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W2017798820 abstract "The lipopolysaccharide of certain strains ofHelicobacter pylori was recently shown to contain the Lewis X (Lex) trisaccharide (Galβ-1,4-(Fucα(1,3))-GlcNAc). Lex is an oncofetal antigen which appears on human gastric epithelium, and its mimicry by carbohydrate structures on the surface of H. pylori may play an important part in the interaction of this pathogen with its host. Potential roles for bacterial Lex in mucosal adhesion, immune evasion, and autoantibody induction have been proposed (Moran, A. P., Prendergast, M. M., and Appelmelk, B. J. (1996) FEMS Immunol. Med. Microbiol. 16, 105–115). In mammals, the final step of Lex biosynthesis is the α(1,3)-fucosylation of GlcNAc in a terminal Galβ(1→4)GlcNAc unit, and a corresponding GDP-fucose:N-acetylglucosaminyl α(1,3) fucosyltransferase (α(1,3)-Fuc-T) activity was recently discovered in H. pylori extracts. We used part of a human α(1,3)-Fuc-T amino acid sequence to search an H. pylori genomic data base for related sequences. Using a probe based upon weakly matching data base sequences, we retrieved clones from a plasmid library of H. pylori DNA. DNA sequence analysis of the library clones revealed a gene which we have named fucT, encoding a protein with localized homology to the human α(1,3)-Fuc-Ts. We have demonstrated that fucT encodes an active Fuc-T enzyme by expressing the gene in Escherichia coli. The recombinant enzyme shows a strong preference for type 2 (e.g. LacNAc) over type 1 (e.g. lacto-N-biose) acceptors in vitro. Certain residues in a short segment of the H. pylori protein are completely conserved throughout the α(1,3)-Fuc-T family, defining an α(1,3)-Fuc-T motif which may be of use in identifying new fucosyltransferase genes. The lipopolysaccharide of certain strains ofHelicobacter pylori was recently shown to contain the Lewis X (Lex) trisaccharide (Galβ-1,4-(Fucα(1,3))-GlcNAc). Lex is an oncofetal antigen which appears on human gastric epithelium, and its mimicry by carbohydrate structures on the surface of H. pylori may play an important part in the interaction of this pathogen with its host. Potential roles for bacterial Lex in mucosal adhesion, immune evasion, and autoantibody induction have been proposed (Moran, A. P., Prendergast, M. M., and Appelmelk, B. J. (1996) FEMS Immunol. Med. Microbiol. 16, 105–115). In mammals, the final step of Lex biosynthesis is the α(1,3)-fucosylation of GlcNAc in a terminal Galβ(1→4)GlcNAc unit, and a corresponding GDP-fucose:N-acetylglucosaminyl α(1,3) fucosyltransferase (α(1,3)-Fuc-T) activity was recently discovered in H. pylori extracts. We used part of a human α(1,3)-Fuc-T amino acid sequence to search an H. pylori genomic data base for related sequences. Using a probe based upon weakly matching data base sequences, we retrieved clones from a plasmid library of H. pylori DNA. DNA sequence analysis of the library clones revealed a gene which we have named fucT, encoding a protein with localized homology to the human α(1,3)-Fuc-Ts. We have demonstrated that fucT encodes an active Fuc-T enzyme by expressing the gene in Escherichia coli. The recombinant enzyme shows a strong preference for type 2 (e.g. LacNAc) over type 1 (e.g. lacto-N-biose) acceptors in vitro. Certain residues in a short segment of the H. pylori protein are completely conserved throughout the α(1,3)-Fuc-T family, defining an α(1,3)-Fuc-T motif which may be of use in identifying new fucosyltransferase genes. The Gram-negative bacterium Helicobacter pylori is a major cause of chronic gastritis and peptic and duodenal ulcers (1Warren J.R. Lancet. 