title: Variants of the beta 1,3-galactosyltransferase CgtB from the bacterium Campylobacter jejuni have distinct acceptor specificities.
abstract: 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 value.
authors.affiliation: Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Bernatchez
authors.forename: Michel
authors.initials: M
authors.lastname: Gilbert
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Blanchard
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Karwaski
authors.forename: Jianjun
authors.initials: J
authors.lastname: Li
authors.forename: Shawn
authors.initials: S
authors.lastname: Defrees
authors.forename: Warren W
authors.initials: WW
authors.lastname: Wakarchuk
mesh_terms: D000595:Amino Acid Sequence; D016123:Campylobacter jejuni / Q000201:enzymology*; D005700:Galactosyltransferases / Q000096:biosynthesis* / Q000737:chemistry*; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D005838:Genotype; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D005810:Multigene Family; D010957:Plasmids / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D011134:Polysaccharides; D016695:Glycosyltransferases; D005700:Galactosyltransferases