Structure and function of the N-linked carbohydrate chains of pig zona pellucida glycoproteins

The N-linked carbohydrate chain of the PZP3 glycoprotein family of pig zona pellucida is shown by competition assay to possess sperm receptor activity. Structural analysis reveals that the N-linked chains comprise neutral and acidic complex-type chains. The tri- or tetra-antennary fraction in the neutral chains possesses the sperm receptor activity, but the activity is markedly reduced compared with that of PZP3, suggesting that the protein moiety participates in the maintenance of proper orientation of the active chain. Two components in the PZP3 family can be separated by reverse-phase HPLC after endo-beta-galactosidase (E beta G) digestion and one of the digests, E beta G-PZP3 alpha, possesses the sperm receptor activity. E beta G-PZP3 alpha deprived of O-linked chains by treatment with alkali retains the activity comparable with E beta G-PZP3 alpha, while E beta G-PZP3 alpha deprived of N-linked chains by N-glycanase digestion loses the activity. Furthermore, competition assay of the digests of E beta G-PZP3 alpha with lysyl-endopeptidase indicates that the active chain is linked to Asn67 or Asn84 of PZP3 alpha or to both residues. We conclude that sperm-egg binding in pigs is mainly mediated by tri- or tetra-antennary, neutral, complex-type N-linked carbohydrate chain(s) localized in the N-terminal region of PZP3 alpha protein.