168D(D)andD621E(E)aremarkedatthetopofthesequences.
SequencedatahavebeendepositedatGenBankunderaccessionno.
EF585976.
BMCVeterinaryResearch2007,3:27http://www.
biomedcentral.
com/1746-6148/3/27Page4of12(pagenumbernotforcitationpurposes)insertionof27aminoacidsatthesameposition(P744EPLQLSSTSSAASATLSRLPLLCAQQ770)[Gen-Bank:DQ474234]whichispredictedtolengthentheα-helixfurther.
Thenucleotidicsequenceofthisinsertioncorrespondstothatofthe3'endoftheadjacentbovineandhumanintron18(79%and70%ofidentityrespec-tively),suggestingthattheinsertionoftheglutamineorofthe27aminoacids-longstretchinposition744ofthethighdomaincomesfromanalternativesplicingratherthanfromdifferentalleles.
AlthoughthesetwoinsertionswerenotobservedintheWbCD11ayetandbecauseofthebetween-speciesconservationofthispotentialalternativesplicingsite,wehypothesizethatitcanhaveabiologicalimportanceforthematureCD11a,forexample,inregu-latingtheligandbindingandsignalingactivity.
WbCD11acomparisonamongspeciesOverall,thegeneralorganizationofwildboar(Susscrofa),porcine(Susscrofadomestica)[31],bovine(Bostaurus)[46],ovine(Ovisaries)[45],caprine(Caprahircus)[47],human[38],simian(Pantroglodytes)[44],canine(Canisfamiliaris)[GenBank:XM_547024],rat(Rattusnorvegicus)[GenBank:NP_001029170],andmurine(Musmusculus)[48]CD11aproteinsisquitesimilar(Fig.
2).
ComparisonbetweenmatureWbCD11asequenceanditsporcine,bovine,ovine,caprine,human,simian,canine,ratandmurinecounterpartsshowsrespectivelyoverall99%,77%,77%,77%,76%,76%,76%,70%and69%identity,and99%,87%,86%,86%,86%,86%,85%,81%,and80%similarity(BLOSUM62table)(Table1).
Thehighestidentityisfoundforthe"G1113FFKR"motif,thegenu,theMIDASmotifandthetransmembraneregionandthelow-estforthecytoplasmictailandtheputativesignalpeptide(Table1).
AlthoughDCBM3presentsaweakidentity,itssimilarityscoreishigh.
The"G1113FFKR"sequenceishighlyconservedwhichisconsistentwiththestabilizingroleofthismotifforthealpha/betacomplex,possiblybecauseofitsdirectinvolvementinheterodimerforma-Comparisonofthewildboar(Wb-),porcine(Po-),bovine(Bo-),ovine(Ov-),caprine(Cap-),human(Hu-),simian(Si-),canine(Can-),rat(Ra-)andmurine(Mu-)αLsubunitsaminoacidssequencesFigure2Comparisonofthewildboar(Wb-),porcine(Po-),bovine(Bo-),ovine(Ov-),caprine(Cap-),human(Hu-),simian(Si-),canine(Can-),rat(Ra-)andmurine(Mu-)αLsubunitsaminoacidssequences.
WbCD11a:-MNSCIIVMRL-LLSGPFLFAAAWSYNLDVRHVQHFSFPLAGRHFGYRVLQVGNGVFVGAPGEGNSMGSLYQCQPDNGQCMQVTPSGSNYTSKYLGMTLATDPTTNSLLACDPGLSRTCDQNIYLSGLCYLFHQGLGGPVLQGRPGYQEC:148PoCD11a:-MNSCIIVMRL-LLSGPFLFAAAWSYNLDVRHVQHFSFPLAGRHFGYRVLQVGNGVFVGAPGEGNSMGSLYQCQPDNGQCMQVTPSGSNYTSKYLGMTLATDPTTNSLLACDPGLSRTCDQNIYLSGLCYLFHQGLGGPVLQGRPGYQEC:148BoCD11a:-MNSCIIVLRL-LLSGPFVFAPAWSYNLDVRHVQNFSFPLAGRHFGYRVLQVGNGVVVGAPSEGNSMGNLYQCQPETGDCLPVTLS-SNYTSKYLGMTLATDPTSDNLLACDPGLSRTCDQNIYLSGLCYLIHENLRGPVLQGHPGYQEC:147OvCD11a:-MNSYTIVMRF-LLSGPFVFAPAWSYNLDVRHVQSFSFPLAGRHFGYRVLQVGNRVVVGAPNEGNRMGNLYQCQPETGDCLPVTLS-SSYTSKYLGMTLATDPTSGNLLACDPGLSRTCDQNIYLSGLCYLIHENLRGPVLQGYPGYQEC:147CapCD11a:-MNSCTIVMRF-LLSGPFVFAPAWSYNLDVRHVQNFSFPLAGRHFGYRVLQVGNRVVVGAPNEGNRMGNLYQCQPETGDCLPVTLS-SSYTSKYLGMTLATDPTSGNLLACDPGLSRTCDQNIYLSGLCYLIHENLRGPVLQGHPGYQEC:147HuCD11a:MKDSCITVMAMALLSGFFFFAPASSYNLDVRGARSFSPPRAGRHFGYRVLQVGNGVIVGAPGEGNSTGSLYQCQSGTGHCLPVTLRGSNYTSKYLGMTLATDPTDGSILACDPGLSRTCDQNTYLSGLCYLFRQNLQGPMLQGRPGFQEC:150SiCD11a:MKDSCITVMAMALLSGFFFFAPASSYNLDVRGARSFSPPRAGRHFGYRVLQVGNGVIVGAPGEGNSTGSLYQCQSGTGHCLPVTLRGSNYTSKYLGMTLATDPTDGSILACDPGLSRTCDQNTYLSGLCYLFRQNLQGPMLQGRPGFQEC:150CanCD11a:-MNSYIIITRL-LLSGPSLFALASGYNLEVRHVQNFSFPHAGRHFGYRVLQVGDRVVVGAPGEGNSTGNLYQCQPGTGHCLPVSLSGSNYTSKYLGMTLATDFISGKLLACDPGLSRTCDQNVYLSGLCYLFHQNLRGVVLQGRPGYQEC:148RaCD11a:-MSFRIPGPRLLLL-GLQLFVNAWSYNLDTRHAQSF-LTQAGRHFGYQVLQFGDGVVVGAPGEGNSTGNLYHCHPSSGSCQPVHL-GSNHTSKYLGMTMVTDAAKGSLLACDPGLSRTCDQNPYLSGLCYLFSQSLGKPILQNRPAYQEC:146MuCD11a:-MSFRIAGPRLLLL-GLQLFAKAWSYNLDTRPTQSF-LAQAGRHFGYQVLQIEDGVVVGAPGEGDNTGGLYHCRTSSEFCQPVSLHGSNHTSKYLGMTLATDAAKGSLLACDPGLSRTCDQNTYLSGLCYLFPQSLEGPMLQNRPAYQEC:147WbCD11a:IKGNVDLVFLFDGSNSLSPDEFRKIVDFMKDVMKKLSNTSYQFAAVQFSTTYRTEFTFLDYAKQ-KNPDVLLNNVKHMRELTNTFGAIDYVASHVFQQKLGARPDATKVLIIITDGEATDGKDISSAKDIVRYIIGVGKHFKTTKSQETL:297PoCD11a:IKGNVDLVFLFDGSNSLSPEEFRKIVDFMKDVMKKLSNTSYQFAAVQFSTTYRTEFTFLDYAKQ-KNPDVLLNNVKHMRELTNTFGAIDYVASHVFQQKLGARPDATKVLIIITDGEATDGKDISSAKDIVRYIIGVGKHFKTTKSQETL:297BoCD11a:IKGNVDLVFLFDGSMSLQQDEFEKIVDFMKDVMKKLSNSSYQFAAVQFSTYFRTEFTFLDYIRQ-KDPDALLAGVKHMRLLTNTFGAINYVAKEVFRPDLGARPDATKVLIIITDGEATDEHNIDAAKDIIRYIIGIGKNFKTKESQEAL:296OvCD11a:IKGNVDLVFLFDGSMSLQQDEFEKIVDFMKDVMKKLSNSSYQFAAVQFSTDFKTEFTFLDYNRQ-KDPDALLAGVKHMRLLTNTFGAINYVAKEVFRQELGARPDATKVLIIITDGEATDEQNIDAAKDIIRYIIGIGKNFKTKESQEAL:296CapCD11a:IKGNVDLVFLFDGSMSLQQDEFGKIVDFMKDVMKKLSNSSYQFAAVQFSTDFKTEFTFLDYNRQ-KDPDALLAGVKHMRLLTNTFGAINYVAKEVFRQELGARPDATKVLIIITDGEATDEQNIDAAKDIIRYIIGIGKNFKTKESQEAL:296HuCD11a:IKGNVDLVFLFDGSMSLQPDEFQKILDFMKDVMKKLSNTSYQFAAVQFSTSYKTEFDFSDYVKR-KDPDALLKHVKHMLLLTNTFGAINYVATEVFREELGARPDATKVLIIITDGEATDSGNIDAAKDIIRYIIGIGKHFQTKESQETL:299SiCD11a:IKGNVDLVFLFDGSMSLQPDEFQKILDFMKDVMKKLSNTSYQFAAVQFSTSYKTEFDFSDYVKR-KDPDALLKHVKHMLLLTNTFGAINYVATEVFREELGARPDATKVLIIITDGEATDSGNIDAAKDIIRYIIGIGKHFQTKESQETL:299CanCD11a:IKGNVDLVFLFDGSMSLQQDEFQKILDFMKDVMKKLSNSSYQFAAVQFSTDCKTEFNFLDYVKV-KNPDVLLGKVVHMRLLTNTFGAINYVAREVFRQDLGARPDATKVLIIITDGEATDRANIDSAKDIIRYIIGIGKHFATKRSQETL:297RaCD11a:MKGNVDLVFLFDGSQSLDKKDFEKIVDFMKDVMRKLSNTSYQFAAVQFSTECKTEFTFLDYIKLNKNPDVLLGNVTPMFLLTNTFRAINYVVTHVFKEESGARPDATKVLVIITDGEASDNGNIDAAQDITRYIIGIGKHFSTTQKQEKL:296MuCD11a:MKGKVDLVFLFDGSQSLDRKDFEKILEFMKDVMRKLSNTSYQFAAVQFSTDCRTEFTFLDYVKQNKNPDVLLGSVQPMFLLTNTFRAINYVVAHVFKEESGARPDATKVLVIITDGEASDKGNISAAHDITRYIIGIGKHFVSVQKQKTL:297#¤$#WbCD11a:HQFASKPVEEFVKILDTFEKLKDLFTELQKKIYDIEGTSKQDLTSFDMELSSSGISADLSKGHGIVGAVGAEDWAGGFLDLKADLQGDTFVGNVPLTPEVRAGYLGYTVTWLPSRGTMSLLAAGAPRYQHVGRVLLFQKPEDKGPWRQIQ:447PoCD11a:HQFASKPVEEFVKILDTFEKLKDLFTELQKKIYDIEGTSKQDLTSFDMELSSSGISADLSKGHGIVGAVGAEDWAGGFLDLKADLQGDTFVGNVPLTPEVRAGYLGYTVTWLPSRGTMSLLAAGAPRYQHVGRVLLFQKPEDKGPWRQIQ:447BoCD11a:HQFASKPVEEFVKILDTFEKLKDLFTELQKKIYVIEGTSKQDLTSFNMELSSSGISADLSEGHGVVGAVGAKDWAGGFLDLKADLKSSTFVGNEPLTVESRAGYLGYTVTWLPSRGTMSLLATGAPRYQHVGRVLLFQQPKRGGPWSQIQ:446OvCD11a:HQFASKPVEEFVKILDTFEKLKDLFTELQKKIYVIEGTSKQDLTSFNMELSSSGISADLSEGHGVVGAVGAKDWAGGFLDLKADLQSSTFVGNEQPTVESREGYLGYTVTWLPSRGMMSLLATGAPRYQHVGRVLLFQQPKRGGPWSQIQ:446CapCD11a:HQFASKPVEEFVKILDTFEKLKDLFTELQKKIYVIEGTSKQDLTSFNMELSSSGISADLSEGHGVVGAVGAKDWAGGFLDLKADLQSSTFVGNEQLTVESREGYLGYTVTWLPSRGTMSLLATGAPRYQHVGRVLLFQQPKRGGPWSQIQ:446HuCD11a:HKFASKPASEFVKILDTFEKLKDLFTELQKKIYVIEGTSKQDLTSFNMELSSSGISADLSRGHAVVGAVGAKDWAGGFLDLKADLQDDTFIGNEPLTPEVRAGYLGYTVTWLPSRQKTSLLASGAPRYQHMGRVLLFQEPQGGGHWSQVQ:449SiCD11a:HKFASKPASEFVKILDTFEKLKDLFTELQKKIYVIEGTSKQDLTSFNMELSSSGISADLSRGHAVVGAVGAKDWAGGFLDLKADLQDDTFIGNEPLTPEVRAGYLGYTVTWLPSREKTSLLASGAPRYQHVGRVLLFQEPQGGGHWSQVQ:449CanCD11a:HQFASKPAEEFVKILDTFEKLKDLFTELQKKIYVIEGTSKQDLTSFNMELSSSGLSADISQDRCIVGAVGAKDWAGGFLDLKADLQDDAFVGNEPLTPEARAGYLGYTVTWLYSRGLTSLLAAGAPRYQHVGRVVLFQESKDKEHWSQIQ:447RaCD11a:HIFASKPVEEFVKILDTFEKLKDLFTDLQRKIYAIEGTSKQDLASFNMELSSSGISADLSKGHAVVGAVGAKDWAGGFLDLHEDLEGATFVGQEPLTSDERGGYLGYTVAWLTSRSSRPLLAAGAPRYQHVGQVLLFQAPEAGGHWNQTQ:446MuCD11a:HIFASEPVEEFVKILDTFEKLKDLFTDLQRRIYAIEGTNRQDLTSFNMELSSSGISADLSKGHAVVGAVGAKDWAGGFLDLREDLQGATFVGQEPLTSDVRGGYLGYTVAWMTSRSSRPLLAAGAPRYQHVGQVLLFQAPEAGGRWNQTQ:447$¤WbCD11a:KIDGTQIGSYFGGELCGVDLNQDGETELLLIGVPLFYGEQRGGRVFVYQRKQLEFQMISELHGDPGYLLGRFGAAITALMDLNGDGLTDVAVGAPLEEQGAVYIFNGKPGGLSAWPSQRIAGAQMFSEIRWFGRSIHGVKDLGGDGLADV:597PoCD11a:KIDGTQIGSYFGGELCGVDLNQDGETELLLIGVPLFYGEQRGGRVFVYQRKQLEFQMISELHGDPGYLLGRFGAAITALMDLNGDGLTDVAVGAPLEEQGAVYIFNGKPGGLSAWPSQRIAGAQMFSEIRWFGRSIHGVKDLGGDGLADV:597BoCD11a:EIDGIQIGSYFGGELCGVDVDRDGETELLLIAAPLYYGEQRGGRVFIYQKIQLEFQMVSELQGETGYPLGRFGAAIAALTDINGDELTDVAVGAPLEEQGAVYIFNGQQGGLSPRPSQRIEGTQMFSGIQWFGRSIHGVKDLGGDGLADV:596OvCD11a:KIDGIQVGSYFGGELCGVDVDRDGETELLLIAAPLYYGEQRGGRVFIYQKIQLGFQMVSELQGETGYPLGRFGAAIAALTDINGDELTDVAVGAPLEDQGAVYIFNGQQGGLSPRPSQRIEGTQMFSGIQWFGRSIHGVKDLGGDGLADV:596CapCD11a:KIDGIQVGSYFGGELCGVDVDRDGETELLLIAAPLYYGEQRGGRVFIYQKIQLGFQMVSELQGETGYPLGRFGAAIAALTDINGDELTDVAVGAPLEEQGAVYIFNGQQGGLSPRPSQRIEGTQMFSGIQWFGRSIHGVKDLGGDGLADV:596HuCD11a:TIHGTQIGSYFGGELCGVDVDQDGETELLLIGAPLFYGEQRGGRVFIYQRRQLGFEEVSELQGDPGYPLGRFGEAITALTDINGDGLVDVAVGAPLEEQGAVYIFNGRHGGLSPQPSQRIEGTQVLSGIQWFGRSIHGVKDLEGDGLADV:599SiCD11a:TIHGTQIGSYFGGELCGVDMDQDGETELLLIGAPLFYGEQRGGRVFIYQRRQLGFEEVSELQGDPGYPLGQFGEAITALTDINGDGLVDVAVGAPLEEQGAVYIFNGRHGGLSPQPSQRIEGTQVLSGIQWFGRSIHGVKDLEGDGLADV:599CanCD11a:KIDGNQIGSYFGGELCGVDIDQDGETELLLIGAPLFYGEQRGGRVFIYQRKQLGFEAVSELQGEPGYPLGRFGAAITALTDINGDGLVDVAVGAPLEEKGAVYIFNGQHGGLSPQPSQRIEGIQVFSGIQWFGRSIHGVKDLGGDGLTDV:597RaCD11a:KIEGTQIGSYFGGELCSVDLHQDGETDLLLIGAPLFYGEQRGGRVSVYQRRRSLFEMVSELQGDPGYPLGRFGAAISALTDINGDGLTDVAVGAPLEEQGAVYIFNGKPGGFSSQSSQRIPGTQVSPGVRWFGRSIHGVKDLGGDRLADV:596MuCD11a:KIEGTQIGSYFGGELCSVDLDQDGEAELLLIGAPLFFGEQRGGRVFTYQRRQSLFEMVSELQGDPGYPLGRFGAAITALTDINGDRLTDVAVGAPLEEQGAVYIFNGKPGGLSPQPSQRIQGAQVFPGIRWFGRSIHGVKDLGGDRLADV:597¤WbCD11a:AVGAEGQVIVLSSRPVVDINTTIEFSPAEIPMHEVECSYSTSNQKKEGVNLIVCFQVKSLISQFQGPLVANLTYTLQLDGHRTRSRGLFPGGTHELSGNTAVTSDQSCIRFKFHIPVCVQDLISPINVSLNYSLWEEEATSRDQRA-DKD:746PoCD11a:AVGAEGQVIVLSSRPVVDINTTIDFSPAEIPMHEVECSYSTSNQKKEGVNLIVCFQVKSLISQFQGPLVANLTYTLQLDGHRTRSRGLFPGGTHELSGNTAVTSDQSCIRFKFHIPVCVQDLISPINVSLNYSLWEEEGTSRDQRAQDKD:747BoCD11a:AVGAEGQVIVLSSRPVVDIITSVSFSPAEIPVHEVECSYSTSNQKKEGVNLTVCFQVKSLISTFQGHLVANLTYTLQLDGHRTRSRGLFPGGKHKLIGNTAVTPVKSCFVFWFHFPICIQDLISPINVSLSYSLWEEEGTPRDPRALDRD:746OvCD11a:AVGAEGQVIVLSSRPVVDIITSMSFSPAEIPVREVECSYSTSNQEKEGVNITVCFQVKSLISTFEGHLVANLTYTLQLDGHRTRSRGLFPGGKHELNGNTAVTSVKSCFMFWFHFPICIQDLISPINVSLSYSLWEEEGTPRDPRAQGRD:746CapCD11a:AVGAEGQVIVLSSRPVVDIITSVSFSPAEIPVREVECSYSTSNQEKEGVNITVCFQVKSLISTFQGHLVANLTYTLQLDGHRTRSRGLFPGGKHELNGNTAVTSVKSCFMFWFHFPICIQDLISPINVSLSYSLWEEEGTPRDPRAQGRD:746HuCD11a:AVGAESQMIVLSSRPVVDMVTLMSFSPAEIPVHEVECSYSTSNKMKEGVNITICFQIKSLIPQFQGRLVANLTYTLQLDGHRTRRRGLFPGGRHELRRNIAVTTSMSCTDFSFHFPVCVQDLISPINVSLNFSLWEEEGTPRDQRAQGKD:749SiCD11a:AVGAESQMIVLSSRPVVDVVTLMSFSPAEIPVHEVECSYSTSNKMKEGVNITICFQIKSLIPQFQGRLVANLTYTLQLDGHRTRRRGLFPGGRHELRRNIAVTTSMSCTDFSFHFPVCVQDLISPINVSLNFSLWEEEGTPRDQRAQGKD:749CanCD11a:AVGAEGQVIVLSSRPVVDILTHISFSPAEIPVHEVECSHSTSNKKKEGVNITVCFQVKPLTLQFRGRLVANLIYTLQLDGHRTRSRGLFPGGRQELSGNIAVTRDWSCIKLWFHFPVCIQDLISPINVSLNFSLWEEEETPRNKRALDRD:747RaCD11a:VVGAEGQVIVLSSRPVVDIVTELSFSPDEIPVHEVECSYSASQEQKEGVKLKVCFQIRPLTSQFQGRLLANLSYTLQLDGHRTRSRGLFPGGSRELGGNTFVTPDRSCMDFHFHFPICIQDLISPINVSLNFSLLEEEGSPRDQKG-GRD:745MuCD11a:VVGAEGRVVVLSSRPVVDVVTELSFSPEEIPVHEVECSYSAREEQKHGVKLKACFRIKPLTPQFQGRLLANLSYTLQLDGHRMRSRGLFPDGSHELSGNTSITPDKSCLDFHFHFPICIQDLISPINVSLNFSLLEEEGTPRDQKG--RA:745WbCD11a:IQPILRPSAHSETREIPFEKNCGEDKKCEADLRVAFSPESSKVLRLTPSTSLAVRLTLQNVKEDAYWVHLSLSFPWGLSFRKVEVLKPHSQMPVSCEELLEETSLQSRAVSCNVSSPIFKANSSVDIQVMFDTLSNSSWEDLVELKADVR:896PoCD11a:IQPILRPSAHSETREIPFEKNCGEDKKCEADLRVAFSPESSKVLRLTPSTSLAVRLTLQNVKEDAYWVHLSLSFPWGLSFRKVEVLKPHSQMPVSCEELLEETSLQSRAVSCNVSSPIFKANSSVDIQVMFDTLSNSSWEDLVELKADVR:897BoCD11a:IPPILKPSPHLETKEIPFEKNCGEDKNCEADLKLAFSDMRSKILRLTPSASLSVRLTLRNTAEDAYWVQVTLSFPQGLSFRKVEILKPHSHVPVGCEELPEEAVVHSRALSCNVSSPIFGEDSMVDIQVMFNTLQKGSWGDFIELQANVS:896OvCD11a:IQPILKPSPHLETKEIPFEKNCGEDKTCEADLKLAFSDMRSKILHLTPSASLSVRLTLRNTAEDAYWVQVTLSFPQGLSFRKVEALKPHSHVPVGCEELPEEAILQSRALSCNVSSPIFGADSMVDIQVMFNTLQKGSWGDLIELHANVS:896CapCD11a:IPPILKPSPHLETKEIPFEKNCGEDKTCEADLKLAFSDMRSKILHLTPSASLSVRLTLRNTAEDAYWVQVTLSFPQGLSFRKVEALKPHSHVPVGCEELPEEAILQSRALSCNVSSPIFGADSMVDIQVMFNTLQKGSWGDLIKLHANVS:896HuCD11a:IPPILRPSLHSETWEIPFEKNCGEDKKCEANLRVSFSPARSRALRLTAFASLSVELSLSNLEEDAYWVQLDLHFPPGLSFRKVEMLKPHSQIPVSCEELPEESRLLSRALSCNVSSPIFKAGHSVALQMMFNTLVNSSWGDSVELHANVT:899SiCD11a:IPPILRPSLHSETWEIPFEKNCGEDKKCEANLRVSFSPARSRALRLTAFASLSVELSLSNLEEDAYWVQLDLHFPPGLSFRKVEMLKPHSQIPVSCEELPEESRLLSRALSCNVSSPIFKAGHSVALQMMFNTLVNSSWGDSVELRANVT:899CanCD11a:IRPILRPLPHVANTEIPFEKNCGEDKKCEADLGLSFSPAGSRVLRLIPSASLSVELILSNLREDAYSVHLNLSFPQGLSFRKVEMLKPHSQIPVSCEELPEVAKLMNRIVSCNVSSPIFRAGSVVAIQVMFHMLLNSSWGDSVELHANVS:897RaCD11a:MQPILRPSIHAVTKEIPFEKNCGEDKKCEADLALS-PPARSGVLRLMSSASLAVEWTLRNLGEDAYWVRLDLDFPRGLSFRKVEMLQPHSQIPVSCEELTEESSLLTKTLKCNVSSPIFKAGKQMTLQVMFNTLLNSSWGDFVELNGTVH:894MuCD11a:MQPILRPSIHTVTKEIPFEKNCGEDKKCEANLTLS-SPARSGPLRLMSSASLAVEWTLSNSGEDAYWVRLDLDFPRGLSFRKVEMLQPHSRMPVSCEELTEGSSLLTKTLKCNVSSPIFKAGQEVSLQVMFNTLLNSSWEDFVELNGTVH:894WbCD11a:CNNEDTGHLIDNWAATSIPVLYPLNILTKDQENSTLYISFTPKGPKTHHVKHSYQVKIQPSVYDHNMPALEALVGVPQPHPKGPITHKWSVQMEPPVTCHQEEVESLPSVAEPLCQLGAQFRCPVVFKQEILVQVIGTVELVGDIEASSV:1046PoCD11a:CNNEDTGHLIDNWAATSIPVLYPLNILTKDQENSTLYISFTPKGPKTHHVKHSYQVKIQPSVYDHNMPALEALVGVPQPHPKGPITHKWSVQMEPPVTCHQEEVESLPSVAEPLCQLGAQFRCPVVFKQEILVQVIGTVELVGDIEASSV:1047BoCD11a:CNNEDSSLLEDNSATTSIPVMYPINVLTKDQENSTLYISFTPKSPKIHHVKHIYQVRIQPSNYD-NMPPLEALVRVPRVHSEGLITHKWSIQMEPPVNCSPRNLESPSDEAES-CSFGTEFRCPIDFRQEILVQVNGMVELRGTIKASSM:1044OvCD11a:CDNENSSLLEDNSATASIPVMYPINILTKDQENSTLYISFTPKSPKIHHVKHIYQVRIQPSNYD-NVPPLEALVRVPRVHSEGLITHRWSIQMEPPVNCSPRNLESPSDVAEP-CSFGTEFRCPIDFRQEILVQVNGTVELRGTIKASSM:1044CapCD11a:CDNENSSLLEDNSATASIPVMYPINILTKDQENSTLYINFTPKSPKIHHVKHIYQVRIQPSNYD-NVPPLEALVRVPQVHSEELITHRWSIQMEPPVNCSSRNLESPSDVAEP-GSFGTEFRCPIDFRQEILVQVNGTVELRGTIKASSM:1044HuCD11a:CNNEDSDLLEDNSATTIIPILYPINILIQDQEDSTLYVSFTPKGPKIHQVKHMYQVRIQPSIHDHNIPTLEAVVGVPQPPSEGPITHQWSVQMEPPVPCHYEDLERLPDAAEP-CLPGALFRCPVVFRQEILVQVIGTLELVGEIEASSM:1048SiCD11a:CNNEDSDLLEDNSATTIIPILYPINILIQDQENSTLYVSFTPKGPKIHQVKHMYQVRIQPSIHDHNIPTLEAVVGVPQPPSQGPITHQWSVQMEPPVPCHYEDLERLPDAAEP-CLPGALLRCPVVFRQEILVQVIGTLELVGEIEASSM:1048CanCD11a:CHNEDSGLLENNLATTTIPVLYPINILTEDKENSTLYVGFTPKGPKMYFVEHFYQVRIQPSIYDHKVPALEALIGVPVSHSEGPITHKWNVEMEPPVTCYREDLDRLPSVAEP-CLPGARFHCPFVYKEEIFVKVIGTIELVEKIEASSM:1046RaCD11a:CENENSSLGKDNSATTRIPVLYPVNILTEDQENSTLYISFTPKGPKTQQVQHIYKVRIQPSAYDHNMPALEALVEVPQPHSEGPIAHTWTVHTDPLVTCHCEDLKKPTREAE-PCLPGVQFRCPIVFRQETFIRVAGTVELSEKIKASST:1043MuCD11a:CENENSSLQEDNSAATHIPVLYPVNILTKEQENSTLYISFTPKGPKTQQVQHVYQVRIQPSAYDHNMPTLEALVGVPRPHSEDLITYTWSVQTDPLVTCHSEDLKRPSSEAEQPCLPGVQFRCPIVFRWEILIQVTGTVELSKEIKASST:1044WbCD11a:FSLCSSLAISFNSSKHFHLYGSNASLAQVNMKVDIIYEKDMLYLYVLSSIGGLLLLLLIFLVLYKVGFFKRNLKEQMEATEEASNDTPEEDSGRPAS-GEDARDPGCLEPLHNDNTQDAVGATEAQA:1172PoCD11a:FSLCSSLAISFNSSKHFHLYGSNASLAQVNMKVDIIYEKDMLYLYVLSSIGGLLLLLLIFLVLYKVGFFKRNLKEQMEATEEASNDTPEEDSGRPAS-GEDARDPGCLEPLHNDNTQDAVGATEAQA:1173BoCD11a:LSLCSSLAISFNSSKHFHLYGRNASMAQVVMKVDLVYEKEMLYLYVLSGIGGLLLLFLIFIALYKVGFFKRNLKEKMEANVDASSEIPGEDAGQPEL-EKECKDPGCLEPLQKTD-EDGSGGD----:1165OvCD11a:LSLCSSLSISFNSSKHFHLYGSNASMAQVVMKVDLVYEKKMLYLYVLSGIGGLLLLLLIFIALYKVGFFKRNLKEKMEANVDASSEIPGEDAGQPEL-EKEFKDPGCLEPLQKED-EDGSGGD----:1165CapCD11a:LSLSSSLSISFNSSKHFHLYGSNASMAQVVMKVDLVYEKKMLYLYVLSGIGGLLLLLLIFIALYKVGFFKRNLKEKMEANVDASSEIPAEDAGQPEL-EKEFKDPGCLEPLQKKD-EDESGGD----:1165HuCD11a:FSLCSSLSISFNSSKHFHLYGSNASLAQVVMKVDVVYEKQMLYLYVLSGIGGLLLLLLIFIVLYKVGFFKRNLKEKMEAGRGVPNGIPAEDSEQLAS-GQEAGDPGCLKPLHEKDSESGGGKD----:1170SiCD11a:FSLCSSLSISFNSSKHFHLYGSNASLAQVVMKVDVVYEKQMLYLYVLSGIGGLLLLLLIFIVLYKVGFFKRNLKEKMEAGRGVPNGIPAEDSEQLAS-GQEAGDPGCLKPLHEKDSESGGGKD----:1170CanCD11a:VSLCSSLSVSFNSIKHFHLYGSNASMAQVVMKVDIIYEKEMLHVYALSSIGGLLLLLLIFVALYKLGFFKRNLKEKMEAVEA-SNEIPKQDSEQPVS-KEEAGDPGCLEPLQEGEAQDEAVKD----:1167RaCD11a:LSLCSSLSVSFNSSKHFHLYGSKASKAQVLMKVDMIYEKEMLHLYVLSGIGGLLLLSLIFLALYKVGFFKRKLKEKMEADGGVPNGSPGEDADPLAVPGEETKDMGCLEPL-RESDKD-1160MuCD11a:LSLCSSLSVSFNSSKHFHLYGSKASEAQVLVKVDLIHEKEMLHVYVLSGIGGLVLLFLIFLALYKVGFFKRNLKEKMEADGGVPNGSPPEDTDPLAVPGEETKDMGCLEPSGRVTRTKA-1163Blackcolumnswithwhiteletterrepresentidentityamongthe10species.
Cysteineresidues(¤),potentialN-glycosylationsites(#)andpotentialcytoplasmic-tailphosphorylationsites(+)aremarkedatthebottomofthesequencesinredfor100%identityandinblueforless.
TheimportantP215,E333andK1120residuesaremarkedby()inredfor100%identityandinblueforlessandtheQ744residueisidentifiedby(=).
Thestripesabovethesequencesrepresentthededuceddifferentconstitutivepartsoftheprotein:thesignalpeptide(),theextracellulardomain(),thesevenrepeatsthatsurroundtheαI-domain(,)andthecentraldivalentcation-bindingmotifsofthethreeCOOH-terminalrepeats(),theαI-domain()anditsmetal-iondependentadhesionsite(),thethighdomain(),thegenu(),theCALF-1()andCALF-2()domains,thetransmembraneregion(),thecytoplasmictail()andthehighlyconserved"GFFKR"motif().
BMCVeterinaryResearch2007,3:27http://www.
biomedcentral.
com/1746-6148/3/27Page5of12(pagenumbernotforcitationpurposes)tion[42].
Thegenuseemstoplayakeyroleintheactiva-tionoftheLFA-1throughthedeploymentofthereceptor[41]anditsgreatconservationisthereforenotsurprising.
ThehighconservationoftheMIDASandtheputativecat-ionbindingmotifsisconsistentwithaninvolvementoftheseregionsinthefunctionalactivityoftheLFA-1αsub-unit,assuggestedbytherequirementofMg2+andCa2+forCD11a/CD18-dependentcellularinteractions[40]orTable1:Between-speciespercentidentitiesandsimilaritiesofCD11aconstitutiveblocksBlockWbvs.
Po(%)Wbvs.
Bo(%)Wbvs.
Ov(%)Wbvs.
Cap(%)Wbvs.
Hu(%)Wbvs.
