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Glycobiology Advance Access published online on November 13, 2007
Glycobiology, doi:10.1093/glycob/cwm126
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© The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Characterization of {alpha}-2,8-Polysialyltransferase from Neisseria Meningitidis with Synthetic Acceptors, and the Development of a Self-Priming Polysialyltransferase Fusion Enzyme

Lisa M. Willis, Michel Gilbert, Marie-France Karwaski, Marie-Claude Blanchard and Warren W. Wakarchuk

Address correspondence to: Warren Wakarchuk, National Research Council Canada, Institute for Biological Sciences, Glycobiology Program, 100 Sussex Drive, Ottawa, ON., Canada K1A 0R6. email: warren.wakarchuk{at}nrc-cnrc.gc.ca Telephone: 613-952-4299, FAX : 613-941-1327

Received on July 27, 2007; accepted on November 6, 2007

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 was 3 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 bi-functional {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 glycopeptides.

Key words: fusion enzyme / glycosyltransferase / polysialic acid / {alpha}-2,8-Polysialyltransferase / synthetic acceptor


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