IDENTIFICATION OF AN N-GLYCOSYLATED REGION OF PIG ZONA-PELLUCIDA GLYCOPROTEIN ZPB THAT IS INVOLVED IN SPERM BINDING

ZPB, one of the pig zona pellucida glycoproteins, can be purified afte r removal of sialylated and/or sulfated N-acetylpolylactosamine from t he nonreducing region of its carbohydrate chains by digestion with end o-beta-galactosidase. Among the components produced, only ZPB shows sp erm-binding activity after the digestion. Recently, we have shown that N-linked carbohydrate chains of endo-beta-galactosidase-digested ZPB (E beta G-ZPB) are predominantly involved in sperm binding [Yonezawa, N., Aoki, H., Hatanaka, Y. & Nakano, M. (1995) Eur J. Biochem. 233, 35 -41]. In this study, to define the sperm-binding region in E beta G-ZP B, glycopeptides were purified from lysyl endopeptidase digests of E b eta G-ZPB and analyzed for sperm-binding activity by an in vitro compe tition assay. The locations of the glycopeptides were determined from partial amino acid sequences, amino acid and sugar composition analyse s, and apparent molecular masses after SDS/PAGE. The N-terminal fragme nt (amino acid residues 137-247), which contains two N-linked carbohyd rate chains, showed a significant inhibition of sperm-egg binding. How ever, the fragment that had one N-linked carbohydrate chain (residues 325-341) and the fragment that had two or three O-linked carbohydrate chains (residues 248-324) did not inhibit sperm-egg binding. Thus, the two N-linked carbohydrate chains in the N-terminal fragment of E beta G-ZPB are important for sperm binding of pig zona pellucida.