1983; i: 1273Google Scholar, 2Marshall B. Lancet. 1983; i: 1273-1275Google Scholar, 3Marshall B.J. Warren J.R. Lancet. 1984; i: 1311-1314Abstract Scopus (4173) Google Scholar, 4Rathbone B.J. Wyatt J.I. Worsley B.W. Shires S.E. Trejdosiewicz L.K. Heatley R.V. Losowsky M.S. Gut. 1986; 27: 642-647Crossref PubMed Scopus (213) Google Scholar, 5NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease JAMA. 1994; 272: 65-69Crossref PubMed Scopus (1063) Google Scholar). It has also been implicated in gastric adenocarcinoma (6Parsonnet J.M.D. Friedman G.D. Vandersteen M.S. Chang Y. Vogelman J.H. Orentreich N. Sibley R.K. N. Engl. J. Med. 1991; 325: 1127-1131Crossref PubMed Scopus (3580) Google Scholar, 7Nomura A. Stemmermann G.N. Chyou P-H. Kato I. Perez-Perez G.I. Blaser M.J. N. Engl. J. Med. 1991; 325: 1132-1136Crossref PubMed Scopus (1701) Google Scholar, 8Hansson L-E. Nyrén O. Hsing A.W. Bergström R. Josefsson S. Chow W-H. Fraumeni J.F. Adami H-O. N. Engl. J. Med. 1996; 335: 242-249Crossref PubMed Scopus (540) Google Scholar, 9Wotherspoon A.C. Ortiz-Hidalgo C. Falzon M.R. Isaacson P.G. Lancet. 1991; 338: 1175-1176Abstract PubMed Scopus (1714) Google Scholar) and gastric lymphoma (10Nakamura S. Yao T. Aoyagi K. Iida M. Fujishima M. Tsuneyoshi M. Cancer. 1997; 79: 3-11Crossref PubMed Scopus (163) Google Scholar), leading to its classification as a type I human carcinogen (11International Agency for Research on Cancer IARC Monogr. Eval. Carcinog. Risks Hum. 1994; 61: 177-240PubMed Google Scholar). H. pylori is a chronic pathogen, and the means by which this organism is able to persist in the stomach and resist or evade destruction by the immune system is central to its involvement in disease. Some aspects of the host-pathogen interaction have been resolved, including the involvement of the Lewis b (Leb) 1The abbreviations used are: Leb, Lewis b; Lex, Lewis X; Ley, Lewis Y; α(1,3)-Fuc-T, α(1,3)-fucosyltransferase, GDP-fucose:β-d-N-acetylglucosaminide 3-α-fucosyltransferase; Gal-T, galactosyltransferase; bp, base pair(s); kb, kilobase(s); PCR, polymerase chain reaction; LacNAc,N-acetyllactosamine, Galβ1→4GlcNAc; LNT, lacto-N-tetraose, Galβ1→ 4GlcNAcβ1→3Galβ1→4Glc; lacto-N-biose, Galβ1→3GlcNAc; HPLC, high performance liquid chromatography; NEM,N-ethylmaleimide; BSA, bovine serum albumin.1The abbreviations used are: Leb, Lewis b; Lex, Lewis X; Ley, Lewis Y; α(1,3)-Fuc-T, α(1,3)-fucosyltransferase, GDP-fucose:β-d-N-acetylglucosaminide 3-α-fucosyltransferase; Gal-T, galactosyltransferase; bp, base pair(s); kb, kilobase(s); PCR, polymerase chain reaction; LacNAc,N-acetyllactosamine, Galβ1→4GlcNAc; LNT, lacto-N-tetraose, Galβ1→ 4GlcNAcβ1→3Galβ1→4Glc; lacto-N-biose, Galβ1→3GlcNAc; HPLC, high performance liquid chromatography; NEM,N-ethylmaleimide; BSA, bovine serum albumin. epitope on epithelial cells in attachment of H. pylori (12Falk P.G. Bry L. Holgersson J. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1515-1519Crossref PubMed Scopus (107) Google Scholar), and characterization of a bacterial cytotoxin responsible for gastric epithelial damage (for a review see Ref. 13Labigne L. de Reuse H. Infect. Agents Dis. 1996; 5: 191-202PubMed Google Scholar), but clearly much remains to be discovered.Recent structural analysis of H. pylori lipopolysaccharides revealed that the O antigen contains fucosylated carbohydrate structures identical to the mammalian Lewis X (Lex) and Lewis Y (Ley) epitopes (14Aspinall G.O. Monteiro M.A. Pang H. Walsh E.J. Moran A.P. Carbohydr. Lett. 1994; 1: 151-156Google Scholar, 15Aspinall G.O. Monteiro M.A. Pang H. Walsh E.J. Moran A.P. Biochemistry. 