Successive glycosyltransfer of sialic acid by Escherichia coli K92 polysialyltransferase in elongation of oligosialic acceptors.
Escherichia coli K92 produces a capsular polysialic acid with alternating alpha2,8 alpha2,9 NeuNAc linkages. This polysaccharide is cross-reactive with the neuroinvasive pathogen Neisseria meningitidis Group C. The K92 polysialyltransferase (PST) catalyzes the synthesis of the polysialic acid with alternating linkages by the transfer of NeuNAc from CMP-NeuNAc to the nonreducing end of the growing polymer. We used a fluorescent-based high-performance liquid chromatography assay to characterize the process of chain extension. The PST elongates the acceptor GT3-FCHASE in a biphasic fashion. The initial phase polymers are characterized by accumulation of product containing 1-8 additional sialic acid residues. This phase is followed by a very rapid formation of high-molecular weight (MW) polymer as the accumulated oligosaccharides containing 8-10 sialic acids are consumed. The high-MW 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 PST forms both alpha2,8 and alpha2,9 linkages in a successive and nonprocessive fashion.
Vionnet
Justine
J
Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, FDA, Bethesda, MD 20892, USA.
Vann
Willie F
WF
D002462:Cell Membrane / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000737:chemistry; D008956:Models, Chemical; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011135:Polysaccharides, Bacterial / Q000737:chemistry; D012794:Sialic Acids / Q000737:chemistry*; D012799:Sialyltransferases / Q000737:chemistry / Q000502:physiology*; D013997:Time Factors
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D009844:Oligosaccharides; D011108:Polymers; D011135:Polysaccharides, Bacterial; D012794:Sialic Acids; C021319:polysialic acid; D012799:Sialyltransferases; D006026:Glycoside Hydrolases; C090534:endo-alpha-sialidase; D019158:N-Acetylneuraminic Acid