Glycoprotein constituents of the vitelline coat of Phallusia mammillata (Ascidiacea) with fertilization inhibiting activity☆

摘要

Vitellinecoats(VCs)ofPhallusiamammillatawereisolatedandpurifiedfollowinghomogenizationofliveeggsinordertoinvestigatethemolecularbasisofsperm-eggrecognition.CleanVCswerepartlysolubilizedbysonicationinH2Oandthesolublefraction(SFVC),derivedfromtheoutersurfaceofVCs,wasusedforfurthercharacterization.ElectrophoreticanalysesofradioiodinatedVCsrevealedthatSFVCconsistsoftwomajorglycoproteincomponentswithapparentaverageMr'sof450,000and180,000,respectively.The450,000Mrcomponentiscomposedofseveralchargeisomers,whereasthe180,000Mrcomponentissupposedtoconsistoftwooligomers,bothwithacidicpI,heldtogetherbyadisulfidelinkage(s).EachofthetwocomponentspossessesWGA-bindingsitesasshownbytransblottingfollowedbyWGA-peroxidasetreatment.TheaminoacidcompositionofSFVCwasdeterminedafteracidhydrolysisanditscarbohydratecompositionwasanalyzedandquantifiedbyGLC.GlcNAcandGalNAcwerefoundtopredominatewith86%byweightoftotalsugarcontentandfucose,mannose,andglucoseaccountedfortheremaining14%.ThesusceptibilityofSFVCtoenzymatic(N-glycosidaseF)andchemical(TFMS)deglycosylationaswellastoprotease(trypsinandchymotrypsin)digestionwasinvestigated.Furthermore,spermreceptoractivityofSFVCwastestedinafertilizationassay.Thefertilizationratedecreasedinaconcentration-dependentmannerwhenspermwerepreincubatedwithSFVC.Additionally,spermtreatedwithSFVCshowedbindingforFITC-WGAorWGA-goldattheapicalportionofthespermhead.Therefore,westronglyassumethatoneorbothoftheidentifiedglycoproteinmacromoleculesofSFVCareinvolvedinsperm-eggrecognition.