1996; 35: 2489-2497Crossref PubMed Scopus (185) Google Scholar, 16Aspinall G.O. Monteiro M.A. Biochemistry. 1996; 35: 2498-2504Crossref PubMed Scopus (158) Google Scholar, 17Sherburne R. Taylor D.E. Infect. Immun. 1995; 63: 4564-4568Crossref PubMed Google Scholar). It was further established that the bacterium contains endogenous galactosyltransferase (Gal-T) and fucosyltransferase (Fuc-T) activities necessary for biosynthesis of these structures (18Chan N.W.C. Stangier K. Sherburne R. Taylor D.E. Zhang Y. Dovichi N.J. Palcic M.M. Glycobiology. 1995; 5: 683-688Crossref PubMed Scopus (68) Google Scholar) suggesting that they are synthesized de novo by H. pylori rather than scavenged from the surface of mammalian cells. Lex is an oncofetal antigen (19Feizi T. Nature. 1985; 314: 53-57Crossref PubMed Scopus (1016) Google Scholar, 20Kannagi R. Nudelman E. Levery S.B. Hakomori S. J. Biol. Chem. 1982; 257: 14865-14874Abstract Full Text PDF PubMed Google Scholar) also expressed on adult human gastric mucosa (21Koyabashi K. Sakamoto J. Kito T. Yamamura Y. Koshikawa T. Fujita M. Watanabe T. Nakazato H. Am. J. Gastroenterol. 1993; 88: 475-479Google Scholar), and its presence on H. pylori lipopolysaccharides may play a role in survival and pathogenesis. H. pylori infection is known to induce antibodies that cross-react with human gastric mucosa (22Negrini R. Lisato L. Zanella I. Cavazzini L. Gullini S. Villanacci V. Poiesil C. Albertini A. Ghielmi S. Gastroenterology. 1991; 101: 437-445Abstract Full Text PDF PubMed Scopus (0) Google Scholar). In a recent report, Appelmelk et al. (23Appelmelk B.J. Simoons-Smit I.M. Negrini R. Moran A.P. Aspinall G.O. Forte J.G. de Vries T. Quan H. Verboom T. Maaskant J.J. Ghiara P. Kuipers E.J. Bloemena E. Tadema T.M. Townsend R.R. Tyagarajan K Crothers Jr., J.M. Monteiro M.A. Savio A. de Graaff J. Infect. Immun. 1996; 64: 2031-2040Crossref PubMed Google Scholar) demonstrated that the targets of this autoimmune response include Lex and/or Ley epitopes and provided evidence that anti-Lex/y antibodies may be involved in H. pylori-associated gastritis. Interestingly, molecular mimicry of Lex is also thought to be responsible for autoantibody production by Schistosoma mansoni (24Ko A.I. Dräger U.C. Harn D.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4159-4163Crossref PubMed Scopus (111) Google Scholar, 25Srivatsan J. Smith D.F. Cummings R.D. J. Biol. Chem. 1992; 267: 20196-20203Abstract Full Text PDF PubMed Google Scholar). In addition, surface carbohydrate antigens containing Lex structures may play a part in the immunopathology of H. pylori infection by promoting Th-1 to Th-2 switching as has been reported in schistosomal infections (26Velupillai P. Harn D.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 18-22Crossref PubMed Scopus (298) Google Scholar). Two recent reports (27Wirth H-P. Yang M. Karita M. Blaser M.J. Infect. Immun. 1996; 64: 4958-4965Crossref Google Scholar, 28Simoons-Smit I.M. Appelmelk B.J. Verboom T. Negrini R. Penner J.L. Aspinall G.O. Moran A.P. Fei S.F. Bi-shan S. Rudnica W. Savio A. de Graaff J. J. Clin. Microbiol. 