Human α1,3/4-Fucosyltransferases

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 ␣1,3/4-fucosyltransferase III (FucT III) conferred a significant increase in ␣1,4-FucT acceptor substrate specificity into ␣1,3-fucosyltransferase V (FucT V), which, under the same assay conditions, has extremely low ␣1,4-FucT acceptor substrate specificity. In the current study, site-directed mutagenesis was utilized to identify which of the eight amino acids, associated with ␣1,4-FucT acceptor substrate specificity, is/ are responsible for conferring this new property. The results demonstrate that increased ␣1,4-FucT activity with both disaccharide and glycolipid acceptors can be conferred on FucT V by modifying as few as two (Asn 86 to His and Thr 87 to Ile) of the eight amino acids originally swapped from FucT III into the FucT V sequence. Neither single amino acid mutant had increased ␣1,4-FucT activity relative to that of FucT V. Kinetic analyses of FucT V mutants demonstrated a reduced K m for Gal␤1,3GlcNAc (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.␣1,3/4-Fucosyltransferases (FucTs) 1 can bind a wide variety of acceptor substrates (see Ref. 1 and references therein) and catalyze the synthesis of glycoconjugates with different immunological and functional properties including blood group antigens and selectin ligands (2-9). Interestingly, the acceptor substrate specificity of different forms of the human FucTs with highly homologous amino acid sequences can differ significantly (1, 10 -13). For example, FucT III has a very high level of activity with a type 1 disaccharide acceptor (Gal␤1, 3GlcNAc), whereas FucT VI appears to be a true ␣1,3-FucT and utilizes exclusively type 2 acceptors (1, 10 -15 substrate specificities when assayed in vitro with disaccharide acceptors despite the fact that they share Ͼ90% amino acid sequence homology (10 -13). In addition, some of these enzymes can bind the same acceptor substrates but produce different products. For example, mammalian cells transfected with FucT V can produce cell surface antigens recognized by anti-VIM 2 and anti-difucosyl-sLe x , whereas cells transfected with FucT VI express antigens recognized by antidifucosyl-sLe x but not anti-VIM 2 (11, 13). In a previous study, 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). Furthermore, we found that the swapping of an NH 2 -terminal segment of FucT III, containing eight amino acids u...

Highly Conserved Cysteines of Mouse Core 2 β1,6-N-Acetylglucosaminyltransferase I Form a Network of Disulfide Bonds and Include a Thiol That Affects Enzyme Activity

“…3 Interestingly, the disulfide linkage formed between Cys 59 and Cys 413 brings the amino acid residues located near the N and C termini of the catalytic domain within close proximity. This distinctive arrangement is similar to that occurring in ␣(1,3/1,4) fucosyltransferase III (51) and polysialyltransferase ST8Sia IV (52), where site-directed mutagenesis demonstrated that such steric conformation may be associated with acceptor substrate specificity (53,54) and catalytic efficiency (52), respectively. Similarly, the catalytic activity of GM2 synthase depends on the formation of disulfide-bonded homodimers, whose antiparallel orientation also brings the N and C termini in close proximity, most likely to establish a catalytic domain (55).…”

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

“…In particular, expression of the aromatic Trp 111 residue of FucT-III (corresponding to Trp 124 of FucT-V) has been shown to be primarily responsible for the type 1 chain acceptor specificity found in FucT-III and FucT-V (22). Additional evidence has demonstrated that higher type 1 chain specificity can be conferred upon FucT-V by substitution of Asn 86 and Thr 87 with His and Ile, respectively, amino acids found in the corresponding positions in FucT-III (20). Taken together, the results independently indicate that, in addition to having a role in defining type 1 versus type 2 chain specificity, amino acids in positions corresponding to Thr 87 and Trp 124 of FucT-V also function in defining a preference for the fucosylation of an internal versus distal GlcNAc residue of sialylated polylactosamines, leading to VIM2 or sialyl-Le x structures.…”

Human α1,3/4-Fucosyltransferases

“…An increasing amount of information is becoming available to show how specific amino acid residues within the more variable regions of the catalytic domain of these proteins influence specific properties of a given enzyme form (Refs. 17, 21, and 23-32; see also accompanying papers (34,36)). The results presented in this paper address a region of the catalytic domain that is instead rather highly conserved among enzyme forms.…”

“…Western Blot Analysis of pPROTA-expressed Enzymes-The pPROTAexpressed recombinant FucT enzymes were separated on 12% Tris/ glycine polyacrylamide gels, transferred to nitrocellulose membranes, and probed as described in an accompanying paper (34).…”