Cloning of the lipooligosaccharide alpha-2,3-sialyltransferase from the bacterial pathogens Neisseria meningitidis and Neisseria gonorrhoeae.
The genes encoding the alpha-2,3-sialyltransferases involved in lipooligosaccharide biosynthesis from Neisseria meningitidis and Neisseria gonorrhoeae have been cloned and expressed in Escherichia coli. A high sensitivity enzyme assay using a synthetic fluorescent glycosyltransferase acceptor and capillary electrophoresis was used to screen a genomic library of N. meningitidis MC58 L3 in a "divide and conquer" strategy. The gene, denoted lst, was found on a 2. 0-kilobase fragment of DNA, and its sequence was determined and then used to design probes to amplify and subsequently clone the corresponding lst genes from N. meningitidis 406Y L3, N. meningitidis M982B L7, and N. gonorrhoeae F62. Functional sialyltransferase was produced from the genes derived from both L3 N. meningitidis strains and the N. gonorrhoeae F62. However, the N. meningitidis M982B L7 gene contained a frameshift mutation that renders it inactive. The expression of the lst gene was easily detected using the enzyme assay, and the protein expression could be detected when an immunodetection tag was added to the COOH-terminal end of the protein. Using the synthetic acceptor N-acetyllactosamine-aminophenyl-(6-(5-(fluorescein-carboxamido)-hexan oic acid amide), the alpha-2,3 specificity of the enzyme was confirmed by NMR examination of the reaction product. The enzyme could also use synthetic acceptors with lactose or galactose as the saccharide portion. This study is the first example of the cloning, expression, and examination of alpha-2,3-sialyltransferase activity from a bacterial source.
Gilbert
M
M
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada. wakarchu@biologysx.lan.nrc.ca
Watson
D C
DC
Cunningham
A M
AM
Jennings
M P
MP
Young
N M
NM
Wakarchuk
W W
WW
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000096:biosynthesis*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000201:enzymology*; D009345:Neisseria meningitidis / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017384:Sequence Deletion; D012799:Sialyltransferases / Q000235:genetics*; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D000942:Antigens, Bacterial; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; C023023:lipid-linked oligosaccharides; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase