Human α1,3/4-Fucosyltransferases: I. IDENTIFICATION OF AMINO ACIDS INVOLVED IN ACCEPTOR SUBSTRATE BINDING BY SITE-DIRECTED MUTAGENESIS

Anton T Nguyen,Eric H Holmes,Jeffrey M Whitaker,Stephanie Ho,Susan Shetterly,Bruce A Macher

doi:10.1074/jbc.273.39.25244

Anton T Nguyen, Eric H Holmes + Show 4 more

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https://doi.org/10.1074/jbc.273.39.25244

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Abstract

In a previous study (Xu, Z., Vo, L., and Macher, B. A. (1996) J. Biol. Chem. 271, 8818-8823), a domain swapping approach demonstrated that a region of amino acids found in human alpha1, 3/4-fucosyltransferase III (FucT III) conferred a significant increase in alpha1,4-FucT acceptor substrate specificity into alpha1, 3-fucosyltransferase V (FucT V), which, under the same assay conditions, has extremely low alpha1,4-FucT acceptor substrate specificity. In the current study, site-directed mutagenesis was utilized to identify which of the eight amino acids, associated with alpha1,4-FucT acceptor substrate specificity, is/are responsible for conferring this new property. The results demonstrate that increased alpha1,4-FucT activity with both disaccharide and glycolipid acceptors can be conferred on FucT V by modifying as few as two (Asn86 to His and Thr87 to Ile) of the eight amino acids originally swapped from FucT III into the FucT V sequence. Neither single amino acid mutant had increased alpha1,4-FucT activity relative to that of FucT V. Kinetic analyses of FucT V mutants demonstrated a reduced Km for Galbeta1,3GlcNAc (type 1) acceptor substrates compared with native FucT V. However, this was about 20-fold higher than that found for native FucT III, suggesting that other amino acids in FucT III must contribute to its overall binding site for type 1 substrates. These results demonstrate that amino acid residues near the amino terminus of the catalytic domain of FucT III contribute to its acceptor substrate specificity.

Highlights

The results demonstrate that a fucosyltransferase V (FucT V) mutant containing two amino acids characteristic of the FucT III amino acid sequence has significantly higher levels of ␣1,4-FucT activity compared with FucT V
Four FucT V mutants were prepared containing amino acids at the amino (HIS substituted for NTP and HI for NT) or the carboxyl (THRKT substituted for ANSSA and RKT for SSA) terminus of the sequence of the original domain swap
Under the assay conditions used, FucT III has no detectable activity with a type 2 disaccharide, whereas FucT V has very low activity with a type 1 compared with a type 2 acceptor substrate (Fig. 2A)

Summary

The abbreviations used are

FucT, fucosyltransferase; Lc4, lactotetraosylceramide; nLc4, neolactotetraosylceramide; type 1 disaccharide, Gal␤1,3GlcNAc; type 2 disaccharide, Gal␤1,4GlcNAc; MOPS, 4-morpholinepropanesulfonic acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-propane-1,3-diol; Cer, ceramide. We demonstrated that truncated forms of FucT III and FucT V, containing ϳ300 amino acids and differing at less than 25 positions, have dramatically different acceptor substrate specificities when assayed with simple disaccharide substrates [16]. The results demonstrate that a FucT V mutant containing two amino acids characteristic of the FucT III amino acid sequence has significantly higher levels of ␣1,4-FucT activity compared with FucT V. All of these studies were completed with truncated, soluble forms of the enzymes that are fused to the IgG-binding domain of protein A. In a previous study [16], we demonstrated that the attachment of the fusion partner does not significantly alter the acceptor substrate specificity of the enzymes

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION