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Originally published In Press as doi:10.1074/jbc.M207369200 on October 2, 2002

J. Biol. Chem., Vol. 277, Issue 49, 47088-47096, December 6, 2002
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The Lectin Domain of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase 1 Is Involved in O-Glycosylation of a Polypeptide with Multiple Acceptor Sites*

Mari TennoDagger , Aki SaekiDagger , Ferénc J. Kézdy§, Åke P. Elhammer§, and Akira KurosakaDagger ||

From the Dagger  Department of Biotechnology, Faculty of Engineering, and  Institute for Comprehensive Research, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto 603-8555, Japan and § Pharmacia Corp., Kalamazoo, Michigan 49007

Mucin type O-glycosylation begins with the transfer of GalNAc to serine and threonine residues on proteins by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminlytransferases. These enzymes all contain a lectin-like (QXW)3 repeat sequence at the C terminus that consists of three tandem repeats (alpha , beta , and gamma ). The putative lectin domain of one of the most ubiquitous isozymes, GalNAc-T1, is reportedly not functional. In this report, we have reevaluated the role of the GalNAc-T1 lectin domain. Deletion of the lectin domain resulted in a complete loss of enzymatic activity. We also found that GalNAc-T1 has two activities distinguished by their sensitivities to inhibition with free GalNAc; one activity is sensitive, and the other is resistant. In our experiments, the former activity is represented by the O-glycosylation of apomucin, an acceptor that contains multiple glycosylation sites, and the latter is represented by synthetic peptides that contain a single glycosylation site. Site-directed mutagenesis of the lectin domain selectively reduced the former activity and identified Asp444 in the alpha  repeat as the most important site for GalNAc recognition. A further reduction of the GalNAc-inhibitable activity was observed when both Asp444 and the corresponding aspartate residues in the beta  and the gamma  repeats were mutated. This suggests a cooperative involvement of each repeat unit in the glycosylation of polypeptides with multiple acceptor sites.

* This work was in part supported by the Research Foundation for Pharmaceutical Science, Sasakawa Scientific Research Grant, and the Foundation for Bio-venture Research Center from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The 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 Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto 603-8555, Japan. Tel.: 81-75-705-1894; Fax: 81-75-705-1914; E-mail: kurosaka@cc.kyoto-su.ac.jp.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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