1996; 34: 2196-2200Crossref PubMed Google Scholar) that over 85% of H. pylori isolates from geographically widespread locations express Lex and/or Ley antigens would also seem to imply selective pressure for maintenance of these structures, given the considerable structural variability often shown by lipopolysaccharides from Gram-negative bacteria.In mammals, the defining step of Lex biosynthesis is fucosylation of a type 2 core structure (Galβ1→4GlcNAc). This reaction is catalyzed in humans by one or more members of a family of α(1,3)-fucosyltransferases which employ GDP-fucose as an activated sugar donor (29Kukowska-Latallo J.F. Larsen R.D. Nair R.D. Lowe J.B. Genes Dev. 1990; 4: 1288-1303Crossref PubMed Scopus (469) Google Scholar, 30Cameron H.S. Szczepaniak D. Weston B.W. J. Biol. Chem. 1995; 270: 20112-20122Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 31Lowe J.B. Kukowska-Latallo J.F. Nair R.P. Larsen R.D. Marks R.M. Macher B.A. Kelly R.J. Ernst L.K. J. Biol. Chem. 1991; 266: 17467-17477Abstract Full Text PDF PubMed Google Scholar, 32Goelz S.E. Hession C. Goff D. Griffiths B. Tizard R. Newman B. Chi-Rosso G. Lobb R. Cell. 1990; 63: 1349-1356Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 33Kumar R. Potvin B. Muller W.A. Stanley P. J. Biol. Chem. 1991; 266: 21777-21783Abstract Full Text PDF PubMed Google Scholar, 34Weston B.W. Nair R.P. Larsen R.D. Lowe J.B. J. Biol. Chem. 1992; 267: 4152-4160Abstract Full Text PDF PubMed Google Scholar, 35Koszdin K.L. Bowen B.R. Biochem. Biophys. Res. Commun. 1992; 187: 152-157Crossref PubMed Scopus (147) Google Scholar, 36Weston B.W. Smith P.L. Kelly R.J. Lowe J.B. J. Biol. Chem. 1992; 267: 24575-24584Abstract Full Text PDF PubMed Google Scholar, 37Natsuka S. Gersten K.M. Zenita K. Kannagi R. Lowe J.B. J. Biol. Chem. 1994; 269: 16789-16794Abstract Full Text PDF PubMed Google Scholar, 38Sasaki K. Kurata K. Funayama K. Nagata M. Watanabe E. Ohta S. Hanai N. Nishi T. J. Biol. Chem. 1994; 269: 14730-14737Abstract Full Text PDF PubMed Google Scholar). Fuc-T and Gal-T activities have been detected inH. pylori extracts (18Chan N.W.C. Stangier K. Sherburne R. Taylor D.E. Zhang Y. Dovichi N.J. Palcic M.M. Glycobiology. 1995; 5: 683-688Crossref PubMed Scopus (68) Google Scholar), but although the order of sugar transfer appears to follow the same course as in mammalian systems, with galactosylation preceding fucosylation, little is known about the bacterial Fuc-T and how it is related to the mammalian transferases. If, as evidence is beginning to suggest, cell-surface Lex/yepitopes play an important role in H. pylori persistence and pathogenesis (23Appelmelk B.J. Simoons-Smit I.M. Negrini R. Moran A.P. Aspinall G.O. Forte J.G. de Vries T. Quan H. Verboom T. Maaskant J.J. Ghiara P. Kuipers E.J. Bloemena E. Tadema T.M. Townsend R.R. Tyagarajan K Crothers Jr., J.M. Monteiro M.A. Savio A. de Graaff J. Infect. Immun. 1996; 64: 2031-2040Crossref PubMed Google Scholar, 39Moran A.P. Prendergast M.M. Appelmelk B.J. FEMS Immunol. Med. Microbiol. 1996; 16: 105-115Crossref PubMed Google Scholar), the α(1,3)-Fuc-T may offer a nonbactericidal therapeutic target for eradication of H. pylori without otherwise disturbing the balance of gut fauna.Five members of the human α(1,3)-Fuc-T gene family have been cloned (Fuc-TIII–VII) (29Kukowska-Latallo J.F. Larsen R.D. Nair R.D. Lowe J.B. Genes Dev. 1990; 4: 1288-1303Crossref PubMed Scopus (469) Google Scholar, 30Cameron H.S. Szczepaniak D. Weston B.W. J. Biol. Chem. 1995; 270: 20112-20122Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 31Lowe J.B. Kukowska-Latallo J.F. Nair R.P. Larsen R.D. Marks R.M. Macher B.A. Kelly R.J. Ernst L.K. J. Biol. Chem. 1991; 266: 17467-17477Abstract Full Text PDF PubMed Google Scholar, 32Goelz S.E. Hession C. Goff D. Griffiths B. Tizard R. Newman B. Chi-Rosso G. Lobb R. Cell. 1990; 63: 1349-1356Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 33Kumar R. Potvin B. Muller W.A. Stanley P. J. Biol. Chem. 1991; 266: 21777-21783Abstract Full Text PDF PubMed Google Scholar, 34Weston B.W. Nair R.P. Larsen R.D. Lowe J.B. J. Biol. Chem. 1992; 267: 4152-4160Abstract Full Text PDF PubMed Google Scholar, 35Koszdin K.L. Bowen B.R. Biochem. Biophys. Res. Commun. 1992; 187: 152-157Crossref PubMed Scopus (147) Google Scholar, 36Weston B.W. Smith P.L. Kelly R.J. Lowe J.B. J. Biol. Chem. 1992; 267: 24575-24584Abstract Full Text PDF PubMed Google Scholar, 37Natsuka S. Gersten K.M. Zenita K. Kannagi R. Lowe J.B. J. Biol. Chem. 1994; 269: 16789-16794Abstract Full Text PDF PubMed Google Scholar, 38Sasaki K. Kurata K. Funayama K. Nagata M. Watanabe E. Ohta S. Hanai N. Nishi T. J. Biol. Chem. 1994; 269: 14730-14737Abstract Full Text PDF PubMed Google Scholar). Homologs of some of these genes have also been cloned from mouse (40Ozawa M. Muramatsu T. J. Biochem. ( Tokyo ). 1996; 119: 302-308Crossref PubMed Scopus (21) Google Scholar, 41Gersten K.M. Natsuka S. Trinchera M. Petryniak B. Kelly R.J. Hiraiwa N. Jenkins N.A. Gilbert D.J. Copeland N.G. Lowe J.B. J. Biol. Chem. 1995; 270: 25047-25056Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 42Smith P.L. Gersten K.M. Petryniak B. Kelly R.J. Rogers C. Natsuka Y. Alford III, J.A. Scheidegger E.P. Natsuka S. Lowe J.B. J. Biol. Chem. 1996; 271: 8250-8259Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), rat (43Sajdel-Sulkowska E.M. Smith F.I. Wiederschain G. McCluer R.H. Glycoconj. J. 1997; 14: 249-258Crossref PubMed Scopus (32) Google Scholar), and cow (44Oulmouden A. Wierinckx A. Petit J.M. Costache M. Palcic M.M. Mollicone R. Oriol R. Julien R. J. Biol. Chem. 1997; 272: 8764-8773Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) cDNA. The remarkable degree of sequence conservation between mammalian α(1,3)-Fuc-Ts and the recently cloned chicken α(1,3)-fucosyltransferase (CFT1) (45Lee K.P. Carlson L.M. Woodcock J.B. Ramachandra N. Schultz T.L. Davis T.A. Lowe J.B. Thompson C.B. Larsen R.D. J. Biol. Chem. 1996; 271: 32960-32967Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) suggests that other nonmammalian α(1,3)-Fuc-Ts may also show significant homology to the known members of this enzyme family. We describe here the identification and cloning of a gene from H. pylori,fucT, which encodes an active Fuc-T with localized sequence similarity to the α(1,3)-Fuc-Ts.DISCUSSIONBy searching an H. pylori genomic data set with part of the catalytic domain sequence of a human α(1,3)-Fuc-T and sequencing corresponding clones from a plasmid library of H. pylori DNA we were able to identify a gene (fucT) with highly localized similarity to known α(1,3)-Fuc-T enzymes. Cell extracts from library clones containing the H. pylori gene possessed Fuc-T activity, and by subcloning fucT into an E. coliexpression vector we were able to confirm that it encodes an active α(1,3)-Fuc-T. H. pylori fucT is the first Fuc-T gene to be cloned from an invertebrate, although enzyme activity has been detected in the freshwater snail Lymnea stagnalis (56Mulder H. Schachter H. Thomas J.R. Halkes K.M. Kamerling J.P. Vliegenthart F.G. Glycoconj. J. 1996; 13: 107-113Crossref PubMed Scopus (13) Google Scholar) and in the parasite S. mansoni (53DeBose-Boyd R. Nyame A.K. Cummings R.D. Exp. Parasitol. 1996; 82: 1-10Crossref PubMed Scopus (31) Google Scholar).Sequence similarity between the mammalian Fuc-Ts and chick fucosyltransferase (CFT1) provided evidence for evolutionary conservation of α(1,3)-Fuc-T sequences (45Lee K.P. Carlson L.M. Woodcock J.B. Ramachandra N. Schultz T.L. Davis T.A. Lowe J.B. Thompson C.B. Larsen R.D. J. Biol. Chem. 1996; 271: 32960-32967Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Conservation betweenH. pylori FucT and the mammalian enzymes, although limited and highly localized, suggests that aspects of the α(1,3)-Fuc-T