Chemical synthesis of GDP-fucose analogs and their utilization by the Lewis *A(1 → 4) fucosyltransferase

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.

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“…Gal␤1 (38). Sep-Pak Plus C-18 reverse-phase cartridges were purchased from Waters (Mississauga, Ontario).…”

Section: Methodsmentioning

confidence: 99%

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

Ge¹,

Chan²,

Palcic³

et al. 1997

Journal of Biological Chemistry

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“…Trimeric N-acetyllactosamine trifluoroacetamidophenylethyl { (LacNAc),-EtPhNHCOCF, [30] }, monofucosyl(LacNAc),-EtPhNHCOCF, [31] and GDP-fucose [32] were prepared as described previously. alAcid glycoprotein was isolated from human plasma according to [33].…”

Section: Methodsmentioning

confidence: 99%

The use of human milk fucosyltransferase in the synthesis of tumor‐associated trimeric X determinants

Vries

Norberg

Lönn

et al. 1993

European Journal of Biochemistry

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We have studied the fucosylation of a chemically synthesized trimer of N-acetyllactosamine [(LacNAc),-EtPhNHCOCF,] with a fucosyltransferase preparation from normal human milk, which utilizes both type-1 and type-2 structures, whether sialylated or not. When fucose residues were added enzymically to the (LacNAc),-EtPhNHCOCF, hexasaccharide, mono-, di-, or trifucosylated oligosaccharide species were formed, containing the LewisX determinant (Gal~l+4[Fucal+3]GlcNAcPl-3). With excess GDP-fucose and prolonged reaction times, the trifucosylated product was formed in almost quantitative yield. Kinetic analysis of the fucosylation reaction indicated that there is a significant difference in the rate of transfer of the first, second and third fucose residues onto the acceptor molecule. The location of the fucose residues in the monofucosylated and difucosylated intermediate products was assessed by analyzing the digests obtained after endo-P-galactosidase treatment by HPLC and reverse-phase chromatography. In addition, the fucosylated (LacNAc),-EtPhNHCOCF, structures were characterized by HPLC and were identified by 400-MHz 'H-NMR spectroscopy. There is a highly preferred order in which the fucosyl residues are attached to (LacNAc),-EtPhNHCOCF,. In the major pathway, the first two fucose residues are transferred with equal preference to the medial (GN3) and proximal (GN1) GlcNAc residues, whereas the third fucose is attached to the distal (GN5) GlcNAc residue. These results are of relevance in understanding the role of a-3-fucosyltransferase in the biosynthesis of LewisX-related cell-surface carbohydrate structures, that function as ligands for selectin-type cell-adhesion molecules and may play a role in the invasion and metastasis of several carcinoma.

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“…Unlike other sugar nucleotides, the enzymatic preparation of GDP-Fuc has not been established on a large scale, and as such, several groups have reported the chemical synthesis of this substrate (Adelhorst & Whitesides, 1993;Arlt & Hindsgaul 1995;Gokhale et al, 1990;Ichikawa et al, 1992b;Nunez et al, 1981, Veeneman et al, 1991. A key step in most of these procedures is the coupling of -l-fucopyranosyl phosphate with the commercially available guanosine 5′-monophosphomorpholidate (GMP-morpholidate) (Moffatt, 1966;Roseman et al, 1961).…”

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confidence: 99%

Mechanism of Human α-1,3-Fucosyltransferase V: Glycosidic Cleavage Occurs Prior to Nucleophilic Attack

et al. 1997

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R-1,3-Fucosyltransferase V (FucT V) catalyzes the transfer of l-fucose from the donor sugar guanosine 5′-diphospho--l-fucose (GDP-Fuc) to an acceptor sugar. A secondary isotope effect on the fucosyltransfer reaction with guanosine 5′-diphospho-[1-2 H]--l-fucose (GDP-[1-2 H]-Fuc) as the substrate was observed and determined to be D V ) 1.32 ( 0.13 and D V/K ) 1.27 ( 0.07. Competitive inhibition of FucT V by guanosine 5′-diphospho-2-deoxy-2-fluoro--l-fucose (GDP-2F-Fuc) was observed with an inhibition constant of 4.2 µM which represents the most potent inhibitor of this enzyme to date. Incubation of GDP-2F-Fuc with FucT V and an acceptor molecule prior to the addition of GDP-Fuc had no effect on the potency of inhibition, indicating that GDP-2F-Fuc is neither an inactivator nor a slow substrate. Both the observed secondary isotope effect and the inhibition by GDP-2F-Fuc are consistent with a charged, sp 2 -hybridized, transition-state structure. A convenient and efficient synthesis of GDP-[1-2 H]-Fuc and GDP-2F-Fuc and a nonradioactive, fluorescence assay for fucosyltransferase activity have been developed.The R-1,3-fucosylated oligosaccharide structures are central to numerous cell-cell interactions (Ichikawa et al., 1994) such as inflammation, tumor development, and blood clotting (Foxall et al., 1992;Parekh & Edge, 1994). Five distinct human R-1,3-fucosyltransferases have been cloned (KukowskaLatallo et al., 1990;Lowe et al., 1991;McCurley et al., 1995;Reguigne-Arnould et al., 1995;Sasaki et al., 1994; Weston et al., 1992a,b) and shown to have different acceptor sugar specificity. R-1,3-Fucosyltransferase V (FucT V) 1 is responsible for the terminal step in the biosynthesis of Lewis x (Le x ) and sialyl Lewis x (sLe x ) (Figure 1), a tetrasaccharide ligand involved in inflammatory cell adhesion and metastasis (Lasky, 1992;Muramatsu, 1993). Thus inhibition of this enzyme may impede inflammation or cancer progression, and understanding the mechanism of FucT V may lead to the development of effective inhibitors.The R-1,3-fucosyltransferase V-catalyzed reaction proceeds with inversion of configuration at the anomeric center of l-fucose (Weston et al., 1992a). Product inhibition studies have been used to establish that FucT V has an ordered, sequential, bi-bi mechanism with guanosine 5′-diphospho--l-fucose (GDP-Fuc) binding first and the product GDP releasing last (Qiao et al., 1996). FucT V has been shown to have a catalytic residue with pK a ) 4.1, presumably an active-site carboxylate residue . A solvent isotope effect was observed (D V ) 2.9, D V/K ) 2.1) and exploited in a proton inventory study to show that there is a one-proton transfer in the transition state . The transition-state structure of glycosyltransferasecatalyzed reactions has been proposed to have a flattened half-chair conformation with substantial oxocarbenium ion character at the anomeric position (Kim et al., 1988;Murray et al., 1996), analogous to that of the glycosidase reactions (Look et al., 1993;Sinnott, 1990). Consistent with...

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Chemical synthesis of GDP-fucose analogs and their utilization by the Lewis *A(1 → 4) fucosyltransferase

Cited by 94 publications

References 16 publications

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

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

The use of human milk fucosyltransferase in the synthesis of tumor‐associated trimeric X determinants

Mechanism of Human α-1,3-Fucosyltransferase V: Glycosidic Cleavage Occurs Prior to Nucleophilic Attack

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