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Glycobiology Advance Access originally published online on August 30, 2007
Glycobiology 2007 17(12):1333-1343; doi:10.1093/glycob/cwm090
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Published by Oxford University Press 2007.

Variants of the ß1,3-Galactosyltransferase CgtB from the Bacterium Campylobacter Jejuni have Distinct Acceptor Specificities

Stéphane Bernatchez2, Michel Gilbert2, Marie-Claude Blanchard2, Marie-France Karwaski2, Jianjun Li2, Shawn DeFrees3 and Warren W Wakarchuk1,2

2 Institute for Biological Sciences, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada
3 NEOSE Technologies, Inc. 102 Witmer Road, Horsham, PA 19044, USA

1 To whom correspondence should be addressed: Tel. +613 952 4299; Fax +613 952 9092; e-mail: warren.wakarchuk{at}nrc-cnrc.gc.ca

Received on May 17, 2007; revised on July 23, 2007; accepted on August 23, 2007

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 ß1,3-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 CgtBOH4384{Delta}C-MalE was used to galactosylate a glyco-peptide acceptor based on the interferon {alpha}2b 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 IFN{alpha}2b[GalNAc{alpha}]. 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.

Key words: ß1,3-galactosyltransferase / CgtB / enzyme improvement / glycosyltransferase


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L. M Willis, M. Gilbert, M.-F. Karwaski, M.-C. Blanchard, and W. W Wakarchuk
Characterization of the {alpha}-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme
Glycobiology, February 1, 2008; 18(2): 177 - 186.
[Abstract] [Full Text] [PDF]


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