Structure/Function Analysis of the Sea Urchin Sperm Adhesive Protein Bindin

摘要

Thefunctionaldomainstructureofbindin,anadhesiveproteincomponentoftheacrosomalgranuleofseaurchinsperm,hasbeenanalyzedusingaseriesofcarboxyl-andamino-terminaldeletionanalogsofrecombinantbindinexpressedinEscherichiacoli.Bindin,whichself-associatestoformmacromolecularaggregates,mediatesthespecies-specificattachmentofspermtotheeggsurfaceaswellasegg-to-eggagglutinationinvitrobybindingtoeggsurface-sulfatedfucose-containingglycoconjugates.Cross-specieseggagglutinationactivityandspermadhesionweredeterminedfortwoseaurchinspecies,StrongylocentrotuspurpuratusandS.franciscanus.WefoundthatS.franciscanusspermbindinagglutinatesbothS.franciscanusandS.purpuratuseggswhereasS.purpuratusspermbindindisplaysamuchmorerestrictedspecificityforhomologousS.purpuratuseggs.Consistentwiththisresult,cross-speciesspermadhesionexperimentsdemonstratethatS.franciscanusspermalsobindmoreefficientlytoS.purpuratuseggsincomparisontoS.purpuratusspermattachmenttoS.franciscanuseggs.RecombinantS.purpuratusbindindemonstratesthesamespecies-specificeggagglutinationpropertiesobservedforspermbindin.DeletionmutantsofS.purpuratusbindinweretestedfortheirabilitytospeciesspecificallyagglutinateeggs.Wefoundthatresidues1-74orresidues122-236canbedeletedwithoutalossofthespecies-specificagglutinationpropertiesofbindin.Comparisonofthebindinaminoacidsequencesfromthetwospeciesrevealsthatalloftheactivedeletionanalogshaveacentralsegmentincommoncorrespondingtoresidues75through121ofS.purpuratusbindin,wherethesequencesareidenticalexceptforasingleaminoacid,Arg77,whichisanAlaresidueinS.franciscanus.However,substitutionofArg77forAladoesnotalterthespeciesspecificityofrecombinantbindin,suggestingthatthemoredivergentamino-andcarboxyl-terminalsequencesflankingtheconservedcentraldomaindeterminethespeciesspecificityofbindinandeitherendissufficienttoimpartspecificity.Theamino-andcarboxyl-terminalthirdsofS.purpuratusbindineachcontaintworepeatsofthesequenceMGGPP/V.ThissequencedoesnotoccurinS.franciscanus,whichinsteadhasfiverepeatsofthesequenceMGGA/V/QF/V/P.Itisconceivablethattheserepeats,whichvaryinsequenceinbindinsfromdifferentspecies,constituteanimportantpartofthespeciesrecognitionmechanism.