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J. Biol. Chem., Vol. 280, Issue 21, 20189-20196, May 27, 2005

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Recombinant Porcine Zona Pellucida Glycoproteins Expressed in Sf9 Cells Bind to Bovine Sperm but Not to Porcine Sperm^*

Naoto Yonezawa, Katsuyasu Kudo, Hirotomo Terauchi, Saeko Kanai, Naoto Yoda, Masaru Tanokura¶, Kosuke Ito¶, Kin-ichiro Miura||, Toshiyuki Katsumata**, and Minoru Nakano

From the Graduate School of Science and Technology and the Department of Chemistry, Faculty of Science, Chiba University, Inage-ku, Chiba 263-8522, the ^¶Graduate School of Agriculture and Life Science, the University of Tokyo, Hongo, Tokyo 113-8657, the ^||Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171-8588, and the ^**College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Ichikawa, Chiba 272-0827, Japan

The zona pellucida, which surrounds the mammalian oocyte, consists of the ZPA, ZPB, and ZPC glycoproteins and plays roles in species-selective sperm-egg interactions via its carbohydrate moieties. In the pig, this activity is conferred by tri- and tetraantennary complex type chains; in cattle, it is conferred by a chain of 5 mannose residues. In this study, porcine zona glycoproteins were expressed as secreted forms, using the baculovirus-Sf9 insect cell system. The sperm binding activities of the recombinant proteins were examined in three different assays. The assays clearly demonstrated that recombinant ZPB bound bovine sperm weakly but did not bind porcine sperm; when recombinant ZPC was also present, bovine sperm binding activity was greatly increased, but porcine sperm still was not bound. The major sugar chains of ZPB were pauci and high mannose type chains that were similar in structure to the major neutral N-linked chain of the bovine zona. In fact, the nonreducing terminal -mannose residues were necessary for the sperm binding activity. These results show that the carbohydrate moieties of zona glycoproteins, but not the polypeptide moieties, play an essential role in species-selective recognition of porcine and bovine sperm. Moreover, Asn to Asp mutations at either of two of the N-glycosylation sites of ZPB, residue 203 or 220, significantly reduced the sperm binding activity of the ZPB/ZPC mixture, whereas a similar mutation at the third N-glycosylation site, Asn-333, had no effect on binding. These results suggest that the N-glycans located in the N-terminal half of the ZP domain of porcine ZPB are involved in sperm-zona binding.

Received for publication, December 17, 2004 , and in revised form, March 23, 2005.

^* This work was supported by grants-in-aid for Scientific Research, and the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Chemistry, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. Tel.: 81-43-290-2794; Fax: 81-43-290-2874; E-mail: mnakano{at}faculty.chiba-u.jp.

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