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 hind to mZP3, one of three egg zona pellucida glycoproteins, Sperm recognize and bind to s pecific serine/threonine-linked (O-linked) oligosaccharides located at the mZP3 combining site for sperm, Shortly after binding to mZP3, spe rm 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) similar to 65,000-100,000) on its bioactivit y; i.e. its ability to inhibit binding of sperm to eggs and to induce the acrosome reaction in vitro. Low (av. M(r) similar to 70,000), medi um (av. M(r) similar to 82,000), and high (av. M(r) similar to 94,000) M(r) fractions of mZP3 were purified and shown to vary in extent of a sparagine-linked (N-linked) glycosylation. All three size-fractions ex hibited bioactivity, suggesting that the ability of mZP3 to inhibit bi nding of sperm to eggs is not related to the extent of glycosylation o f its polypeptide (M(r) similar to 44,000), Digestion of mZP3 by neura minidase decreased its average M(r) from similar to 83,000 to similar to 77,000 and increased its average pi from similar to 4.7 to similar to 6.0, but did not significantly affect mZP3 bioactivity, Terminal si alic acid largely accounts for the glycoprotein's acidic nature, but i s not an essential element of the mZP3 combining site for sperm, Exper iments with stably transfected embryonal carcinoma (EC) cells that sec rete bioactive EC-mZP3 revealed that, of the sulfate present, similar to 70-75% was located on N-linked and similar to 25-30% on O-linked ol igosaccharides, EC-mZP3 devoid of sulfate inhibited binding of sperm t o eggs and induced the acrosome reaction to the same extent as sulfate d EC-mZP3, These results suggest that sulfation of EC-mZP3 oligosaccha rides is not essential for bioactivity, Overall, these findings contra st with those reported for certain other glycoproteins involved in cel lular adhesion that require sulfate and/or sialic acid for bioactivity .