Si(%)Wbvs.
Can(%)Wbvs.
Ra(%)Wbvs.
Mu(%)Overall99%(1)99%(2)77%87%77%86%77%86%76%86%76%86%76%85%70%81%69%80%Putativesignalpeptide100%100%86%95%78%86%78%86%82%91%56%68%69%73%50%54%54%58%Extracellulardomain99%99%79%87%78%87%78%87%78%87%78%87%77%87%72%82%71%82%FG-GAP1100%100%80%86%74%82%74%82%80%84%80%84%76%86%66%80%56%76%FG-GAP2100%100%86%93%86%91%86%91%84%89%84%89%84%87%77%81%81%82%I-domain99%100%82%89%82%90%82%90%80%90%80%90%82%87%75%85%72%85%MIDAS100%100%85%85%85%85%85%85%85%85%85%85%85%85%85%85%85%85%FG-GAP3100%100%82%90%80%86%82%88%86%94%86%94%78%86%76%84%80%88%FG-GAP4100%100%85%90%83%87%85%89%74%83%76%83%80%85%72%78%70%78%FG-GAP5100%100%78%93%76%91%76%91%83%95%83%95%83%95%80%91%80%90%DCBM1100%100%66%100%66%100%66%100%77%100%77%100%77%100%77%100%77%88%FG-GAP6100%100%82%86%81%86%82%86%79%86%77%86%81%87%81%89%87%91%DCBM2100%100%77%88%77%88%77%88%77%88%77%88%77%88%88%100%77%88%FG-GAP798%100%90%96%90%96%90%96%83%90%83%90%90%92%84%88%81%88%DCBM3100%100%100%100%100%100%100%100%88%88%100%100%88%88%88%88%88%88%Thighdomain98%98%80%87%79%87%79%87%79%84%79%84%74%84%71%84%67%81%Genu100%100%87%87%87%87%87%87%100%100%100%100%100%100%100%100%100%100%CALF1100%100%67%83%67%81%67%81%67%80%67%81%67%79%61%73%62%73%CALF2100%100%71%81%72%83%71%81%77%89%77%89%71%88%68%83%69%83%Transmembraneregion100%100%83%91%87%91%87%91%91%95%91%95%79%91%83%87%75%91%Cytoplasmictail100%100%50%69%50%70%50%69%51%64%51%64%54%67%42%52%44%52%"GFFKR"motif100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%Wb,Po,Bo,Ov,Cap,Hu,Si,Can,Ra,andMu:wildboar,porcine,bovine,ovine,caprine,human,simian,canine,rat,andmurineCD11a,respectively;FG-GAP:β-propellerrepeat;MIDAS:metal-iondependentadhesionsite;DCBM:divalent-cationbindingmotif;vs:versus.
(1)identity;(2)similarity(BLOSUM62table).
BMCVeterinaryResearch2007,3:27http://www.
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com/1746-6148/3/27Page6of12(pagenumbernotforcitationpurposes)bindingtopurifiedICAM-1[49,50].
Thetransmembraneregionalsoshowsahighdegreeofconservation,probablyduetosharedphysicochemicalandfunctionalcon-straints.
Indeed,residueslyinginthemembranefirsthavetopossessahydrophobiccharactertowarrantliposolubil-ity,whichisconfirmedbythepresenceofmanyleucineresidues(Fig.
2).
Secondly,bidirectionalintegrinsignal-ling(inside-outandoutside-in)isaccomplishedbytrans-missionofinformationacrosstheplasmamembrane[51].
Bycontrast,thelowconservationoftheCOOH-ter-minalpartofthecytoplasmictailsuggeststhatitisnotrequiredtoguaranteeadequatefunctioningofLFA-1.
ThisisinagreementwiththeobservationthattruncationoftheLFA-1αsubunitcytoplasmicdomainhasnoeffectonbindingtoICAM-1,whereasbindingismarkedlydimin-ishedbyβsubunitcytoplasmicdomaintruncation[52].
The"I149KGN"motifknowntoparticipateinthebindingtoICAM-3[53]showsahighdegreeofconservation(Fig.
2).
TheaminoacidP215,participatingtothebindingtoICAM-1[36]ishighlyconservedtoo(Fig.
2).
ResidueE333,locatedinthelinkerfollowingtheIdomainwhichiscrit-icalforcommunicationwiththeβ2I-likedomain,rolling,integrinextensionandactivationbyMn2+[16]islogicallystrictlyconservedtoo.
TheK1120residue,criticalforRap1-dependentLFA-1activationandaffinityup-regulation[5]isalsostronglyconserved(Fig.
2).
EverycysteineresidueinthematureWbCD11aispresentatthesamelocationinbovine,ovine,human,simian,andmurineCD11a,whichisconsistentwitharoleinmaintainingtheglobalstruc-tureoftheprotein.
Finally,ofsixpotentialAsn-glycosyla-tionsitesinWbCD11a,theonespresentataminoacids186and724arestrictlyconserved(Fig.
2).
Inaddition,althoughWbCD11asequenceswereobtainedfromonlyfourwildboars,oneofthemwasheterozygous.
Bothalle-lesdifferedfromthosefoundinpigsbyaG736Asubstitu-tion.
Onealleledisplayed2additionalsubstitutionscomparedtopigs:E168D(intheI-domain)andD621E(intheFG-GAP7,Fig.
1).
AccordingtotheBLOSUM62table,thesesubstitutionsaretheoreticallypredictedtohaveaweakimpactonthegeneralstructureofCD11a.
ThenucleotideanddeducedaminoacidsequencesofwildboarCD18cDNAFigure3ThenucleotideanddeducedaminoacidsequencesofwildboarCD18cDNA.
1:MLCRCSPLLLLVGLLTLRSALSQECAKYKVSTCRDCIESGPGCAWCQKLNFSGQGG:551:ATGCTGTGCCGGTGCTCCCCGCTGCTCCTCCTGGTGGGCCTGCTCACCCTCCGGTCCGCCCTCTCCCAGGAGTGTGCCAAGTACAAGGTCAGCACCTGCCGGGACTGCATTGAGTCGGGACCCGGCTGTGCCTGGTGCCAGAAGCTGAACTTCTCTGGGCAAGGG:165¤¤56:EPDSVRCDTREQLLAKGCVADDIVDPRSLAETQEDQAGGQKQLSPQKVTLYLRPGG:110166:GAGCCCGACTCCGTCCGCTGTGACACGCGGGAGCAGCTGCTTGCAAAGGGCTGTGTCGCGGATGACATCGTGGATCCCAGGAGCCTGGCCGAGACCCAGGAGGACCAGGCAGGGGGCCAGAAGCAGCTGTCCCCACAGAAAGTGACACTCTACCTGAGACCAGGT:330#β1β1β1β1MIDASADMIDASα1α1α1α1α1α1α1α1'111:QAATFNVTFRRAKGYPIDLYYLMDLSYSMLDDLINVKKLGGDLLRALNEITESGRR:165331:CAGGCGGCCACGTTCAACGTGACCTTCCGGCGCGCCAAGGGCTACCCCATCGACCTGTACTACCTGATGGACCTGTCCTACTCCATGCTCGACGACCTCATCAACGTGAAGAAGCTGGGGGGCGACCTGCTCAGGGCTCTCAACGAGATCACCGAGTCTGGCCGC:495β2β2β2β2LIMBS¤¤β3β3β3β3α2α2α2α2166:IGFGSFVDKTVLPFVNTHPEKLRNPCPNKEKECQAPFAFRHVLKLTDNSNQFQTEE:220496:ATCGGCTTTGGGTCTTTCGTGGACAAGACGGTGCTTCCCTTCGTCAACACGCACCCCGAGAAGCTGCGGAACCCCTGCCCCAACAAAGAGAAGGAGTGCCAGGCCCCGTTCGCCTTCCGGCACGTGCTCAAGCTCACGGACAACTCCAACCAGTTCCAGACGGAG:660LIMBSα3α3α3α3¤α4α4α4α4#β4β4β4β4MIDAS221:VGKQLISGNLDAPEGGLDAMMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLL:275661:GTCGGGAAGCAGCTGATCTCGGGGAACCTGGACGCCCCCGAGGGCGGGCTGGATGCCATGATGCAGGTGGCCGCGTGCCCGGAGGAGATCGGCTGGCGCAACGTCACCAGGCTGCTGGTGTTCGCCACGGACGATGGCTTCCACTTTGCGGGCGACGGGAAGCTG:825¤α5α5α5α5β5β5β5β5276:GAILTPNDGRCHLEDNLYKSSNEFDYPSVGQLAHKLAESNIQPIFAVTKKMVKTYY:330826:GGCGCCATCCTGACCCCCAACGACGGCCGCTGCCACCTGGAAGACAACTTATACAAAAGCAGCAATGAATTCGACTACCCATCAGTGGGACAGCTGGCACACAAACTGGCCGAGAGCAACATCCAGCCCATCTTTGCCGTGACCAAGAAAATGGTGAAAACGTAT:990α6α6α6α6β6β6β6β6ADMIDASα7α7α7α7331:EKLTDIIPKSAVGELSEDSSNVVQLIKNAYNKLSSRVFLDHNALPDTLKVTYDSFF:385991:GAGAAGCTCACAGACATCATCCCCAAGTCCGCCGTCGGGGAGCTGTCGGAGGATTCCAGCAACGTGGTCCAGCTCATTAAGAACGCCTACAATAAACTGTCCTCCAGAGTGTTTTTGGATCACAACGCCCTCCCTGACACCCTGAAGGTCACGTACGACTCCTTC:1155¤¤¤386:CSNGVSQVNQPRGDCDGVQINVPITFQVKVTASECIQEQSFVIRALGFTDTVTVRR:4401156:TGCAGCAACGGGGTGTCGCAGGTGAACCAGCCCAGAGGGGACTGCGACGGCGTCCAGATCAACGTCCCGATCACCTTCCAGGTGAAGGTCACCGCCTCCGAGTGCATCCAGGAGCAGTCGTTCGTCATCCGGGCGCTGGGCTTCACCGACACGGTGACCGTGCGG:1320441:VLPQCECRCGDSSKERSLCGNKGSMECGVCRCDAGYIGKHCECQTQGRSSQELEGG:4951321:GTCCTCCCCCAGTGTGAGTGCCGCTGCGGGGACAGCAGCAAGGAGCGCAGCCTCTGCGGCAACAAGGGCTCCATGGAGTGCGGGGTCTGCAGGTGCGATGCCGGCTACATCGGGAAGCACTGCGAGTGCCAGACGCAGGGCCGGAGCAGCCAGGAGCTGGAAGGA:1485496:SCRKDNSSIICSGLGDCICGQCVCHTSDVPNKKIYGQFCECDNMNCERFDGQVCGG:5501486:AGCTGCCGCAAGGACAACAGCTCCATCATCTGCTCGGGGCTGGGCGACTGCATCTGCGGGCAGTGCGTGTGCCACACGAGCGACGTGCCCAACAAGAAGATTTACGGCCAGTTCTGCGAGTGTGACAACATGAACTGCGAGCGCTTCGATGGCCAAGTCTGCGGG:1650551:GEKRGLCFCGTCRCQEGFEGSACQCLKSTQGCLNLQGVECSGRGRCRCNVCQCDFF:6051651:GGCGAGAAGCGGGGCCTCTGCTTCTGCGGCACCTGCAGGTGCCAAGAAGGCTTCGAGGGCTCGGCGTGCCAGTGCCTCAAGTCCACGCAGGGCTGCCTCAACCTGCAGGGCGTCGAGTGCAGCGGCCGCGGCCGGTGCCGCTGCAACGTGTGCCAGTGCGACTTT:1815606:GYQPPLCTDCPSCQVPCARYAKCAECLKFDTGPFAKNCSAECGTTKLLPSRMSGRR:6601816:GGCTACCAGCCGCCCCTGTGCACCGACTGCCCCAGCTGCCAGGTGCCCTGCGCCCGCTATGCCAAATGCGCCGAGTGCCTGAAGTTCGACACCGGCCCCTTCGCCAAAAACTGCAGCGCGGAGTGCGGGACCACCAAGCTGCTGCCCAGCCGGATGTCGGGCCGC:1980¤¤¤661:KCNERDSEGCWMTYFLVQRDGRDNYDLHVEETRECVKGPNIAAIVGGTVGGVVLVV:7151981:AAGTGCAATGAGCGGGACTCCGAGGGCTGCTGGATGACCTACTTCCTGGTGCAGCGCGACGGCCGGGACAACTACGACCTGCACGTGGAGGAGACGCGCGAGTGTGTGAAAGGCCCCAACATCGCCGCCATCGTGGGGGGCACCGTGGGGGGAGTCGTGCTCGTG:2145716:GIFLLAIWKVLTHLSDLREYKRFEKEKLKSQWNNDNPLFKSATTTVMNPKFAER*:7702146:GGCATCTTCCTGCTGGCCATCTGGAAGGTCCTGACCCACCTGAGTGACCTCAGGGAGTACAAGCGCTTCGAGAAGGAGAAGCTCAAGTCCCAGTGGAACAACGATAACCCCCTTTTCAAGAGCGCCACCACGACAGTTATGAACCCCAAGTTTGCTGAGCGCTAG:2310Boxesframingthesequencesrepresentthededuceddifferentconstitutivepartsoftheprotein:theputativeleaderpeptide(),PSIdomain(),spacer(hybriddomain)(),βI-likedomain()withitsMIDAS(),ADMIDAS()andLIMBS()sites,mid-region(hybriddomain)(),linker(),fourEGFdomains(),β-taildomain(),transmembraneregion(),bindingsiteofcytohesin(),Rack1andα-actinin().
