Alpha 1,3-fucosyltransferases (FucT) share a conserved amino acid sequence designated the alpha 1,3 FucT motif that has been proposed to be important for nucleotide sugar binding. To evaluate the importance of the amino acids in this motif, each of the alpha 1,3 FucT motif amino acids was replaced with alanine (alanine scanning mutagenesis) in human FucT VI, and the resulting mutant proteins were analyzed for enzyme activity and kinetically characterized in those cases in which the mutant protein had sufficient activity. Two of the mutant proteins were inactive, six had less than 1% of wild-type activity, and four had approximately 10-50% of wild-type enzyme activity. Three of the mutant proteins with significant enzyme activity had substantially larger Km (5 to 15 times) for GDP-fucose than FucT VI wild-type enzyme. The fourth mutant protein with significant enzyme activity (S249A) had a Km at least 10 times larger than wild-type FucT VI for the acceptor substrate, with only a slightly larger (2-3 times) Km for GDP-fucose. Thus mutation of any of the amino acids within the alpha 1,3 FucT motif to Ala affects alpha 1,3-FucT activity, and substitution of Ala for some of the alpha 1,3 FucT motif amino acids results in proteins with altered kinetic constants for both the acceptor and donor substrates. Secondary structure prediction suggests a helix-loop-helix fold for the alpha 1,3 FucT motif, which can be used to rationalize the effects of mutations in terms of 3D structure.
Fucose transfer from GDP-fucose to GlcNAc residues of the sialylated polylactosamine acceptor NeuAc␣2-3Gal1-4Glc-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 ϳ12-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. ␣1,3/4-Fucosyltransferases (FucTs)3 catalyze transfer of fucose to GlcNAc residues present in lacto or neolacto series structures of cell-surface glycolipids and glycoproteins. Fucosylated structures are known to accumulate in many human cancers (1-4), function as ligands for leukocyte adhesion during inflammation (4, 5), and undergo developmental regulation (6 -8). The diversity of naturally occurring ␣1,3/4-fucosylated structures found in human cells is controlled by a family of six distinct yet related FucTs with individual tissue distribution properties and acceptor substrate preferences. Among these six FucTs, FucT-III, FucT-V, and FucT-VI share Ͼ85% amino acid sequence homology and originated from a common ancestral gene via gene duplication (9 -11). Genes for these enzymes form a cluster on chromosome 19 in humans (12-15). FucT-IV, FucT-VII, and FucT-IX share a lower amino acid sequence homology both between each other and with FucT-III, FucT-V, and FucT-VI. Additionally, they have distinct chromosomal localization. Genes for FucT-IV, FucT-VII, and FucT-IX have been mapped to chromosomes 11, 9, and 6, respectively (16 -18).Although all of these enzymes catalyze fucose transfer to (LacNAc)Gal1-4GlcNAc chain acceptors to produce ...
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