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J. Biol. Chem., Vol. 282, Issue 34, 24882-24892, August 24, 2007

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Site-specific Fucosylation of Sialylated Polylactosamines by 1,3/4-Fucosyltransferases-V and -VI Is Defined by Amino Acids Near the N Terminus of the Catalytic Domain^*

Susan Shetterly¹, Franziska Jost, Susan R. Watson, Ronald Knegtel^¶, Bruce A. Macher, and Eric H. Holmes²

From the Department of Chemistry and Biochemistry, San Francisco State University, and the Department of Microbiology and Immunology, University of California, San Francisco, California 94132 and ^¶Vertex Pharmaceuticals (Europe) Limited, Abingdon, Oxfordshire OX14 4RY, United Kingdom

Fucose transfer from GDP-fucose to GlcNAc residues of the sialylated polylactosamine acceptor NeuAc2-3Gal1-4Glc-NAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-ceramide leads to two isomeric monofucosyl antigens, VIM2 and sialyl-Le^x. Human 1,3/4-fucosyltransferase (FucT)-V catalyzes primarily the synthesis of VIM2, whereas human FucT-VI catalyzes primarily the synthesis of sialyl-Le^x. Thus, these two enzymes have distinct "site-specific fucosylation" properties. Amino acid sequence alignment of these enzymes showed that there are 24 amino acid differences in their catalytic domains. Studies were conducted to determine which of the amino acid differences are responsible for the site-specific fucosylation properties of each enzyme. Domain swapping (replacing a portion of the catalytic domain from one enzyme with an analogous portion from the other enzyme) demonstrated that site-specific fucosylation was defined within a 40-amino acid segment containing 8 amino acid differences between the two enzymes. Site-directed mutagenesis studies demonstrated that the site-specific fucosylation properties of these enzymes could be reversed by substituting 4 amino acids from one sequence with the other. These results were observed in both in vitro enzyme assays and flow cytometric analyses of Chinese hamster ovary cells transfected with plasmids containing the various enzyme constructs. Modeling studies of human FucT using a structure of a bacterial fucosyltransferase as a template demonstrated that the amino acids responsible for site-specific fucosylation map near the GDP-fucose-binding site. Additional enzyme studies demonstrated that FucT-VI has 12-fold higher activity compared with FucT-V and that the Trp¹²⁴/Arg¹¹⁰ site in these enzymes is responsible primarily for this activity difference.

Received for publication, March 20, 2007 , and in revised form, June 27, 2007.

^* This work was supported in part by Grant MCB-9816780 from the National Science Foundation and Grant P20 MD000262 from the National Center on Minority Health and Health Disparities (to B. A. M.) and by Grant R01 CA70740 from the National Institutes of Health (to E. H. H.). 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.

¹ Present address: Amgen, 1120 Veterans Blvd., South San Francisco, CA 94080.

² To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132. Tel.: 425-373-0171 (ext. 1026); Fax: 425-373-0181; E-mail: eholmes{at}sfsu.edu.

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