Characterization of the Binding of Recombinant Mouse Sperm Fertilin β Subunit to Mouse Eggs: Evidence for Adhesive Activity via an Egg β1Integrin-Mediated Interaction ☆

摘要

Thespermproteinfertilin(alsoknownasPH-30)isacandidateformediatingtheinteractionsbetweenspermandeggplasmamembranes.Fertilinisaheterodimer.Theβsubunit,whichhasaregionwithhomologytothefamilyofintegrinligandsknownasdisintegrins,hasbeenhypothesizedtobeinvolvedinthebindingofspermtotheeggsurface.Toinvestigatethishypothesisanddeterminewhatrolefertilinβplaysinfertilization,wehaveexpressedtheputativeextracellulardomainofmousefertilinβinbacteriaasafusionproteinwithmaltose-bindingprotein(hereafterreferredtoasrecombinantfertilinβ-EC)andusedtwoassaystocharacterizeitsbindingtomouseeggs.Immunocytochemistrywasusedtoexaminethelocalizationofrecombinantfertilinβ-ECbinding.Aluminometricassaywasalsodevelopedtoquantifylevelsofbindingofrecombinantfertilinβ-ECtosingleeggs.Wefindthatrecombinantfertilinβ-ECbindstotheregionoftheplasmamembraneoftheeggtowhichspermbind,thusprovidingthefirstdirectevidencethatfertilinβhasadhesiveproperties.Peptidescorrespondingtothedisintegrindomainoffertilinβreduceitsbindingtoeggs,suggestingthatthisdomainisatleastpartiallyinvolvedintherecognitionoffertilinβbybindingsitesontheegg.Treatmentofzonapellucida-freeeggswithchymotrypsinreducestheabilityoftheeggstosupportthebindingofrecombinantfertilinβ-EC,implicatinganeggsurfaceproteinasabindingsiteforrecombinantfertilinβ-EC.Bindingofrecombinantfertilinβ-ECtoeggsisalsoreducedintheabsenceofdivalentcationsandissupportedby2.0memM/emCasup2+/sup,Mgsup2+/sup,orMnsup2+/sup.Furthermore,eggsincubatedinrecombinantfertilinβ-ECpriortoeminvitro/emfertilizationshowreducedlevelsofspermbinding.Finally,wehaveexaminedthepossibleroleofintegrinsoneggsasreceptorsforfertilinβ,sinceananti-αsub6/subintegrinsubunitmonoclonalantibody,GoH3,hasbeenshowntoinhibitspermbinding(E.A.C.Almeidaemetal./em(1995)emCell/em81,1095ndash;1104).Wefindthat:(a)anincreasedamountofGoH3epitopeontheeggsurfacedoesnotcorrelatewithanincreasedabilityoftheeggstobindspermorrecombinantfertilinβ-EC;(b)theGoH3antibodyhasvirtuallynoinhibitoryeffectonrecombinantfertilinβ-ECbinding;and(c)recombinantfertilinβ-ECbindingisreducedinthepresenceofanti-βsub1/subintegrinantibodies.Theseresultssuggestthataβsub1/sub-containingintegrinparticipatesinthebindingofrecombinantfertilinβ-ECtomouseeggs./p/div