Characterization of the alpha-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme

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TypeArticle
Journal titleGlycobiology
Volume18
Issue2
Pages177186; # of pages: 10
SubjectACID; ACID RESIDUES; Amino Acid Sequence; bacterial; Bacterial Proteins; Campylobacter; Campylobacter jejuni; Canada; Capillaries; capillary; chemistry; Comparative Study; DISACCHARIDE; Electrophoresis; ENZYMATIC-SYNTHESIS; ENZYME; enzymes; enzymology; Escherichia; Escherichia coli; Escherichia coli Proteins; ESCHERICHIA-COLI; Galactose; genetics; Glycoconjugates; glycopeptide; Glycopeptides; In Vitro; isolation & purification; Kinetics; Magnesium; metabolism; Mixture; Molecular Sequence Data; MOLECULE; N-Acetylneuraminic Acid; Neisseria; Neisseria meningitidis; POTENTIAL; PROGRAM; protein; Proteins; Recombinant Fusion Proteins; Recombinant Proteins; RESIDUES; SIALIC; SIALIC-ACID; Sialyltransferases; Synthesis; synthetic; TARGET
AbstractGlycoconjugates 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
Publication date
LanguageEnglish
AffiliationNRC Institute for Biological Sciences; National Research Council Canada
Peer reviewedNo
NRC numberWILLIS2008
NPARC number9373450
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Record identifier0b5742eb-85f8-4bb3-bc68-9e7aaf2b2469
Record created2009-07-10
Record modified2016-05-09
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