High-level expression of recombinant Neisseria CMP-Neu5Ac synthetase and its use in the gram-scale synthesis of CMP-Neu5Ac: Prot.Expr.Pur.

NRC publication: Ye

Characterization of the alpha-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme

Glycoconjugates containing polysialic acid have many biological activities and represent target molecules for therapeutic interventions. Enzymatic synthesis of these glycoconjugates should give access to these important molecules to evaluate their potential. The polysialyltransferases from both Neisseria meningitidis and Escherichia coli were cloned and expressed as recombinant proteins in E. coli. We have used synthetic acceptors to probe the acceptor requirement of these enzymes and to examine the basic enzymology. The minimum number of sialic acid residues (Neu5Ac) on the acceptor for activity in vitro was shown to be 2 for both enzymes, but a large increase in activity was seen if the acceptor had three Neu5Ac residues. The polysialyltransferase from N. meningitidis generated longer reaction products than the enzyme from E. coli on FCHASE acceptors. Examination of the products showed them to be a heterogeneous mixture, but products with >50 Neu5Ac residues could be seen using capillary zone electrophoresis analyses. In addition we made fusion proteins of these polysialyltransferase enzymes with the bifunctional alpha-2,3/alpha-2,8-sialyltransferase from Campylobacter jejuni to create self priming polysialyltransferases. These bifunctional sialyltransferases utilized various synthetic disaccharide acceptors with a terminal galactose, and we demonstrate here that the PST enzyme from N. meningitidis and its fusion protein with the C. jejuni sialyltransferase can be used to create polysialic acid on O-linked glycopeptidesNRC publication: Ye

Variants of the beta 1,3-galactosyltransferase CgtB from the bacterium Campylobacter jejuni have distinct acceptor specificities

The gene clusters encoding the lipooligosaccharide biosynthesis glycosyltransferases from Campylobacter jejuni have previously been divided in eight classes based on their genetic organization. Here, three variants of the beta1,3-galactosyltransferase CgtB from two classes were purified as fusions with the maltose-binding protein (MalE) from Escherichia coli and their acceptor preference was determined. The acceptor preference of each CgtB variant was directly related to the presence or absence of sialic acid in the acceptor, which correlated with the core oligosaccharide structure in vivo. The three variants were evaluated for their ability to use a derivitized monosaccharide, a GM2 ganglioside mimic, a GA2 ganglioside mimic as well as a peptide containing alpha-linked GalNAc. This characterization shows the flexibility of these galactosyltransferases for diverse acceptors. The CgtB variants were engineered via carboxy-terminal deletions and inversion of the gene fusion order. The combination of a 20 to 30 aa deletion in CgtB followed by MalE at its carboxy terminus significantly improved the glycosyltransferase activity (up to a 51.8-fold increase of activity compared to the full length enzyme) in all cases regardless of the acceptor tested. The improved enzyme CgtB(OH4384)DeltaC-MalE was used to galactosylate a glyco-peptide acceptor based on the interferon alpha2b protein O-linked glycosylation site as confirmed by the CE-MS analysis of the reaction products. This improved enzyme was also used successfully to galactosylate the human therapeutic protein IFNalpha2b[GalNAcalpha]. This constitutes the first report of the in vitro synthesis of the O-linked T-antigen glycan on a human protein by a bacterial glycosyltransferase and illustrates the potential of bacterial glycosyltransferases as tools for in vitro glycosylation of human proteins of therapeutic valueNRC publication: Ye

Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)h and (13)c NMR analysis: J.Biol.Chem.

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barre syndrome. We first cloned a gene encoding an alpha-2, 3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a beta-1, 4-N-acetylgalactosaminyl-transferase (cgtA), a beta-1, 3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked alpha-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of (3)J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two related C. jejuni strains that express shorter ganglioside mimics in their LOSNRC publication: Ye

Evidence for the acquisition of the lipooligosaccharide biosynthesis locus in Campylobacter jejuni GB11, a strain isolated from a Guillain-Barre syndrome patient, by horizontal exchange.: Infect.Immun.

NRC publication: Ye

Identification of a sialate-O-acetyltransferase from Campylobacter jejuni: demonstration of direct transfer to the C9 position of terminal alpha-2,8-linked sialic acid: J.Biol.Chem.

We have identified a sialate-O-acetyltransferase in the lipo-oligosaccharide biosynthesis locus of Campylobacter jejuni. Strains possessing this locus are known to produce sialylated outer core structures that mimic host gangliosides, and have been implicated in triggering the onset of Guillain-Barre syndrome. The acetyltransferase, which was cloned and expressed as a fusion construct in Escherichia coli, is soluble and homologous with members of the NodL-LacA-CysE family of O-acetyltransferases. This enzyme catalyzes the transfer of O-acetyl groups onto oligosaccharide-bound sialic acid, with a high specificity for terminal a2,8-linked residues. The modification is directed to C9, and not C7, as is believed to occur more commonly in other organisms. Despite their wide prevalence and importance in both eukaryotes and prokaryotes, this is the first report to describe the characterization of a purified sialate-O-acetyltransferaseNRC publication: Ye

Identification of a sialate O-acetyltransferase from Campylobacter jejuni: Demonstration of direct transfer to the C-9 position of terminal α-2,8-linked sialic acid

We have identified a sialate O-acetyltransferase in the lipo-oligosaccharide biosynthesis locus of Campylobacter jejuni. Strains possessing this locus are known to produce sialylated outer core structures that mimic host gangliosides, and have been implicated in triggering the onset of Guillain-Barré syndrome. The a

GM3, GM2 and GM1 mimics designed for biosensing: chemoenzymatic synthesis, target affinities and 900 MHz NMR analysis

Undec-10-enyl, undec-10-ynyl and 11-azidoundecyl glycoside analogues corresponding to the oligosaccharides of human gangliosides GM3, GM2 and GM1 were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. Due to poor water solubility of the substrates, the reactions were carried out in methanol–water media, which for the first time were shown to be compatible with the C. jejuni α-(2→3)-sialyltransferase (CST-06) and β-(1→4)-N-acetylgalactosaminyltransferase (CJL-30). Bioequivalence of our synthetic analogues and natural gangliosides was examined by binding to Vibrio cholerae toxin and to the B subunit of Escherichia coli heat-labile enterotoxin. This bioequivalence was confirmed by binding mouse and human monoclonal antibodies to GM1 and acute phase sera containing IgM and IgG antibodies to GM1 from patients with the immune-mediated polyneuropathy Guillain–Barré syndrome. The synthesized compounds were analyzed by 1D and 2D 900 MHz NMR spectroscopy. TOCSY and DQF-COSY experiments in combination with 13C–1H correlation measurements (HSQC, HMBC) were carried out for primary structural characterization, and a complete assignment of all 1H and 13C chemical shifts is presented