Katsuyasu Kudo

Publications (2)4.77 Total impact

• Article: 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, Minoru Nakano
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  ABSTRACT: 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 alpha-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.
  Journal of Biological Chemistry 06/2005; 280(21):20189-96. · 4.77 Impact Factor
• Article: Identification of an N‐Glycosylated Region of Pig Zona Pellucida Glycoprotein ZPB that is involved in Sperm Binding

  Naoto Yonezawa, Sachiko Mitsui, Katsuyasu Kudo, Minoru Nakano
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  ABSTRACT: ZPB, one of the pig zona pellucida glycoproteins, can be purified after removal of sialylated and/or sulfated N-acetylpolylactosamine from the nonreducing region of its carbohydrate chains by digestion with endo-β-galactosidase. Among the components produced, only ZPB shows sperm-binding activity after the digestion. Recently, we have shown that N-linked carbohydrate chains of endo-β-galactosidase-digested ZPB (EβG-ZPB) are predominantly involved in sperm binding [Yonezawa, N., Aoki, H., Hatanaka, Y. & Nakano, M. (1995) Eur. J. Biochem. 233, 35–41]. In this study, to define the sperm-binding region in EβG-ZPB, glycopeptides were purified from lysyl endopeptidase digests of EβG-ZPB and analyzed for sperm-binding activity by an in vitro competition assay. The locations of the glycopeptides were determined from partial amino acid sequences, amino acid and sugar composition analyses, and apparent molecular masses after SDS/PAGE. The N-terminal fragment (amino acid residues 137–247), which contains two N-linked carbohydrate chains, showed a significant inhibition of sperm-egg binding. However, the fragment that had one N-linked carbohydrate chain (residues 325–341) and the fragment that had two or three O-linked carbohydrate chains (residues 248–324) did not inhibit sperm-egg binding. Thus, the two N-linked carbohydrate chains in the N-terminal fragment of EβG-ZPB are important for sperm binding of pig zona pellucida.
  European Journal of Biochemistry. 07/1997; 248(1):86 - 92.