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Volume 271, Number 39, Issue of September 27, 1996 pp. 24069-24074
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Site-directed Mutagenesis of Rabbit Proacrosin
IDENTIFICATION OF RESIDUES INVOLVED IN ZONA PELLUCIDA BINDING

(Received for publication, April 4, 1996, and in revised form, June 24, 1996)

From the Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, North Carolina 27599

The mammalian acrosomal sperm protease proacrosin plays a role in fertilization by proteolysis of the oocyte's outer investments. In addition to its serine protease activity, acrosin from several species is known to have binding activity for the zona pellucida, and this action may serve to anchor sperm during zona penetration. In this study, proacrosin was purified from acid extracts of rabbit sperm and shown to bind to homologous zona pellucida using an in vitro assay. Measurement of this binding activity indicated a high affinity saturable interaction with a K_D = 1.4 × 10⁸ M.

Using cDNAs obtained from previously cloned and sequenced rabbit proacrosin and a splice variant that encodes a shorter form of acrosin (Richardson, R. T., and O'Rand, M. G. (1994) Biochim. Biophys. Acta 1219, 215-218), constructs of various sizes were produced using polymerase chain reaction and expressed as recombinant proteins. In the same in vitro zona binding assay, a construct representing residues 1-279 of rabbit proacrosin was found to bind to zona with a high affinity similar to that of native proacrosin, K_D = 2.1 × 10⁸ M. By making smaller recombinant fragments and assaying them for zona binding activity, the location of the binding site was mapped to residues 47-94. Protein modeling of rabbit proacrosin using chymotrypsinogen A as a three-dimensional model indicated that an exposed loop Asp³⁵ to His⁴⁰ in chymotrypsinogen A is extended with an additional five amino acid residues in rabbit proacrosin from Ile⁴³ to His⁵³ containing arginine residues Arg⁴⁷, Arg⁵⁰ and Arg⁵¹. Site-directed mutagenesis of arginine residues Arg⁵⁰ and Arg⁵¹ to alanine produced a recombinant without significant zona binding activity. These results are consistent with the hypothesis that rabbit proacrosin contains a specific zona pellucida binding site and that the loop containing arginine residues 50 and 51 is critical for zona binding activity.

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