Zona pellucida glycoprotein mZP3 bioactivity is not dependent on the extent of glycosylation of its polypeptide or on sulfation and sialylation of its oligosaccharides.

During fertilization in mice, free-swimming sperm bind to mZP3, one of three egg zona pellucida glycoproteins. Sperm recognize and bind to specific serine/threonine-linked (O-linked) oligosaccharides located at the mZP3 combining site for sperm. Shortly after binding to mZP3, sperm undergo the acrosome reaction, a form of cellular exocytosis. Here, we examined the influence of extent of glycosylation, sulfation, and sialylation of mZP3 (M(r) approximately 65,000-100,000) on its bioactivity; i.e. its ability to inhibit binding of sperm to eggs and to induce the acrosome reaction in vitro. Low (av. M(r) approximately 70,000), medium (av. M(r) approximately 82,000), and high (av. M(r) approximately 94,000) M(r) fractions of mZP3 were purified and shown to vary in extent of asparagine-linked (N-linked) glycosylation. All three size-fractions exhibited bioactivity, suggesting that the ability of mZP3 to inhibit binding of sperm to eggs is not related to the extent of glycosylation of its polypeptide (M(r) approximately 44,000). Digestion of mZP3 by neuraminidase decreased its average M(r) from approximately 83,000 to approximately 77,000 and increased its average pI from approximately 4.7 to approximately 6.0, but did not significantly affect mZP3 bioactivity. Terminal sialic acid largely accounts for the glycoprotein's acidic nature, but is not an essential element of the mZP3 combining site for sperm. Experiments with stably transfected embryonal carcinoma (EC) cells that secrete bioactive EC-mZP3 revealed that, of the sulfate present, approximately 70-75% was located on N-linked and approximately 25-30% on O-linked oligosaccharides. EC-mZP3 devoid of sulfate inhibited binding of sperm to eggs and induced the acrosome reaction to the same extent as sulfated EC-mZP3. These results suggest that sulfation of EC-mZP3 oligosaccharides is not essential for bioactivity. Overall, these findings contrast with those reported for certain other glycoproteins involved in cellular adhesion that require sulfate and/or sialic acid for bioactivity.