Site-specific Fucosylation of Sialylated Polylactosamines by α1,3/4-Fucosyltransferases-V and -VI Is Defined by Amino Acids Near the N Terminus of the Catalytic Domain

• Published in: The Journal of biological chemistry Vol. 282; no. 34; p. 24882
• Main Authors: Susan Shetterly, Franziska Jost, Susan R. Watson, Ronald Knegtel, Bruce A. Macher, Eric H. Holmes
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 24-08-2007
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M702395200

Fucose transfer from GDP-fucose to GlcNAc residues of the sialylated polylactosamine acceptor NeuAcα2-3Galβ1-4Glc-NAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-ceramide
leads to two isomeric monofucosyl antigens, VIM2 and sialyl-Le x . Human α1,3/4-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 Trp 124 /Arg 110 site in these enzymes is responsible primarily for this activity difference.