Glycobiology Advance Access published online on August 30, 2007
Glycobiology, doi:10.1093/glycob/cwm090
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Variants of the ß-1,3-galactosyltransferase CgtB from the bacterium Campylobacter jejuni have distinct acceptor specificities
1 Institute for Biological Sciences, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada
2 NEOSE Technologies, Inc. 102 Witmer Road, Horsham, PA, 19044, United States of America
* To whom correspondence should be addressed: Warren.wakarchuk{at}nrc-cnrc.gc.ca
Received on May 17, 2007; accepted on August 23, 2007
The gene clusters encoding the lipooligosaccharide biosynthesis glycosyltransferases from C. 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 3 variants were evaluated for their ability to use a derivitized mono-saccharide, a GM2 ganglioside mimic, a GA2 ganglioside mimic as well as a peptide containing 
-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 CgtBBOH4384B
C-MalE was used to galactosylate a glyco-peptide acceptor based on the interferon 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 IFN2b[GalNAc]. 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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