Thesix-strandedβ-sheetsandsevenα-helicesoftheβI-likedomainareunderlined.
Theimportant"C191PNKEKEC",L732,S756,T758TT,F766areframedinblackboxes.
Cysteineresidues(¤),potentialN-glycosylationsites(#)andpotentialcytoplasmic-tailphosphorylationsites(+)aremarkedatthetopofthesequences.
SequencedatahavebeendepositedatGenBankunderaccessionno.
EF585977.
BMCVeterinaryResearch2007,3:27http://www.
biomedcentral.
com/1746-6148/3/27Page7of12(pagenumbernotforcitationpurposes)However,thesetwowildboar-specificCD11aisoformsmightdisplayanaltered/improvedfunctioncomparedtothosedescribedamongdomesticpigs[45].
CharacterizationofWbCD18-encodingcDNAanddeducedamino-acidsequenceThecDNAsequenceofWbCD18containsanORFof2310bp[GenBank:EF585977]thatcodesfor769aa(Fig.
3).
ThematureWbCD18containsa22aaputativeleaderpeptide(M1-S22),anextracellulardomainof679residues(Q23-N700),asinglehydrophobictransmembraneregionof23residues(I701-W723)andashortcytoplasmictailof46residues(K724-R769)(Fig.
3).
StartingfromtheN-termi-nalend,theextracellularregionsuccessivelycontainsacysteine-richrepeats-containinganalogueoftheso-called[54]plexin-semaphorinintegrindomain(Q23-V74)[55],aspacer(A75-K123)[55,56],aninsertedI-likedomainof240aminoacids(G124-L363),amid-region(S364-C445)thatfold-upwiththespacertoformthehybriddomain[55,56],acysteine-richregioncontainingfourEGF-likeregions(C449-E482,C483-E535,C536-Q574,andC575-T613)[39,55,57],andaC-terminalβ-taildomain[5](D614-N700)(Fig.
3).
Aputativemetalion-dependentadhesionsite(MIDAS)-likeDXSXSmotif(D134-M139),anadjacentsitetoMIDAS(ADMIDAS)(D141,D142,E347),andalig-and-inducedmetal-bindingsite(LIMBS)(D173,N229,D231,P233,E234)motifarepredictedwithintheI-likedomain[58].
Thecytoplasmictailcontainsthebindingsitesforcytohesin,Rack1(K724VLT),andα-actinin(K736RFEKEKLKSQ)[59].
Overall,theproteincontains58cysteineresiduesand5N-linkedputativeglycosylationsites(Asn-X-Thr/Ser),alllocatedwithintheextracellularregion(Fig.
3).
WbCD18comparisonamongspeciesOverall,thegeneralorganizationofwildboar(Susscrofa),porcine(Susscrofadomestica)[30],bovine(Bostaurus)[60],waterbuffalo(Bubalusbubalis)(GenPeptAAW29104),caprine(Caprahircus)[61],ovine(OvisariesandOviscanadensis)[62,63],human[64],canine(Canisfamiliaris)[65],murine(Musmusculus)[66],rat(Rattusnorvegicus)[GenBank:NM_001037780],chicken(Gallusgallus)[67],carpandchannelcatfish(CyprinuscarpioandIctaluruspunctatus)[GenBank:AB031070][68]CD18pro-teinsisquitesimilar(Fig.
4).
SequencecomparisonsbetweenWbCD18anditsporcine,bovine,waterbuffalo,caprine,ovine,human,canine,murine,rat,chicken,carpandchannelcatfishcounterpartsshowsrespectively,99%,88%,88%,88%,88%,87%,83%,81%,81%,80%,62%,49%and48%identity,and99%,93%,93%,93%,93%,93%,90%,89%,88%,88%,76%,64%and63%similar-ity(BLOSUM62table)(Table2).
TheMIDAS-like,ADMI-DAS,LIMBSmotifs,theI-likedomain,theEGF-2domainandthecytoplasmictailhavethehighestidentitywhiletheputativepeptidesignal,theβ-taildomain,andtheEGF-1showthelowestidentity(Table2).
TheveryhighinterspeciesconservationoftheputativeMIDAS-like,ADMIDAS,LIMBS,I-likedomainsandthecytoplasmictailisconsistentwithaninvolvementoftheseregionsinthefunctionalactivitiesofβ2-integrins.
Overall,thehighevolutionaryconservationoftheI-likedomainconfirmsitsimportanceinβ2-integrinsfunctions,whichiscompat-iblewiththeobservationthatmonoclonalantibodiesbindingepitopesmappedwithinthisregioninhibitbind-ingofLFA-1toICAMs1–3[56].
Themaximumconserva-tionbeingobservedfortheCD18MIDAS-likemotif,itistemptingtospeculatethatitplaysafundamentalroleinβ2-integrinfunction.
Inthisway,itwasdemonstratedthattheC169PNKEKECsequenceconservedamongmamma-lianspecies(Fig.
4)constitutivelyactivatesLFA-1bindingtoICAM-1[69].
LIMBSandADMIDASsitesmodulatebindingofligandtotheMIDAS-likeintheintegrinsthatlacktheαIdomain[70-72]buttheADMIDASseemsalsotoregulateαLIdomainaffinityandtoparticipateintheoutside-insignalling[58].
Thehighdegreeofconserva-tioninthecytoplasmictail,withmanySer,Thr,andTyrresidues,iscompatiblewiththeimportantrolethatphos-phorylationoftheseresiduesplaysinregulatingadhesiveactivity[73]andwiththeobservationthatcytoplasmicdomaintruncationofCD18markedlydiminishesbind-ingofLFA-1toICAM-1[52].
Importantly,itwasshownthatthephosphorylationofthehighlyconservedresiduesT758,T759andT760playsacrucialroleintheactivationofthereceptorandthebindingtoICAM-1[59,74].
Inaddi-tion,thekeyresidueF766forbindingtoICAM-1isstrictlyconserved[59].
AlthoughEGF-1possessesaweakeriden-tity,itssimilarityisveryhigh(Table2).
Theweakercon-servationoftheβ-taildomaincouldtranslatealessdegreeofimportanceofthisdomainfortheCD18function.
Everycysteineresidueinthewildboarextracellularpor-tionofmatureCD18ispresentatthesamelocationinCD18fromotherspecies,whichisconsistentwitharoleinmaintainingtheglobalstructureoftheprotein.
Simi-larly,allfivepotentialAsn-glycosylationsitesobservedinwildboararepresentatthesamelocationinothermam-malianspecies.
Wildboar-specificCD18isoformischar-acterizedbytwoaminoacidsubstitutionscomparedtodomesticpigs:G560SandA721V,whichshouldnotresultinstructuraldifferences,accordingtoBLOSUM62table,butmightimpactCD18function.
ConclusionThisstudyreportsthesequencingofthewildboarβ2-integrinCD11a/CD18subunitscDNAs.
PredictedCD11aandCD18subunitsshareallthemainstructuralcharacter-isticsoftheirmammalianhomologues,withalargerinter-speciesconservationfortheCD18thantheCD11a.
Besidesthesestrongoverallsimilarities,wildboaranddomesticpigLFA-1differby2(CD18)and1or3(CD11a)substitutions,ofwhichoneislocatedinthecru-BMCVeterinaryResearch2007,3:27http://www.
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com/1746-6148/3/27Page8of12(pagenumbernotforcitationpurposes)Table2:Between-speciespercentidentitiesandsimilaritiesofCD18constitutiveblocksBlockWbvs.
Po(%)Wbvs.
Bo(%)Wbvs.
Bu(%)Wbvs.
Cap(%)Wbvs.
OvAr(%)Wbvs.
OvCan(%)Wbvs.
Hu(%)Wbvs.
Can(%)Wbvs.
Mu(%)Wbvs.
Ra(%)Wbvs.
Gal(%)Wbvs.
Cyp(%)Wbvs.
