INACTIVATION OF THE MOUSE SPERM RECEPTOR, MZP3, BY SITE-DIRECTED MUTAGENESIS OF INDIVIDUAL SERINE RESIDUES LOCATED AT THE COMBINING SITE FOR SPERM

To initiate fertilization, mouse sperm bind to Ser-(O-) linked oligosa ccharides located at the sperm combining site of zona pellucida glycop rotein mZP3. Apparently, the oligosaccharides are present on one or mo re of five Ser residues clustered in the carboxyl-terminal region of t he mZP3 polypeptide. Here, each of the Ser residues, as well as an int ervening Asn residue, was converted to a small, nonhydroxy amino acid by site-directed mutagenesis. Mouse embryonal carcinoma (EC) cells wer e then stably transfected with the wild-type and mutated mZP3 genes. I n each case, transfected cells synthesized and secreted recombinant EC -mZP3 into the culture medium. The glycoproteins were partially purifi ed and assayed for their ability to inhibit binding of sperm to ovulat ed eggs in vitro. As compared with wild-type EC-mZP3, mutations of Ser -329, Ser-331, or Ser-333 had no effect on sperm receptor activity. Mu tation of Asn-330, a potential N-linked glycosylation site, also had n o effect on sperm receptor activity. On the other hand, mutation of ei ther Ser-332 or Ser-334, or mutation of Ser-332, Ser-333, and Ser-334, resulted in complete inactivation of EC-mZP3 as a sperm receptor. The se results suggest that Ser-332 and Ser-334, residues conserved in mou se, hamster, and human ZP3, are essential for sperm receptor activity.