Characterization of the alpha-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme.
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 had three 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 bifunctional 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.
Willis
Lisa M
LM
National Research Council Canada, Institute for Biological Sciences, Glycobiology Program, 100 Sussex Drive, Ottawa, ON, K1A 0R6, Canada.
Gilbert
Michel
M
Karwaski
Marie-France
MF
Blanchard
Marie-Claude
MC
Wakarchuk
Warren W
WW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D009345:Neisseria meningitidis / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D012799:Sialyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C403313:NeuS protein, E coli; C046778:CMP-N-acetylneuraminate-poly-alpha-2,8-sialosyl sialyltransferase; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D008274:Magnesium