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Glycobiology Advance Access published online on March 23, 2007
Glycobiology, doi:10.1093/glycob/cwm032
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© The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Successive glycosyltransfer of sialic acid by Escherichia coli K92 polysialyltransferase in elongation of oligosialic acceptors

Justine Vionnet and Willie F. Vann*

Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland 20892

* Corresponding author: Willie F. Vann, Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, Building 29, Room 103, 8800 Rockville Pike, Bethesda, MD 20892, E-mail: wvann{at}helix.nih.gov, Phone: 301 496 2008, Fax: 301 402 2776

Received on November 10, 2007; revised on February 19, 2007; accepted on March 12, 2007

Escherichia coli K92 produces a capsular polysialic acid with alternating {alpha}2,8 {alpha}2,9 NeuNAc linkages. This polysaccharide is cross reactive with the neuroinvasive pathogen Neisseria meningitidis Group C. The K92 polysialyltransferase catalyzes the synthesis of the polysialic acid with alternating linkages by the transfer of NeuNAc from CMP-NeuNAc to the non-reducing end of the growing polymer. We used a fluorescent based HPLC assay to characterize the process of chain extension. The polysialyltransferase elongates the acceptor GT3-FCHASE in a biphasic fashion. The initial phase polymers are characterized by accumulation of product containing 1 to 8 additional sialic acid residues. This phase is followed by a very rapid formation of high molecular weight polymer as the accumulated oligosaccharides containing 8-10 sialic acids are consumed. The high molecular weight polymer contains 90-100 sialic acids and is sensitive to degradation by periodate and K1-5 endoneuraminidase suggesting that the polymer contains the alternating structure. The polymerization reaction does not appear to be strictly processive, since oligosaccharides of each intermediate size were detected before accumulation of high molecular weight polymer. Synthesis can be blocked by CMP-9-azido-NeuNAc. These results suggest that the K92 polysialyltransferase forms both {alpha}2,8 and {alpha}2,9 linkages in a successive and non-processive fashion.

Key words: capsular polysaccharide / chain extension / polysialyltransferase / processivity / sialic acid


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