Heterogeneous processing and zona pellucida binding activity of pig zonadhesin

Zonadhesin is a mosaic protein in sperm membrane fractions that binds direc tly and in a species-specific manner to the extracellular matrix (zona pell ucida) of the oocyte. The active form of pig zonadhesin from capacitated, e pididymal spermatozoa comprises two covalently associated polypeptide chain s 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 cell s. 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 polyp eptides in particulate fractions of uncapacitated cells. Although the p105/ 45 form constituted a minority of all zonadhesin forms in sperm membrane fr actions, it was the predominant form capable of binding to the pig zona pel lucida. Zonadhesin-binding sites were distributed over the entire zona pell ucida. Anion exchange chromatography resolved active, p105/45 zonadhesin fr om the p60-90 inactive forms. Without disulfide bond reduction some zonadhe sin 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. Expr essed Dl and D3 domain fragments containing the CG(L/V)CG sequence motif sp ontaneously formed multimers at -246 mV E-h in vitro. Double Cys --> Ser mu tants of the D1 fragment formed multimers with the same apparent kinetics a s the wild type protein. Zonadhesin localized to the apical head of pig spe rmatozoa. We conclude that a heterogeneous combination of specific proteoly sis and intermolecular disulfide bond formation in the sperm head generates multiple forms of zonadhesin with differing avidities for the zona pelluci da.