Lewis X-containing glycans are specific and potent competitive inhibitors of the binding of ZP3 to complementary sites on capacitated, acrosome-intact mouse sperm

Abstract

Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Lex)-containing glycan (Galβ4 [Fucα3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888-1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Lex-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa568)-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC50 ∼180 nM), which at saturation, reduced Alexa568-ZP3 binding by ∼70%. A Lewis A (Lea)-capped glycan (Galβ3 [Fucα4]GlcNAc) was also a potent inhibitor (IC 50 ∼150-200 nM), but at saturation, it reduced Alexa 568-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAcβ4 or Galβ4 residues did not compete; neither did sialyl-Lex (Neu5Acα 3Galβ4[Fucα3]GlcNAc-Lewis X) nor sulfo-Lex (3′-O-SO3-Lewis X). However, at saturation, Galα3Galβ4 GlcNAcβ3Galβ4Glc reduced Alexa 568-ZP3 binding by ∼70% but with moderate apparent affinity (IC50 ∼3000 nM). Fluorescence microscopy revealed that Alexa 568-labeled Lex-Lac-BSA, Lea-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Lea-Lac did not inhibit binding of Alexa568-Lex-Lac-BSA, and Le x-Lac did not inhibit binding of Alexa568-Le a-Lac-BSA. Finally, Lex-Lac and Lea-Lac had an additive inhibitory effect on Alexa568-ZP3 binding. Thus, Le x is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Lea binding defines a different but less-abundant class of sites.