sequence have survived unchanged through evolution from bacteria to higher mammals and man. The lack of overall sequence similarity to human α(1,3)-Fuc-Ts would seem to preclude the idea that H. pylori acquired the Fuc-T gene from a mammalian source. The base composition of the gene (35% GC) is also much closer to the average for H. pylori (36%) than to mammalian and avian α(1,3)-Fuc-T genes, which are typically GC-rich (e.g. CFT-1, 69% GC).Unlike eukaryotic Fuc-Ts which have a hydrophobic transmembrane domain near their N terminus and share a common type II membrane protein topology, the H. pylori enzyme contains no recognizable membrane insertion elements. Aligned on the basis of the short, highly conserved region of homology (Fig. 2), the bacterial enzyme appears to lack a region corresponding to the transmembrane and stem domains of other Fuc-Ts. Most of the “hypervariable region” previously implicated in acceptor binding specificity in human Fuc-TIII and -V (residues 34 to 161 in Fuc-TIII) (57Legault D.J. Kelly R.J. Natsuka Y. Lowe J.B. J. Biol. Chem. 1995; 270: 20987-20996Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) is also absent, suggesting that the architecture of the H. pylori protein is substantially different from the rest of the enzyme family. The alignment also reveals that the C terminus of the bacterial sequence extends for approximately 100 amino acids beyond that of the other Fuc-Ts, half of this C-terminal extension being taken up by a periodic 7-amino acid leucine zipper-like motif. The function of this region, which has no counterpart in mammalian or avian Fuc-T sequences, is unknown. One possibility is that it mediates homo- or heteromultimer formation through coiled-coil type interactions, but at present the subunit structure of the H. pylori protein is unknown and further work will be necessary to establish the role of the zipper-like region.Recombinant H. pylori Fuc-T has a strong preference for type 2 acceptors, and analysis of oligosaccharides generated by fucosidase digestion of the product generated by this Fuc-T with LacNAc indicates that H. pylori Fuc-T is indeed capable of synthesizing the Lex epitope. Some type 1 structures are also fucosylated, but our studies suggest that with these acceptors fucose may be transferred predominantly to glucose rather than GlcNAc, implying that the enzyme has little α(1,2)- or α(1,4)-Fuc-T activity, as has been reported for human Fuc-TV (51de Vries T. Srnka C.A. Palcic M. Swiedler S.J. van den Eijnden D.H. Macher B.A. J. Biol. Chem. 1995; 270: 8712-8722Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Biosynthesis of the Leyepitope found on the surface of many H. pylori isolates is therefore likely to involve a separate α(1,2)-Fuc-T activity. Overall, the acceptor specificity of H. pylori Fuc-T does not match that reported for any of the human enzymes or indeed that ofS. mansoni α(1,3)-Fuc-T (53DeBose-Boyd R. Nyame A.K. Cummings R.D. Exp. Parasitol. 1996; 82: 1-10Crossref PubMed Scopus (31) Google Scholar). Like the schistosome enzyme and human Fuc-Ts IV and VII, however, H. pylori Fuc-T shows only slight sensitivity to NEM inhibition. Interestingly, 3′-sialyl-LacNAc is an efficient acceptor (although 6′-sialyl-LacNAc is not), implying that H. pylori Fuc-T may be capable of synthesizing the sialyl-Lex (sLex) structure which was recently detected in a small number of H. pyloriisolates by Wirth et al. (27Wirth H-P. Yang M. Karita M. Blaser M.J. Infect. Immun. 