Localization of neutral N-linked carbohydrate chains in pig zona pellucidaglycoprotein ZPC

Zona pellucida, a transparent envelope surrounding the mammalian oocyte, pl ays important roles in fertilization and consists of three glycoproteins; Z PA, ZPB and ZPC. In pig, neutral complex-type N-linked chains obtained from a ZPB/ZPC mixture possess sperm-binding activity. We have recently reporte d that among neutral N-linked chains triantennary and tetraantennary chains have a sperm-binding activity stronger than that of diantennary chains. Tr iantennary and tetraantennary chains are localized at the second of the thr ee N-glycosylation sites of ZPB. In this study, we focused on the localizat ion of neutral N-linked chains in ZPC. ZPB and ZPC can not be separated fro m each other unless the acidic N-acetyllactosamine regions of their carbohy drate chains are removed by endo-beta-galactosidase digestion. A large part of the acidic N-linked chains becomes neutral by the digestion, but the ma in neutral N-linked chains are not susceptible to the enzyme. N-glycanase d igestion indicated that ZPC has three N-glycosylation sites. Three glycopep tides each containing one of the N-glycosylation sites were obtained by try ptic digestion of ZPC and the N-glycosylation sites were revealed as Asn124 , Asn146 and Asn271. The carbohydrate structures of the neutral N-Linked ch ains from each glycopeptide were characterized by two-dimensional sugar map ping analysis taking into consideration the structures of the main, intact neutral N-linked chains of ZPB/ZPC mixture reported previously. Triantennar y and tetraantennary chains were found mainly at Asn271 of ZPC, whereas dia ntennary chains were present at all three N-glycosylation sites. Thus, ZPC has tri-antennary and tetra-antennary chains as well as ZPB, but the locali zation of the chains is different from that in ZPB.