LOCALIZATION OF CARBOHYDRATE CHAINS OF PIG SPERM LIGAND IN THE GLYCOPROTEIN ZPB OF EGG ZONA-PELLUCIDA

The three glycoproteins of pig zona pellucida (ZPA, ZPB and ZPC) can b e separated into ZPA and a mixture of ZPB/ZPC by gel-filtration HPLC. We have shown previously that the neutral complex-type N-linked carboh ydrate chains obtained from ZPB/ZPC possess sperm-binding activity. In tact ZPB and ZPC cannot be separated from each other unless acidic N-a cetyllactosamine regions of their carbohydrate chains are removed by e ndo-beta-galactosidase digestion. The endo-beta-galactosidase-digested ZPB retains the sperm-binding activity. Recently, we have reported th at N-linked carbohydrate chains of N-terminal fragment (residues 137-2 47) obtained from endo-beta-galactosidase-digested ZPB are involved ma inly in sperm binding [Yonezawa, N., Mitsui, S., Kudo, K. & Nakano, M. (1997) fur: J. Biochem. 248, 86-92]. In this study, we separated the intact neutral N-linked chains from the ZPB/ZPC mixture into diantenna ry chains and triantennary and tetraantennary chains by affinity chrom atography on Concanavalia ensiformis agglutinin. An in vitro competiti on assay revealed that triantennary and tetraantennary chains possess a sperm-binding activity stronger than that of diantennary chains. Thr ee glycopeptides, having one Asn residue to which the carbohydrate cha in is linked, were obtained by lysyl endopeptidase digestion of the he at-solubilized zonae containing intact ZPB and lysyl endopeptidase and chymotrypsin A digestion of endo-beta-galactosidase-digested ZPB. Fro m sugar-mapping analysis of the carbohydrate chains from these glycope ptides and comparison with the carbohydrate structures of the main int act neutral N-linked chains of ZPB/ZPC, the triantennary and tetraante nnary chains were shown to be localized mainly at Asn220 of ZPB, and d iantennary chains were present on all the three potential residues (As n203, Asn220 and Asn333). These results suggest that the carbohydrate chains linked to Asn220 of ZPB participate predominantly in sperm-egg binding.