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

Abstract

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...

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

“…This suggests that both mammalian and H. pylori ␣-(1,3/1,4)-FucTs bind the Type II and Type I compounds in a similar manner. In our previous work, we have proposed that the GlcNAc moiety in Type I structure is rotated by 180°relative to its location in Type II compound, bringing the 4-OH of GlcNAc in Type I to the same position as the 3-OH of GlcNAc in Type II (44). The 6-CH 2 OH and acetamido groups in Type I are also rotated 180°relative to those in Type II structures.…”

“…The functional similarities that have been shown previously with respect to domain architecture and the region and residues that discriminate Type I from Type II acceptors (24,44) have now been extended to include the minimal catalytic domain, kinetic parameters, and key polar groups of acceptors that are essential for enzyme recognition. Our data suggest that all ␣-(1,3/1,4)-FucTs very likely share a conserved mechanistic and structural basis for fucose transfer.…”

“…Single Substrate Kinetics of WT and 11639 and UA948 in Cell Crude Extracts-The acceptor and donor kinetics for the fulllength and 11639FucT and UA948FucT in cell crude extracts were carried out as described previously (24,44). Kinetic parameters were obtained by fitting the initial rate data to the Michaelis-Menten equation using nonlinear regression analysis with Prism 4.0 software (GraphPad, San Diego).…”

“…Domain swapping and site-directed mutagenesis studies show that acceptor specificity is determined by hypervariable regions that connect the catalytic domain to the membrane anchor, either preceding or following the catalytic domain for mammalian and H. pylori FucTs, respectively (12,13,24). In particular, a single aromatic residue has been reported to be critical for Type I acceptor specificity for both mammalian and H. pylori FucTs (10,11,44).…”

Lipopolysaccharide Diversity Evolving in Helicobacter pylori Communities through Genetic Modifications in Fucosyltransferases

“…All three FucTs are subjected to translational frame-shifting in homopolymeric tracts within the genes, thereby causing the enzymes to switch between an active and silent stage [40], [41]. The specificity of FutA and FutB is, moreover, directed by the nature of a short variable domain that dictates the enzymes towards either α1,3- and/or α1,4-FucT activity [36], [42]. The enzymatic activity of FutA and FutB is also affected by diversity in the heptad-repeat region.…”