Lewis X-containing neoglycoproteins mimic the intrinsic ability of zona pellucida glycoprotein ZP3 to induce the acrosome reaction in capacitated mouse sperm

The binding of zona pellucida (ZP) glycoprotein ZP3 to mouse sperm surface receptors is mediated by protein-carbohydrate interactions. Subsequently, ZP3 induces sperm to undergo the acrosome reaction, an obligatory step in fertilization. We have previously identified Lewis X (Le^x; Galβ4[Fucα3] GlcNAc) as a potent inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273:1888-1895). This glycan is recognized by ∼70% of the ZP3 binding sites on capacitated, acrosome-intact mouse sperm, whereas Lewis A (Le^a; Galβ3[Fucα4]GlcNAc) is recognized by most of the remaining sites (Kerr et al. Biol Reprod 2004; 71:770-777). Herein, we test the hypothesis that Le^x- and Le ^a-containing glycans, when clustered on a neoglycoprotein, bind ZP3 receptors on sperm and induce sperm to undergo the acrosome reaction via the same signaling pathways as ZP3. Results show that a Le^x-containing neoglycoprotein induced the acrosome reaction in a dose-dependent and capacitation-dependent manner. A Le^a-containing neoglycoprotein also induced sperm to undergo the acrosome reaction but was less potent than Le ^x-containing neoglycoproteins. In contrast, neoglycoproteins containing β4-lactosamine (Galβ4GlcNAc), Lewis B (Fucα2Galβ 3[Fucα4]GlcNAc), and sialyl-Le^x glycans were inactive, as were four other neoglycoproteins with different nonfucosylated glycans. Consistent with these results, unconjugated Le^x- and Le^a-capped glycans were dose-dependent inhibitors, which at saturation, reduced the ZP-induced acrosome reaction by about 60% and 30%, respectively. Experiments utilizing pharmacological inhibitors suggest that induction of the acrosome reaction by solubilized ZP and Le^x- and Le^a-containing neoglycoproteins require the same calcium-dependent pathway. However, only the ZP-induced acrosome reaction requires a functional G_i protein. Thus, Le^x-containing neoglycoproteins bind to a major class of ZP3 receptors on capacitated sperm. A Le^a-containing neoglycoprotein binds a second ZP3 receptor but is a less-potent inducer of the acrosome reaction.