Cloning and Heterologous Expression of an α1,3-Fucosyltransferase Gene from the Gastric PathogenHelicobacter pylori

Abstract

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 (Le x ) 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 ␣1,3-fucosyltransferase gene (HpfucT) involved in the biosynthesis of Le x 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 ␣1,3/1,4-fucosyltransferases. The ϳ52-kDa protein encoded by HpfucT was expressed in Escherichia coli CSRDE3 cells and gave rise to ␣1,3-fucosyltransferase activity but neither ␣1,4-fucosyltransferase nor ␣1,2-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 ␣1,3-fucosyltransferases. These lines of evidence indicate that the HpFuc-T represents the bacterial ␣1,3-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 ␣1,3-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.

Novel Helicobacter pylori α1,2-fucosyltransferase, a key enzyme in the synthesis of Lewis antigens

“…2b, lane 1). The yield of the protein, in terms of the fraction of the FucT protein in the total proteins, was much higher than that reported previously (Ge et al, 1997), in which the gene was expressed in E. coli CSRDE3 cells with the induction by IPTG. Correspondingly, we detected a specific α1,3-FucT activity of 1480 µU (mg protein) −" (in the wholecell extract) by using LacNAc as an acceptor (Table 1, A), which is much higher than those obtained before [26 µU (mg cytoplasmic protein) −" and 700 µU (mg membrane protein) −" ].…”

“…Recently, a gene encoding α1,3-FucT in H. pylori was identified and characterized (Ge et al, 1997 ;Martin et al, 1997), and the whole genome sequences (Alm et al, 1999 ;Tomb et al, 1997) (Wang et al, 1999). Here we report the development of a sensitive assay system for detection of Hp α1,2-FucT enzyme activity, and the characterization of its properties and functions in the synthesis of Lewis antigens.…”

“…As described in Methods, the plasmid containing the Hp fucT2 gene under the control of the T7 promoter was transferred into E. coli CLM4(pGP1-2), and the expression of the gene was induced by shifting the host cells from 30 to 42 mC (Tabor & Richardson, 1985). As a reference, we included a previously cloned Hp α1,3-FucT-encoding gene carried on the plasmid pBKHp763fucT39 (Ge et al, 1997). Using the E. coli CLM4(pGP1-2) gene expression system, we obtained the overexpressed Hp α1,3-FucT protein with an expected molecular mass of 52 kDa (Fig.…”

Phase Variation in Helicobacter Pylori Lipopolysaccharide

“…Thus, ␣2-FucT may be involved in the switch from parental type to variant 1c. H. pylori FucT genes have been identified (8,16,23) and expressed (8,16); these genes contain poly(C) tracts. Interestingly, in strain NCTC 11637 (expressing Lewis x), the reported FucT gene contains a C9 tract (gene off, truncated enzyme), while in strain 826695 (expressing Lewis xy [1a]), C13 is present (gene on, full-length enzyme).…”

“…Often, those glycosyltransferase genes contained oligonucleotide repeats in the 5Ј end, and one mechanism for phase variation involved replication errors due to slipped-strand base pairing in the repeat region. Recently, oligonucleotide repeats have been identified in the fucosyltransferase (FucT) genes of H. pylori (8,16,23). The result of phase variation is a more versatile, more heterogeneous microorganism that can cope better with a variety of different environments: with one set of genes switched on, the microorganism may be able to adhere to mucosal cells of the nasopharynx but not to survive complement attack in serum; with that particular set switched off, the reverse may apply.…”