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J. Biol. Chem., Vol. 276, Issue 44, 41502-41509, November 2, 2001

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Heterogeneous Processing and Zona Pellucida Binding Activity of Pig Zonadhesin^*

John R. Hickox, Ming Bi, and Daniel M. Hardy

From the Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

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 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 D1 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.

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