Recognition of β-d-Gal p-(1 → 3)-β-d-Glc pNAc-OR acceptor analogues by the Lewis α-(1 → 34)-fucosyltransferase from human milk

The minimal catalytic domain of ␣-(1,3/1,4)-fucosyltransferases (FucTs) from Helicobacter pylori strains NCTC11639 and UA948 was mapped by N-and C-terminal truncations. Only the C terminus could be truncated without significant loss of activity. 11639FucT and UA948FucT contain 10 and 8 heptad repeats, respectively, which connect the catalytic domain with the C-terminal putative amphipathic ␣-helices. Deletion of all heptad repeats almost completely abolished enzyme activity. Nevertheless, with only one heptad repeat 11639FucT is fully active, whereas UA948FucT is partially active. Removal of the two putative amphipathic ␣-helices dramatically increased protein expression and solubility, enabling purification with yields of milligrams/liter. Steady-state kinetic analysis of the purified FucTs showed that 11639FucTs possessed slightly tighter binding affinity for both Type II acceptor and GDPfucose donor than UA948FucT, and its k cat of 2.3 s ؊1 was double that of UA948FucT, which had a k cat value of 1.1 s ؊1 for both Type II and Type I acceptors. UA948FucT strongly favors Type II over the Type I acceptor with a 20-fold difference in acceptor K m . Sixteen modified Type I and Type II series acceptors were employed to map the molecular determinants of acceptors required for recognition by H. pylori ␣-(1,3/1,4)-FucTs. Deoxygenation at 6-C of the galactose in Type II acceptor caused a 5000-fold decrease in ␣1,3 activity, whereas in Type I acceptor this completely abolished ␣1,4 activity, indicating that this hydroxyl group is a key polar group.

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confidence: 99%

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confidence: 99%

Purification, Kinetic Characterization, and Mapping of the Minimal Catalytic Domain and the Key Polar Groups of Helicobacter pylori α-(1,3/1,4)-Fucosyltransferases

Audette

Lin

et al. 2006

“…This was confirmed in several glycosyltransferases with resolved crystal structures as reviewed previously (43). Using a panel of monodeoxygenated Type I and Type II acceptor substrates, the 6-OH of the galactose moiety in both Type I and Type II acceptors and the reactive hydroxyl group (the OH-4 and OH-3 of GlcNAc in Type I and Type II, respectively) were found to be essential for the recognition by human FucT III, IV, V (44), VI (45), and human milk ␣1,3 and ␣1, 3/4 FucTs (46,47). This suggests that Type I and Type II acceptors, when they bind to human ␣1,3 and ␣1,3/4 FucTs, most likely adopt a conformation so that the OH-4 of GlcNAc in Type I acceptor is placed in the same position relative to the 6-OH of the galactose moiety as is the OH-3 of GlcNAc in Type II acceptor.…”

Section: Discussionmentioning

confidence: 99%

A Single Aromatic Amino Acid at the Carboxyl Terminus of Helicobacter pylori α1,3/4 Fucosyltransferase Determines Substrate Specificity

Lau

Palcic

et al. 2005

Fucosyltransferases (FucT) from differentHelicobacter pylori strains display distinct Type I (Gal␤1,3GlcNAc) or Type II (Gal␤1,4GlcNAc) substrate specificity. FucT from strain UA948 can transfer fucose to the OH-3 of Type II acceptors as well as to the OH-4 of Type I acceptors on the GlcNAc moiety, so it has both ␣1,3 and ␣1,4 activities. In contrast, FucT from strain NCTC11639 has exclusive ␣1,3 activity. Our domain swapping study ( Helicobacter pylori is associated with gastritis and peptic ulcer formation and is a risk factor for the development of gastric cancer and mucosa-associated lymphoid tissue lymphoma. One of the virulence factors of H. pylori is the lipopolysaccharide, which contains lipid A, core oligosaccharide and O-antigens. The O-antigens of H. pylori lipopolysaccharide contain fucosylated oligosaccharides, predominantly the Type II blood group antigens, Lewis X (Gal␤1,4(Fuc␣1,3)GlcNAc) and Lewis Y (Fuc␣1,2Gal␤1,4(Fuc␣1,3)GlcNAc) (1), but a small number of H. pylori strains also express the Type I blood group antigens, Lewis A (Gal␤1,3(Fuc␣1,4)GlcNAc), and Lewis B (Fuc␣1,2Gal␤1,3(Fuc␣1,4)-GlcNAc) (2).The role of Lewis antigens in H. pylori pathogenesis is still ambiguous. It has been suggested that Lewis antigens play a role in H. pylori adhesion to (3, 4) or internalization by (5) the gastric epithelial cells. Nevertheless, conflicting evidence argues that Lewis X and Lewis Y are not required for colonization of human gastric epithelium (6) or mouse stomach (7,8). Lewis antigens may also play an important role in the persistence of H. pylori infection by molecular mimicry, helping the bacteria to evade the host immune response (2, 9, 10). Environmental changes such as pH influence the expression of H. pylori O-antigens, particularly Lewis X and Lewis Y. This may aid in adaptation of the bacterium to its niche in the stomach (10).Fucosyltransferases (FucTs) 3 are enzymes responsible for the last steps in the synthesis of Lewis antigens in H. pylori (11,12). ␣1,2 and ␣1,3 or ␣1,3/4 FucTs have been identified and characterized in H. pylori (13-18). These FucTs catalyze the transfer of the L-fucose moiety from guanosine diphosphate ␤-L-fucose (GDP-Fuc) to the OH-2 of the galactose moiety and the OH-3 or the OH-3 and the OH-4 positions of the GlcNAc moiety in glycoconjugate acceptors, respectively. The H. pylori genome contains two homologous ␣1,3 or ␣1,3/4 FucT genes, futA and futB (19,20), but they do not always encode functional proteins. For instance, only the futA gene encodes an active FucT in H. pylori strains NCTC11639 and UA948 (13,17). Bacterial ␣1,3/4 FucTs are functionally equivalent to the mammalian ␣1,3/4 FucTs, which have been well characterized. Mammalian FucTs are Type II membrane proteins with a short N-terminal cytoplasmic tail, transmembrane domain, stem region, and C-terminal catalytic domain. H. pylori FucTs share weak homology with their mammalian counterparts in two small segments within the catalytic domain, called ␣1,3 FucT motifs (14, 21). H. pylori ␣1,3/4 FucTs lack the N...

