Cloning and heterologous expression of an alpha1,3-fucosyltransferase gene from the gastric pathogen Helicobacter pylori.
Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and is also associated with gastric cancer and lymphoma. This microorganism has been shown to express cell surface glycoconjugates including Lewis X (Lex) and Lewis Y. These bacterial oligosaccharides are structurally similar to tumor-associated carbohydrate antigens found in mammals. In this study, we report the cloning of a novel alpha1,3-fucosyltransferase gene (HpfucT) involved in the biosynthesis of Lex within H. pylori. The deduced amino acid sequence of HpfucT consists of 478 residues with the calculated molecular mass of 56,194 daltons, which is approximately 100 amino acids longer than known mammalian alpha1,3/1,4-fucosyltransferases. The approximately 52-kDa protein encoded by HpfucT was expressed in Escherichia coli CSRDE3 cells and gave rise to alpha1,3-fucosyltransferase activity but neither alpha1,4-fucosyltransferase nor alpha1,2-fucosyltransferase activity as characterized by radiochemical assays and capillary zone electrophoresis. Truncation of the C-terminal 100 amino acids of HpFuc-T abolished the enzyme activity. An approximately 72-amino acid region of HpFuc-T exhibits significant sequence identity (40-45%) with the highly conserved C-terminal catalytic domain among known mammalian and chicken alpha1,3-fucosyltransferases. These lines of evidence indicate that the HpFuc-T represents the bacterial alpha1,3-fucosyltransferase. In addition, several structural features unique to HpFuc-T, including 10 direct repeats of seven amino acids and the lack of the transmembrane segment typical for known eukaryotic alpha1,3-fucosyltransferases, were revealed. Notably, the repeat region contains a leucine zipper motif previously demonstrated to be responsible for dimerization of various basic region-leucine zipper proteins, suggesting that the HpFuc-T protein could form dimers.
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Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Chan
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Palcic
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Taylor
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D005647:Fucosyltransferases / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D016256:Lewis X Antigen / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D001426:Bacterial Proteins; D016256:Lewis X Antigen; D011994:Recombinant Proteins; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase