Heterogeneous processing and zona pellucida binding activity of pig zonadhesin

Zonadhesin is a mosaic protein in sperm membrane fractions that binds directly and in a species-specific manner to the extracellular matrix (zona pellucida) of the oocyte. The active form of pig zonadhesin from capacitated, epididymal spermatozoa comprises two covalently associated polypeptide chains of M-r 105,000 (p105) and M-r 45,000 (p45). Here we report detection and characterization of multiple zonadhesin isoforms in freshly ejaculated cells. Antibodies to the predicted von Willebrand D0-D1, D1, and D3 domains of pig zonadhesin recognized p105, p45, and additional M-r 60,000-90,000 polypeptides in particulate fractions of uncapacitated cells. Although the p105/45 form constituted a minority of all zonadhesin forms in sperm membrane fractions, it was the predominant form capable of binding to the pig zona pellucida. Zonadhesin-binding sites were distributed over the entire zona pellucida. Anion exchange chromatography resolved active, p105/45 zonadhesin from the p60-90 inactive forms. Without disulfide bond reduction some zonadhesin was M-r greater than or equal to 300,000, including M-r 300,000 and 900,000 proteins comprising in part multimers of p105/45. The multimeric forms did not bind the zona pellucida as avidly as did the p105/45 monomer. Expressed Dl and D3 domain fragments containing the CG(L/V)CG sequence motif spontaneously formed multimers at -246 mV E-h in vitro. Double Cys --> Ser mutants of the D1 fragment formed multimers with the same apparent kinetics as the wild type protein. Zonadhesin localized to the apical head of pig spermatozoa. We conclude that a heterogeneous combination of specific proteolysis and intermolecular disulfide bond formation in the sperm head generates multiple forms of zonadhesin with differing avidities for the zona pellucida.