1996; 64: 4958-4965Crossref Google Scholar). The absence of sLex from the majority of H. pylori isolates may therefore reflect a lack of sialyltransferase activity in these strains.Mammalian α(1,3)-Fuc-Ts are a closely-related family of enzymes, making it difficult to identify residues of potential structural and/or catalytic importance from sequence alignments. The recently cloned avian α(1,3)-Fuc-T, CFT-1 (45Lee K.P. Carlson L.M. Woodcock J.B. Ramachandra N. Schultz T.L. Davis T.A. Lowe J.B. Thompson C.B. Larsen R.D. J. Biol. Chem. 1996; 271: 32960-32967Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), also shows a high level of sequence similarity to the corresponding mammalian proteins, with 46.3% sequence identity to human Fuc-TIV. This is not the case with the H. pylori enzyme, which shows significant homology to the other α(1,3)-Fuc-Ts only in one short region. A consensus motif derived from this local area of homology (Fig. 2 B) is unique to members of the α(1,3)-Fuc-T family, including the H. pylori enzyme and an open reading frame from aCaenorhabditis eleganscosmid 4GenPept accession number 1041349. (58Wilson R. Ainscough R. Anderson K. Baynes C. Berks M. Bonfield J. Burton J. Connell M. Copsey T. Cooper J. Coulson A. Craxton M. Dear S. Du Z. Durbin R. Favello A. Fulton L. Gardner A. Green P. Hawkins T. Hillier L. Jier M. Johnston L. Jones M. Kershaw J. Kirsten J. Laister N. Latreille P. Lightning J. Lloyd C. McMurray A. Mortimore B. O'Callaghan M. Parsons J. Percy C. Rifken L. Roopra A. Saunders D. Shownkeen R. Smaldon N. Smith A. Sonnhammer E. Staden R. Sulston J. Thierry-Mieg J. Thomas K. Vaudin M. Vaughan K. Waterston R. Watson A. Weinstock L. Wilkinson-Sproat J. Wohldman P. Nature. 1994; 368: 32-38Crossref PubMed Scopus (1439) Google Scholar). This highly conserved α(1,3)-Fuc-T motif may be useful in identifying novel α(1,3)-Fuc-T genes in genomic and expressed sequence tag sequence data, since its appearance seems to be a reliable predictor of membership of this enzyme family. It may also provide a tool for cloning α(1,3)-Fuc-Ts in a manner similar to the demonstrated utility of the l- and S-sialyl motifs in cloning novel sialyltransferases (59Tsuji S. J. Biochem. ( Tokyo ). 1996; 120: 1-13Crossref PubMed Scopus (217) Google Scholar).The functional significance of the α(1,3)-Fuc-T motif is at present unclear. Marked differences in acceptor preferences among members of the α(1,3)-Fuc-T family would seem to argue against a role in acceptor binding. Human Fuc-TIV and -VII for example both contain the α(1,3)-Fuc-T motif, but while Fuc-TVII uses 2,3-sialylated acceptors almost exclusively, Fuc-TIV strongly prefers neutral type 2 substrates (41Gersten K.M. Natsuka S. Trinchera M. Petryniak B. Kelly R.J. Hiraiwa N. Jenkins N.A. Gilbert D.J. Copeland N.G. Lowe J.B. J. Biol. Chem. 1995; 270: 25047-25056Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) in in vitro assays. The behavior of Fuc-TIV in vivo is apparently more complex (60Goelz S. Kumar R. Potvin B. Sundaram S. Brickelmaier M. Stanley P. J. Biol. Chem. 1994; 269: 1033-1040Abstract Full Text PDF PubMed Google Scholar). The α(1,3)-Fuc-T motif lies outside sequence regions implicated by efforts to define acceptor-discriminating residues in α(1,3)-Fuc-Ts (51de Vries T. Srnka C.A. Palcic M. Swiedler S.J. van den Eijnden D.H. Macher B.A. J. Biol. Chem. 1995; 270: 8712-8722Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 57Legault D.J. Kelly R.J. Natsuka Y. Lowe J.B. J. Biol. Chem. 1995; 270: 20987-20996Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 61Xu Z. Vo L. Macher B.A. J. Biol. Chem. 1996; 271: 8818-8823Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Given that the enzymes transfer fucose from a common sugar nucleotide donor, it