Localization of sperm ligand carbohydrate chains in pig zona pellucida glycoproteins

Neutral N-linked carbohydrate chains from pig ZPB/ZPC mixture are shown to possess sperm ligand activity. Of these complex-type chains, triantennary/tetraantennary chains exhibit the activity stronger than that of diantennary chains. Intact ZPB and ZPC cannot be separated from each other unless acidic N-acetyllactosamine regions of their carbohydrate chains are removed by endo-beta-galactosidase digestion. The endo-beta-galactosidase-digested ZPB and its N-terminal fragment of 111 residues retain the sperm ligand activity. Three glycopeptides, having one Asn residue to which the carbohydrate chain is linked, are obtained by lysylendopeptidase digestion of the heat-solubilized zonae containing intact ZPB, and lysylendopeptidase and chymotryptic digestions of endo-beta-galactosidase-digested ZPB. On the basis of sugar mapping analysis of the N-linked chains from these glycopeptides and comparison with the carbohydrate structures of the main intact neutral N-linked chains of ZPB/ZPC, the triantennary and tetraantennary chains are shown to be localized mainly at Asn220 of ZPB, whereas diantennary chains are present on all the three N-glycosylation sites (Asn203, Asn220 and Asn333). These results suggest that the carbohydrate chains linked to Asn220 of ZPB participate predominantly in sperm-egg binding. ZPC has been shown to support the expression of sperm ligand activity of ZPB. Three glycopeptides, each having one of the N-glycosylation sites, are obtained by tryptic digestion of endo-beta-galactosidase-digested ZPC. Triantennary and tetraantennary chains are found mainly at Asn271 of ZPC, whereas diantennary chains are present at all three N-glycosylation sites (Asn124, Asn146 and Asn271). Thus, the localization of triantennary and tetraantennary chains in ZPC is different from that in ZPB.