“…(30) suggested that oligosaccharide-reactive acceptor hydroxyl groups are involved in a critical hydrogen bond donor interaction with the glycosyltransferases. The main key polar groups on the oligosaccharide acceptors have been identified as the reactive hydroxyls at C-3 or C-4 of GlcNAc and C-6 of Gal (31,32). On the enzyme side, different amino acids can be involved in this hydrogen bond, but typical hydrogen bond acceptors are His and carboxylates (26), as those found in the conserved amino acid positions flanking the candidate Arg or Trp residue for the definition of the type 1/type 2 enzyme activity.…”

Section: Discussionmentioning

confidence: 99%

A Single Amino Acid in the Hypervariable Stem Domain of Vertebrate α1,3/1,4-Fucosyltransferases Determines the Type 1/Type 2 Transfer

Dupuy¹,

Petit²,

Mollicone³

et al. 1999

Alignment of 15 vertebrate ␣1,3-fucosyltransferases revealed one arginine conserved in all the enzymes employing exclusively type 2 acceptor substrates. At the equivalent position, a tryptophan was found in FUT3-encoded Lewis ␣1,3/1,4-fucosyltransferase (Fuc-TIII) and FUT5-encoded ␣1,3/1,4-fucosyltransferase, the only fucosyltransferases that can also transfer fucose in ␣1, Fucosyltransferases are type II transmembrane proteins catalyzing fucose transfer from GDP-fucose to different oligosaccharide acceptors in ␣1,2-, ␣1,3-, ␣1,4-, and ␣1,6-linkages. The fucosylated glycoconjugates they produce are blood group and oncodevelopmental antigens (1) and are involved in tumorigenesis (2), embryogenesis (3), normal leukocyte trafficking (4), and leukocyte extravasation in inflammatory reactions (5-7). Since all fucosyltransferases utilize GDP-fucose as donor substrate, their specificity depends on the recognition of the acceptor substrate and the type of linkage formed. Although the primary sequences of six human ␣1,3-fucosyltransferases are known, the amino acids involved in the recognition of different acceptor substrates as type 1 (Gal␤1,3GlcNAc) and type 2 (Gal␤1,4GlcNAc) disaccharides or blood group H-type 1 (Fuc␣1,2Gal␤1,3GlcNAc) and H-type 2 (Fuc␣1,2Gal␤1, 4GlcNAc) trisaccharides are not yet known. These last trisaccharides are better acceptors than the disaccharides because they give higher values of fucose incorporation with a better K m and they are unambiguous in the sense that C-2 of Gal is already substituted by Fuc, and therefore, they can accept fucose only on C-3 or C-4 of GlcNAc. This is particularly relevant for Fuc-TIII 1 since up to 4% of ␣1,2-fucosyltransferase activity has been found with this enzyme (8, 9).Two enzymes (Fuc-TIII and Fuc-TV) are able to use both H-type 1 and H-type 2 trisaccharide acceptors, and consequently, they have been called ␣1,3/1,4-fucosyltransferases. The Lewis or Fuc-TIII enzyme is ϳ100 times more efficient on H-type 1 compared with H-type 2 acceptor substrates (10). The Fuc-TV enzyme is more efficient on H-type 2 than on H-type 1 substrates, although the relative type 1/type 2 activities are of the same order of magnitude (10). Finally, the remaining four ␣1,3-fucosyltransferases (Fuc-TIV, Fuc-TVI, Fuc-TVII, and Fuc-TIX) are able to use only type 2 acceptor substrates and are consequently called ␣1,3-fucosyltransferases.The Fuc-TIII, Fuc-TV, and Fuc-TVI enzymes constituting the primate Lewis subfamily of fucosyltransferases have appeared by two successive duplications of an ancestral Lewis gene, which occurred rather late in evolution (Fig. 1), after the great mammalian radiation and before the separation of higher apes and man from their common evolutionary path (10). These three enzymes share ϳ85% sequence identity; the main differences among them are located in the stem amino-terminal region (hypervariable region), whereas their carboxyl-terminal regions are almost identical. Therefore, the differences in type 1/type 2 specificity among these three enzymes are expecte...