Ict(%)Overall99%(1)99%(2)88%93%88%93%88%93%88%93%87%93%83%90%81%89%81%88%80%88%62%76%49%64%48%63%Putativesignalpeptide100%100%59%68%54%68%59%68%59%72%59%72%56%60%56%60%52%60%60%60%28%44%28%32%22%50%Extracellulardomain99%99%88%94%89%94%89%94%88%94%88%93%84%91%82%91%81%89%80%88%63%77%50%64%48%63%PSI100%100%84%92%86%94%88%92%88%92%86%90%75%88%88%92%80%90%78%88%65%78%55%69%61%75%Spacer(Hybriddomain)100%100%77%91%79%91%77%93%77%93%77%93%89%93%83%93%79%87%67%79%38%56%25%42%30%50%I-likedomain99%100%96%98%96%98%95%98%95%98%95%98%96%98%95%99%95%97%95%97%79%89%65%75%63%75%MIDAS100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%ADMIDAS100%100%100%100%100%100%100%100%100%100%100%100%100%100%66%100%66%100%66%100%100%100%100%100%66%66%LIMBS100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%Mid-region(Hybriddomain)100%100%91%96%91%96%93%96%93%96%92%95%92%97%89%95%84%90%82%90%58%71%41%53%32%54%EGF-197%100%73%91%70%91%76%94%73%94%70%91%67%82%61%79%70%88%67%85%35%52%47%64%30%52%EGF-2100%100%100%100%100%100%98%100%98%100%98%100%88%96%81%94%88%98%88%98%79%94%41%58%41%50%EGF-397%97%87%94%87%94%87%92%82%92%79%92%76%87%69%87%66%79%79%84%64%76%48%66%51%74%EGF-4100%100%92%94%92%94%92%92%92%92%92%92%69%82%76%92%66%76%69%79%61%66%43%58%38%48%β-taildomain100%100%74%81%74%81%73%80%73%80%73%80%57%71%54%65%56%69%56%70%42%64%39%52%39%56%Transmembraneregion95%95%91%95%91%95%91%95%95%100%95%100%78%91%82%91%73%91%69%91%56%73%52%78%52%78%Cytoplasmictail100%100%93%93%93%93%93%93%93%93%93%93%91%93%89%95%91%95%89%93%65%82%50%68%50%68%Cytohesin,Rack1bindingsite100%100%75%75%75%75%75%75%75%75%75%75%50%50%75%75%75%75%75%75%50%100%25%50%50%50%α-actininbindingsite100%100%90%90%90%90%90%90%90%90%90%90%90%100%90%100%90%100%90%100%63%90%45%72%54%72%Wb,Po,Bo,Bu,Cap,OvAr,OvCa,Hu,Can,Mu,Ra,Gal,Cyp,andIct:wildboar,porcine,bovine,waterbuffalo,caprine,ovine(ovisaries),ovine(oviscanadensis),human,canine,murine,rat,chicken,carp,andchannelcatfishCD18,respectively;PSI:plexin-semaphorinintegrin;MIDAS:metal-iondependentadhesionsite;ADMIDAS:adjacenttoMIDAS;LIMBS:ligand-inducedmetal-bindingsite;EGF:epidermalgrowthfactorsite;vs:versus.
(1)identity;(2)similarity(BLOSUM62table).
BMCVeterinaryResearch2007,3:27http://www.
biomedcentral.
com/1746-6148/3/27Page9of12(pagenumbernotforcitationpurposes)Comparisonofthewildboar(Wb-),porcine(Po-),bovine(Bo-),waterbuffalo(Bu-),caprine(Cap-),ovine(Ovisariesandcanadensis)(OvAr-andOvCan-),human(Hu-),canine(Can-),murine(Mu-),rat(Ra-),chicken(Gal-),carp(Cyp-)andchannelcatfish(Ict-)β2subunitsaminoacidssequencesFigure4Comparisonofthewildboar(Wb-),porcine(Po-),bovine(Bo-),waterbuffalo(Bu-),caprine(Cap-),ovine(Ovisariesandcanadensis)(OvAr-andOvCan-),human(Hu-),canine(Can-),murine(Mu-),rat(Ra-),chicken(Gal-),carp(Cyp-)andchannelcatfish(Ict-)β2subunitsaminoacidssequences.
WbCD18:MLCRCSPLLL-LVG--LLTLRSALSQE--CAKYKVSTCRDCIESGPGCAWCQKLNFSGQGEPDSVRCDTREQLLAKGCVADDIVDP-RSLAETQEDQAGGQK------QLSPQKVTLYLRPGQAATFNVTFRRAKGYPIDLYYLMDLSYS:138PoCD18:MLCRCSPLLL-LVG--LLTLRSALSQE--CAKYKVSTCRDCIESGPGCAWCQKLNFSGQGEPDSVRCDTREQLLAKGCVADDIVDP-RSLAETQEDQAGGQK------QLSPQKVTLYLRPGQAATFNVTFRRAKGYPIDLYYLMDLSYS:138BoCD18:MLRQRPQLLL-LAG--LLALQSVLSQE--CTNYKVSTCRDCIESGPGCAWCQKLNFTGQGEPDSIRCDTRAELLSKGCPADDIMEP-KSLAETRDSQAGSRK------QLSPQEVTLYLRPGQAVAFNVTFRRAKGYPIDLYYLMDLSYS:138BuCD18:MLRQRPQLLF-LSG--LLALQSVLSQE--CTKYKVSTCRDCIESGPGCAWCQKLNFTGQGEPDSLRCDTRAELLSKGCPADDIMEP-KSLAETRDSQADRQK------QLSPQEVTLYLRPGQAAAFNVTFRRAKGYPIDLYYLMDLSYS:138CapCD18:MLPQRPQLLL-LAG--LLALQSVLSQE--CTKYKVSTCRDCIESGPGCAWCQKLNFTGQGEPDSTRCDTRAQLLSKGCPADDIMEP-KSLAETRQSQAGKQK------QLSPEEVTLYLRPGQAAAFNVTFQRAKGYPIDLYYLMDLSYS:138OvArCD18:MLPQRPQLLL-LAG--LLSLQSVLSQE--CTKYKVSTCRDCIESGPGCAWCQKLNFTGQGEPDSTRCDTRAQLLSKGCPADDIMEP-KSLAETRQSQAGKQK------QLSPEEVTLYLRPGQAAAFNVTFQRAKGYPIDLYYLMDLSYS:138OvCanCD18:MLPQRPQLLL-LAG--LLSLQSVLSQE--CTKYKVSTCRDCIESGPSCAWCQKLNFTGQGEPDSTRCDTRAQLLSKGCPADDIMEP-KSLAETRQSQAGRQK------QLSPEEVTLYLRPGQAAAFNVTFQRAKGYPIDLYYLMDLSYS:138HuCD18:MLGLRPPLLA-LVG--LLSLGCVLSQE--CTKFKVSSCRECIESGPGCTWCQKLNFTGPGDPDSIRCDTRPQLLMRGCAADDIMDP-TSLAETQEDHNGGQK------QLSPQKVTLYLRPGQAAAFNVTFRRAKGYPIDLYYLMDLSYS:138CanCD18:ML-RHSSLLLTLEG--LLFLWAASCQE--CTKYKVSTCRDCVESGPGCAWCQKLNFTGLGEPDSVRCDTREQLLLKGCAADDIMDP-QSLAEIQEDKKGGRQ------QLSPQKVTLYLRPGQAAAFNVTFRRAKGYPIDLYYLMDLSYS:132MuCD18:MLGLRPSLLLALAG--LFFLGSAVSQE--CTKYKVSSCRDCIQSGPGCSWCQKLNFTGPGEPDSLRCDTRAQLLLKGCPADDIMDP-RSIANPEFDQRGQRK------QLSPQKVTLYLRPGQAAAFNVTFRRAKGYPIDLYYLMDLSYS:139RaCD18:MLGPH-TLLLILAG--LLFLGSALSEE--CTKYKVSNCRDCIQSGPGCSWCQKLNFTGPGEPDSLRCDTRAQLLLKGCPADDIMDP-KSFADLHPQYQVQRS------QLSPQKVTLNLRPGQAAAFNVTFRRAKGYPIDLYYLMDLSYS:138GalCD18:-MPRDCCLWLPAMTWVLLLLTTAFAAE--CPKIKVGTCKNCIQSGPGCAWCKKLSFTKAGEPDSNRCDTIEQLQQRGCPENEIEFPVNEIKRTQDSAFSNKI------QLTPQEVHLKLRIREPAEFDVKFRRATGYPIDIYYLMDLSYS:141CypCD18:-MLQAVCLRGLLL---LLVGRQVYTEQ--CLKASVNTCGDCIKSGPGCAWCKDLNFTKTGEQEAARCDTPAVLKEKGCPPTDIINPKNDFIKISNKPLKAGE---NPVQIQPQEIQLDLRPGMPYTFQLHFKRAQGYPVDLYYLMDLSYS:141IctCD18:-MPQAAKI--VLL---LLFGQYVYSQEKECIKSSVVSCKDCINSGPGCAWCQELNFTKTGEPEAVRCDTEEKLKEKGCSQQKIISPSSHLENVANKPLSGSDRTKNPVQVQPQEIKLNLRPGQAFTFNLQFKRAVGYPVDLYYLMDLSYS:144WbCD18:MLDDLINVKKLGGDLLRALNEITESGRIGFGSFVDKTVLPFVNTHPEKLRNPCPNKEKECQAPFAFRHVLKLTDNSNQFQTEVGKQLISGNLDAPEGGLDAMMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHL:288PoCD18:MLDDLINVKKLGGDLLRALNEITESGRIGFGSFVDKTVLPFVNTHPEKLRNPCPNKEKECQAPFAFRHVLKLTDNSNQFQTEVGKQLISGNLDAPEGGLDAMMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHL:288BoCD18:MVDDLVNVKKLGGDLLRALNGITESGRIGFGSFVDKTVLPFVNTHPEKLRNPCPNKEKECQPPFAFRHVLKLTDNSKQFETEVGKQLISGNLDAPEGGLDAMMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHL:288BuCD18:MVDDLINVKKLGGDLLRALNDITESGRIGFGSFVDKTVLPFVNTHPEKLRNPCPNKEKECQPPFAFRHVLKLTDNSKQFETEVGKQLISGNLDAPEGGLDAMMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHL:288CapCD18:MVDDLANVKKLGGDLLRALNDITESGRIGFGSFVDKTVLPFVNTHPEKLRNPCPNKEKQCQPPFAFRHVLKLTDNSKQFETEVGKQLISGNLDAPEGGLDAMMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHL:288OvArCD18:MVDDLANVKKLGGDLLRALNDITESGRIGFGSFVDKTVLPFVNTHPEKLRNPCPNKEKECQPPFAFRHVLKLTDNSKQFETEVGKQLISGNLDAPEGGLDAMMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHL:288OvCanCD18:MVDDLANVKKLGGDLLRALNDITESGRIGFGSFVDKTVLPFVNTHPEKLRNPCPNKEKECQPPFAFRHVLKLTDNSKQFETEVGKQLISGNLDAPEGGLDAMMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHL:288HuCD18:MLDDLRNVKKLGGDLLRALNEITESGRIGFGSFVDKTVLPFVNTHPDKLRNPCPNKEKECQPPFAFRHVLKLTNNSNQFQTEVGKQLISGNLDAPEGGLDAMMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHL:288CanCD18:MLDDLINVKKLGGDLLRALNEITESGRIGFGSFVDKTVLPFVNTHPEKLKNPCPNKEKECQAPFAFRHVLKLTNNSNKFQTEVGKQLISGNLDAPEGGLDAMMQVAACPEQIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHL:282MuCD18:MLDDLNNVKKLGGDLLQALNEITESGRIGFGSFVDKTVLPFVNTHPEKLRNPCPNKEKACQPPFAFRHVLKLTDNSNQFQTEVGKQLISGNLDAPEGGLDAIMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHL:289RaCD18:MLDDLNNVKKLGGDLLQALNEITESGRIGFGSFVDKTVLPFVNTHPEKLRNPCPNKEKACQPPFAFRHVLKLTDNSNQFQTEVGKQLISGNLDAPEGGLDAIMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHL:288GalCD18:MLDDLENVKKLGGQLLRALESTTPSRRIGFGSFVDKTVLPFVNTHPEKLKNPCPNKDSNCQPPFAFKHILSLTDNAEKFESEVGKQFISGNLDAPEG-LDAMMQAAVCGDLIGWRNVTRLLVYATDDGFHFAGDGKLGGILTPNDGQCHL:290CypCD18:MKDDLENVKNLGNDLLKELNKITGSARIGFGSFVDKTLLPYTDTNEAKLKRPCSDKNEPCQPAFGFQHVLPLTKDGDKFKSMVAEQHISGNLDPPEGSLDAIMQAAVCVNEIGWGNSTRLLVLATDDGFHMAGDGKLAAILEPNRETCQL:291IctCD18:MEDDLTNVKDLGIQILKRLQQITGNARIGFGSFVDKTLLPFTDTTEEKLKKPCPAKRKQCQPAFGYHHVLSLTQNETLFSTMVSGQEISGNLDTPEGGLDAIMQVATCVNKIGWGNSTRLLVLATDAGFHKAGDGKLAGILEPNQETCQL:294WbCD18:ED-NLYKSSNEFDYPSVGQLAHKLAESNIQPIFAVTKKMVKTYEKLTDIIPKSAVGELSEDSSNVVQLIKNAYNKLSSRVFLDHNALPDTLKVTYDSFCSNGVSQVNQPRGDCDGVQINVPITFQVKVTASECIQEQSFVIRALGFTDTV:437PoCD18:ED-NLYKSSNEFDYPSVGQLAHKLAESNIQPIFAVTKKMVKTYEKLTDIIPKSAVGELSEDSSNVLELIKNAYNKLSSRVFLDHNALPDTLKVTYDSFCSNGVSQVNQPRGDCDGVQINVPITFQVKVTASECIQEQSFVIRALGFTDTV:437BoCD18:ED-NLYKSSNEFDYPSVGQLAHKLAESNIQPIFAVTKKMVKTYEKLTEIIPKSAVGELSEDSRNVVELIKNAYNKLSSRVFLDHSTLPDTLKVTYDSFCSNGKSQVDQPRGDCDGVQINVPITFQVKVTATECIQQQSFTIRALGFTDTV:437BuCD18:ED-NLYKSGNEFDYPSVGQLAHKLAESNIQPIFAVTKKMVKTYEKLTEIIPKSAVGELSEDSKNVVELIKNAYNKLSSRVFLDHSTLPDTLKVTYDSFCSNRVSQVDQPRGDCDGVQINVPITFQVKVTATECIQQQSFTIRALGFTDTV:437CapCD18:ED-NLYKSSNEFDYPSVGQLAHKLAESNIQPIFAVTKKMVKTYEKLTEIIPKSAVGELSEDSKNVVELIKSAYNKLSSRVFLDHNTLPDTLKVAYDSFCSNGVSQVDQPRGDCDGVQINVPITFQVKVTATECIQEQSFTIRALGFTDTV:437OvArCD18:ED-NLYKSSNEFDYPSVGQLAHKLAESNIQPIFAVTKKMVKTYEKLTEIIPKSAVGELSEDSKNVVELIKSAYNKLSSRVFLDHNTLPDTLKVAYDSFCSNGVSQVDQPRGDCDGVQINVPITFQVKVTATECIQEQSFTIRALGFTDTV:437OvCanCD18:ED-NLYKSSNEFDYPSVGQLAHKLAESNIQPIFAVTKKMVKTYEKLTEIIPKSAVGELSEDSKNVVELIKSAYNKLSSRVFLDHNTLPDTLKVAYDSFCSNRVSQVDQPRGDCDGVQINVPITFQVKVTATECIQEQSFTIRALGFTDTV:437HuCD18:ED-NLYKRSNEFDYPSVGQLAHKLAENNIQPIFAVTSRMVKTYEKLTEIIPKSAVGELSEDSSNVVQLIKNAYNKLSSRVFLDHNALPDTLKVTYDSFCSNGVTHRNQPRGDCDGVQINVPITFQVKVTATECIQEQSFVIRALGFTDIV:437CanCD18:ED-NMYKRSNEFDYPSVGQLAHKLAESNIQPIFAVTKRMVTTYEKLTEVIPKSAVGELSDDSSNVVQLIKNAYNKLSSRVFLDHSLAPSTLKVTYDSFCSNGVSQVDQPRGDCDGVQINVPITFQVKVTATECIQEQSFIIRALGFTDTV:431MuCD18:ED-NMYKRSNEFDYPSVGQLAHKLSESNIQPIFAVTKKMVKTYEKLTEIIPKSAVGELSDDSSNVVQLIKNAYYKLSSRVFLDHSTLPDTLKVTYDSFCSNGASSIGKSRGDCDGVQINNPVTFQVKVMASECIQEQSFVIRALGFTDTV:438RaCD18:ED-NMYKRSNEFDYPSVGQLAHKLSESNIQPIFAVTKKMVKTYEKLTEIIPKSAVGELSDDSSNVVQLIKKAYYKLSSRVFLDHTTIPDTLKVTYDSFCNNRVSSIGKSRGDCDGVQINNPVTFQVKVTASECIQEQSFVIRALGFTDTV:437GalCD18:ED-NMYKKSNEFDYPSVGQLVQKLAENNIQPIFAVTSKMVDVYKKLSDMIPKSAVGELNEDSSNIIELIQVAYNNLSSRIILDHSTLPDVLDVKYDSICNNNTGAKNEARGQCDNVKINDEVTFKVKVTANECIKSQSFTIRPLGFTDTL:439CypCD18:VDRK-YSKSTLWDYPSVGQVARKLEEQNIQPIFAVTKKMETVYTELSKLIPKSAVGVLSEDSGNVVNLIVDAYNSLSSEVIMAHEALPEFISVKYTSNCKDGEKPSD--RGKCDNVNIGTEVVFDVTVTVDRCINSQSFLIGPLGFNEKL:438IctCD18:DNKNIYSMSNNWDYPSVGQIARKLGEETIQPIFAVTEDVSHIHQELSKLIPKSEVGVLSSDSSNVVKLIVDAYNKLSSNIIVTHDGLPEDISVTFTSNCPGGEKPSD--RGTCNNVAIGQGVTFSVTVKAAKCLDKAYFQTGPLGFSEKL:442¤¤¤WbCD18:TVRVLPQCECRCGDSSKE--RSLCGNKGSMECGVCRCDAGYIGKHCECQTQGRSSQELEGSCRKDNSSIICSGLGDCICGQCVCHTSDVPNKKIYGQFCECDNMNCERFDGQVCGGEKRGLCFCGTCRCQEGFEGSACQCLKSTQGCLNL:585PoCD18:TVRVLPQCECRCGDSSKE--RTLCGNKGSMECGVCRCDAGYIGKHCECQTQGRSSQELEGSCRKDNSSIICSGLGDCICGQCVCHTSDVPNKKIYGQFCECDNMNCERFDGQVCGGEKRGLCFCSTCRCQEGFEGSACQCLKSTQGCLNL:585BoCD18:TVRVLPQCECQCRDASRD--GSICGGRGSMECGVCRCDAGYIGKNCECQTQGRSSQELEGSCRKDNSSIICSGLGDCICGQCVCHTSDVPNKKIYGQFCECDNVNCERYDGQVCGGEKRGLCFCGTCRCDEQYEGSACQCLKSTQGCLNL:585BuCD18:TVRVLPQCECQCRDASRD--GSICGGRGSMECGVCKCDAGYIGKNCECQTQGRSSQELEGSCRKDNSSIICSGLGDCICGQCVCHTSDVPNKKIYGQFCECDNVNCERYDGQVCGGEKRGLCFCGTCRCDEQYEGSACQCLKSTQGCLNL:585CapCD18:TVRVLPQCECQCRDASRD--RSVCGGRGSMECGVCRCDAGYIGKNCECQTHGRSSQELEGSCRKDNSSIICSGLGDCICGQCVCHTSDVPNKKIYGQFCECDNVNCERYDGQVCGGEKRGLCFCGTCRCNEQHEGSACQCLKSTQGCLNL:585OvArCD18:TVRVLPQCECQCREASRD--RSVCGGRGSMECGVCRCDAGYIGKNCECQTHGRSSQELEGSCRKDNSSIICSGLGDCICGQCVCHTSDVPNKKIYGQFCECDNVNCERYDGQVCGGDKRGLCFCGTCRCNDQHEGSACQCLKSTQGCLNL:585OvCanCD18:TVRVLPQCECQCREASRD--RGVCGGRGSMECGVCRCDAGYIGKNCECQTHGRSSQELEGSCRKDNSSIICSGLGDCICGQCVCHTSDVPNKKIYGQFCECDNVNCERYDGQVCGGDKRGLCFCGACRCNDQYEGSACQCLKSTQGCLNL:585HuCD18:TVQVLPQCECRCRDQSRD--RSLCHGKGFLECGICRCDTGYIGKNCECQTQGRSSQELEGSCRKDNNSIICSGLGDCVCGQCLCHTSDVPGKLIYGQYCECDTINCERYNGQVCGGPGRGLCFCGKCRCHPGFEGSACQCERTTEGCLNP:585CanCD18:TVHVIPQCECQCRDVGQD--HGLCSGKGSLECGICRCEAGYIGKNCECLTHGRSSQELEGSCRRDNSSLICSGLGDCLCGQCVCHRSDVPNKNIFGRYCECDNVNCERYDGQVCGGKVRGSCNCGKCQCEQNYEGSACQCVKSTQGCLST:579MuCD18:TVQVRPQCECQCRDQSRE--QSLCGGKGVMECGICRCESGYIGKNCECQTQGRSSQELERNCRKDNSSIVCSGLGDCICGQCVCHTSDVPNKEIFGQYCECDNVNCERYNSQVCGGSDRGSCNCGKCSCKPGYEGSACQCQRSTTGCLNA:586RaCD18:TVQVHPQCECQCRDQSRM--RNLCGGKGVMECGICRCESGYIGKNCECQTQGRSSQELEGNCRKDNSSIVCSGLGDCICGQCVCHTSDIPNKVIFGQYCECDNFNCERYDGQVCGGLKRGSCSCGQCNCKEGFEGSACQCQRSTTGCLNA:585GalCD18:TVHLDSICDCDCREQP-D--PTACSGNGKVVCGICSCNLSYTGKNCECDTKGKTSKELEGSCRKDNSSVICSGLGDCVCGQCVCHTSDVPGKEIYGTFCDCDNMNCEFHNGSLCGGEERGRCDCGECKCTPKYEGSACQCKKSTDGCRNS:586CypCD18:KVNVRTRCDCECDDINQP--HPHCKEKGKVVCGSCRCDPGFLGQHCECQVGQKDVASLKAQCQKDNGTE-CEGKGDCVCGRCQCHLTE-GGNNFYGEFCECDDEHCEKYANKQCAGH--GKCKCGRCECNEGYEGAACQCAKSDEDC-KI:581IctCD18:KVNIKTRCECECD--NQPGVQLYCNQHGTIVCGTCSCVPGFLGQRCECSLGQKDEATLKAQCRKDNGTE-CEGRGDCECGVCKCHQTE-GGKSYYGPHCECDDEHCEKYQNKLCGGN--GDCRCGVCKCNEGYEGTACQCKKSNEFC-ET:585WbCD18:QGVECSGRGRCRCNVCQCDFGYQPPLCTDCPSCQVPC-ARYAKCAECLKFDTGPFAKNCSAECGTTKLLPSRMSG-RKCNERDSEGCWMTYFLVQRDGRDNYDLHVEETRECVKGPNIAAIVGGTVGGVVLVGIFLLAIWKVLTHLSDLR:733PoCD18:QGVECSGRGRCRCNVCQCDFGYQPPLCTDCPSCQVPC-ARYAKCAECLKFDTGPFAKNCSAECGTTKLLPSRMSG-RKCNERDSEGCWMTYFLVQRDGRDNYDLHVEETRECVKGPNIAAIVGGTVGGVVLVGIFLLVIWKVLTHLSDLR:733BoCD18:DGVECSGRGRCRCNVCQCDPGYQPPLCSECPGCPVPC-AGFAPCTECLKFDKGPFAKNCSAACGQTKLLSSPVPG-RKCKERDSEGCWMTYTLVQRDGRDRYDVHVDDMLECVKGPNIAAIVGGTVGGVVLVGILLLVIWKALTHLSDLR:733BuCD18:DGVECSGRGRCRCNVCQCDPGYQPPLCSECPGCPVPC-AGFAPCTECLKFDKGPFAKNCSAACGQTKLLSSPVPG-RKCKERDSEGCWMTYTLVQRDGRDRYDVHVDDMLECVKGPNIAAIVGGTVGGVVLVGILLLVIWKALTHLSDLR:733CapCD18:DGVECSGRGRCRCNVCQCDPGYQPPLCIDCPGCPVPC-AGFAPCTECLKFDKGPFAKNCSAACGQTKLLSSPVPGGRKCKERDSEGCWMTYTLVQRDGRNRYDVHVDDMLECVKGPNIAAIVGGTVGGVVLVGILLLVIWKALTHLSDLR:734OvArCD18:DGVECSGRGRCRCNVCQCDPGYQPPLCIDCPGCPVPC-AGFAPCTECLKFDKGPFAKNCSAACGQTKLLSSPVPGGRKCKERDSEGCWMTYTLVQRDGRNRYDVHVDDMLECVKGPNIAAIVGGTVGGVVLVGILLLAIWKALTHLSDLR:734OvCanCD18:NGVECSGRGRCRCNVCQCDPGYQPPLCIDCPGCPVPC-AGFAPCTECLKFDKGPFAKNCSAACGQTKLLSSPVPGGRKCKERDSEGCWMTYTLVQRDGRNRYDVHVDDMLECVKGPNIAAIVGGTVGGVVLVGILLLAIWKALTHLSDLR:734HuCD18:RRVECSGRGRCRCNVCECHSGYQLPLCQECPGCPSPC-GKYISCAECLKFEKGPFGKNCSAACPGLQLSNNPVKG-RTCKERDSEGCWVAYTLEQQDGMDRYLIYVDESRECVAGPNIAAIVGGTVAGIVLIGILLLVIWKALIHLSDLR:733CanCD18:EGIECNGRGRCRCNVCECDGGYQPPLCGDCLGCPSPC-GRYITCAQCLKFKQGPSGRNCSVECGNVGLLSKPPEKGRRCKERDLEGCWITYTLRQRAGWDSYEIHVDDSRECVGGPQIAPIVGGTVSGVVLIGILLLAIWKALTHLSDLR:728MuCD18:RLVECSGRGHCQCNRCICDEGYQPPMCEDCPSCGSHCRDNHTSCAECLKFDKGPFEKNCSVQCAGMTLQTIPLKK-KPCKERDSEGCWITYTLQQKDGRNIYNIHVEDSLECVKGPNVAAIVGGTVVGVVLIGVLLLVIWKALTHLTDLR:735RaCD18:RLVECSGRGRCQCNRCICEKGYQPPLCEECPGCPLPC-STYVFCAECLKFDKGPFQKNCSVQCANVTLQTVPFKK-KPCKERDSEGCWITYTLQQKDG-NAYNIHVDDDRECVKGPNVAAIIGGTVAGVVLIGVLLLVIWKALTHLTDLN:732GalCD18:RQNECSLRGSCPCNRCQCRGGYQPPFCEECPGCPSPC-GRHISCVECKSFNSGPLAKNCSVACTSIQLADEPRAGSRQCKEKDSENCWISFYMAQDDGEEMYTVTVDPKKECPEPPNIALIVGSTIAGVALIGLLLLLTWRLLTEIFDRR:735CypCD18:DGIVCHERGKCVCNQCECQRGYKGPSCKVCPTCQRPCQES-GSCVECLAFGTGPFEKSCIENCKHLRHVMVEKLIKHDCRVKDKEGCWMLFTMTEELGFDKYIVNVLKDRECPEGPNIAAIVGGSIAAVALIGLLILLIIKAVLYASDLR:730IctCD18:GKTICHGRGKCVCNQCVCERRYKGITCETCPTCELPCQES-GSCVECNAFGTGPFEKTCNASCYHLDVEVVEKLAKRECQVKDNEGCRMIFSMTEQDGFDKYIVKVLKDRECPEGPNVALIVGGTLAGVALIGLLLLILIKALFYFKDLK:734WbCD18:EYKRFEKEKLKSQWNN-DNPLFKSATTTVMNPKFAER-:769PoCD18:EYKRFEKEKLKSQWNN-DNPLFKSATTTVMNPKFAER-:769BoCD18:EYHRFEKEKLKSQWNN-DNPLFKSATTTVMNPKFAES-:769BuCD18:EYHRFEKEKLKSQWNN-DNPLFKSATTTVMNPKFAES-:769CapCD18:EYHRFEKEKLKSQWNN-DNPLFKSATTTVMNPKFAES-:770OvArCD18:EYHRFEKEKLKSQWNN-DNPLFKSATTTVMNPKFAES-:770OvCanCD18:EYHRFEKEKLKSQWNN-DNPLFKSATTTVMNPKFAES-:770HuCD18:EYRRFEKEKLKSQWNN-DNPLFKSATTTVMNPKFAES-:769CanCD18:EFKRFEKEKLRSQWNN-DNPLFKSATTTVMNPRFAES-:764MuCD18:EYRRFEKEKLKSQWNN-DNPLFKSATTTVMNPKFAES-:771RaCD18:EYRRFEKEKLKSQWNN-DNPLFKSATTTVMNPKFAES-:768GalCD18:EYRRFEKEKSKAKWNEADNPLFKSATTTVMNPRFDGQ-:772CypCD18:EWKRFEKD-RKHEKTSGTNPLFQNATTTVQNPTFSGDS:767IctCD18:EWKKFEKEAQRRQWAKGENPLFQNATTTVANPAFTGDS:772Blackcolumnswithwhiteletterrepresentidentityamongthe14species.
Cysteineresidues(¤),potentialN-glycosylationsites(#)andpotentialcytoplasmic-tailphosphorylationsites(+)aremarkedatthebottomofthesequencesinredfor100%identityandinblueforless.
Theimportant"C191PNKEKEC",L732,S756,T758TTandF766residuesaremarkedby()inredfor100%identityandinblueforless.
Thestripesabovethesequencesrepresentthededuceddifferentconstitutivepartsoftheprotein:thesignalpeptide(),PSIdomain(),spacer(hybriddomain)(),βI-likedomain()withitsMIDAS(),ADMIDAS()andLIMBS()sites,mid-region(hybriddomain)(),linker(),fourEGFdomains(,),β-taildomain(),transmembraneregion(),bindingsiteofcytohesin(),Rack1andα-actinin().
BMCVeterinaryResearch2007,3:27http://www.
biomedcentral.
com/1746-6148/3/27Page10of12(pagenumbernotforcitationpurposes)cialI-domain(CD11a,E168D).
AsmostwildboarsareseropositivetoActinobacilluspleuropneumoniaeandbecausetheyhavesustainedcontinuousnaturalselection,futurestudiesofthefunctionalimpactofthesepolymor-phismscouldbringinterestingnewinformationonthephysiopathologyofpneumoniaindomesticpigs.
MethodsRNAisolationTotalRNAfromspleenoffreshlyslaughteredwildboars(Susscrofa)wasextractedwithTRIzol(Invitrogen,USA)asdescribedbythemanufacturer.
AmplificationandsequencingofthefulllengthcDNATotalRNAfromspleenwasreversetranscribedusingImpromII(Promega).
Thefull-lengthcDNAwasthengeneratedbylongdistancePCRusingTaqandPfuDNApolymerasefromtheHighFidelityPCREnzymeMix(Fer-mentas)withprimersdesignedfromthe5'-and3'UTRofPoCD11a[31]andPoCD18[30].
ThecoupleofprimersusedforamplificationofWbCD11aandWbCD18arethenext:5'GGTATGGTCCCTCCAGAAGC-3'(CD11afor-ward),5'-GCAGGCTGAGTCCAGTCCTG-3'(CD11areverse),5'-GAGGTCTCCAGGACATCAAG-3'(CD18for-ward)and5'-TAGGGGTGCTTGGTGAAGAC-3'(CD18reverse).
Theproceduresrecommendedbythemanufac-turerwereapplied,withthefollowingcyclingparameters:5minat94°C,then35cyclesincluding(i)30sat94°C,(ii)45sat60°Cand(iii)3min45sat72°C,andafinalextensionat72°Cfor10min.
ResultingPCRproductswerepurifiedusingtheNucleoSpinExtractIIkit(Mach-erey-Nagel),andsequencedonaABI-3100GeneticAna-lyzerusingtheBigDyeterminatorchemistry(AppliedBiosystems).
TheCD11a/CD18cDNAsequenceswerededucedfromsequencesobtainedfromfourindependentwildboars.
SequencesdatahavebeendepositedatGen-Bankunderaccessionnos.
EF585976andEF585977.
BioinformaticsPrimersdesignwasperformedwithNetprimer[75]andPrimer3[76].
NucleotidicsequenceandidentityanalyseswerecarriedoutusingrespectivelyChromasv.
2.
21[77]andBLASTprograms[78].
AlignmentofaminoacidssequenceswasdrawnbyGeneDocv.
2.
6.
002[79]follow-ingtheBLOSUM62matrix.
SignalPv.
2.
0.
b2[80]andNetNGlycv.
1.
0[81]providedpeptidesignalandN-glyco-sylationsitesprediction,respectively.
Thesecondarystruc-tureswereresolvedbytheGORsecondarystructurepredictionmethodversionIV[82].
Listofabbreviationsaa,aminoacid;ADMIDAS,adjacenttoMIDAS;Bo,bovine;Can,canine;Cap,caprine;CD,clusterofdiffer-entiation;CR,complementreceptor;Cyp,Cyprinus;DCBM,divalent-cationbindingmotif;EGF,epidermalgrowthfactor;Gal,Gallus;Hu,human;ICAM,intercellu-laradhesionmolecule;Ict,Ictalurus;LFA,lymphocytefunction-associatedantigen;LIMBS,ligand-inducedmetal-bindingsite;MIDAS,metal-iondependentadhe-sionsite;Mu,murine;Ov,ovine;OvAr,Ovisaries;OvCan,OvisCanadensis;Po,porcine;Ra:rat;PSI,plexin-semaphorinintegrin;Si:simian,Wb:wildboar.
Authors'contributionsPVBcarriedoutamplification,sequencing,sequencesalignmentanddraftingofthemanuscript.
TFandLZpar-ticipatedinthedesignofthestudyandhelpedtostructureanalysis.
DDparticipatedinthedesignofthestudyandcoordinationandhelpedtodraftthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsPhilippeVandenBerghistherecipientofastudentshipfromthe"FondspourlaformationàlaRecherchedansl'Industrieetl'Agriculture",rued'Egmont5,B-1000Bruxelles.
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