seems more likely that the α(1,3)-Fuc-T motif is involved in binding GDP-fucose or Mn2+. The motif lies some considerable distance from a cysteine residue implicated in GDP-fucose protectable inhibition by NEM (62Holmes E.H. Xu Z. Sherwood A.L. Macher B.A. J. Biol. Chem. 1995; 270: 8145-8151Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), although it may be much closer in space within the folded protein than the primary sequence suggests. The corresponding position in the H. pylori Fuc-T is occupied by tyrosine (Fig. 2 A), in keeping with observations that enzymes with Cys at this location are inhibited by NEM while those with other amino acids (Fuc-TIV has Ser, Fuc-TVII has Thr) are resistant to NEM inhibition (62Holmes E.H. Xu Z. Sherwood A.L. Macher B.A. J. Biol. Chem. 1995; 270: 8145-8151Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). 5C. Britten and M. I. Bird, manuscript in preparation.Interestingly, the conserved motif contains a lysine residue (Fig. 2 A), possibly a candidate for the so far unidentified GDP-fucose-protected lysine residue identified by pyridoxal phosphate labeling of a human fucosyltransferase (63Holmes E.H. Arch. Biochem. Biophys. 1992; 296: 562-568Crossref PubMed Scopus (12) Google Scholar). Further work is clearly needed to test these speculations, but in this respect the lack of overall similarity between the H. pylori and mammalian transferase sequences may be advantageous. The relatively small number of conserved residues inside and outside the α(1,3)-Fuc-T motif may provide a useful focus for mutagenesis experiments to probe structural and mechanistic aspects of the α(1,3)-fucosyltransferases. The dissimilarity of H. pylori and human Fuc-T enzymes would also seem to auger well for the design of selective inhibitors of the bacterial enzyme.The H. pylori enzyme, which lacks a transmembrane domain and is, presumably, nonglycosylated, is devoid of some of the features which make eukaryotic Fuc-Ts difficult to work with. The possibility of bacterial expression also makes this enzyme a promising candidate for chemoenzymatic glycoconjugate synthesis. The same features may simplify the task of structural determination. Given the conserved motif, it seems reasonable to assume that this enzyme shares at least some structural features in common with its mammalian counterparts which have so far resisted structural elucidation.Mounting evidence appears to point to a role for Lewis antigen mimicry in H. pylori pathogenesis. Identification and cloning of a Fuc-T gene from this organism will allow us to probe the biosynthesis of Lex by H. pylori in vivo via disruption offucT and may make it possible to test the role of Lex directly in models of H. pyloripathogenesis. The Gram-negative bacterium Helicobacter pylori is a major cause of chronic gastritis and peptic and duodenal ulcers (1Warren J.R. Lancet. 1983; i: 1273Google Scholar, 2Marshall B. Lancet. 1983; i: 1273-1275Google Scholar, 3Marshall B.J. Warren J.R. Lancet. 1984; i: 1311-1314Abstract Scopus (4173) Google Scholar, 4Rathbone B.J. Wyatt J.I. Worsley B.W. Shires S.E. Trejdosiewicz L.K. Heatley R.V. Losowsky M.S. Gut. 1986; 27: 642-647Crossref PubMed Scopus (213) Google Scholar, 5NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease JAMA. 1994; 272: 65-69Crossref PubMed Scopus (1063) Google Scholar). It has also been implicated in gastric adenocarcinoma (6Parsonnet J.M.D. Friedman G.D. Vandersteen M.S. Chang Y. 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W2017798820 title "Lewis X Biosynthesis in Helicobacter pylori" @default.
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