Functional characterization of Lpt3 and Lpt6, the inner-core lipooligosaccharide phosphoethanolamine transferases from Neisseria meningitidis.
The lipooligosaccharide (LOS) of Neisseria meningitidis contains heptose (Hep) residues that are modified with phosphoethanolamine (PEtn) at the 3 (3-PEtn) and/or 6 (6-PEtn) position. The lpt3 (NMB2010) and lpt6 (NMA0408) genes of N. meningitidis, which are proposed to encode the required HepII 3- and 6-PEtn transferases, respectively, were cloned and overexpressed as C-terminally polyhistidine-tagged fusion proteins in Escherichia coli and found to localize to the inner membrane, based on sucrose density gradient centrifugation. Lpt3-His(6) and Lpt6-His(6) were purified from Triton X-100-solubilized membranes by nickel chelation chromatography, and dot blot analysis of enzymatic reactions with 3-PEtn- and 6-PEtn-specific monoclonal antibodies demonstrated conclusively that Lpt3 and Lpt6 are phosphatidylethanolamine-dependent LOS HepII 3- and 6-PEtn transferases, respectively, and that both enzymes are capable of transferring PEtn to both fully acylated LOS and de-O-acylated (de-O-Ac) LOS. Further enzymatic studies using capillary electrophoresis-mass spectrometry (MS) demonstrated that both Lpt3 and Lpt6 are capable of transferring PEtn to de-O-Ac LOS molecules already containing PEtn at the 6 and 3 positions of HepII, respectively, demonstrating that there is no obligate order of PEtn addition in the generation of 3,6-di-PEtn LOS moieties in vitro.
Institute for Biological Sciences, 100 Sussex Drive, National Research Council, Ottawa, ON, Canada K1A 0R6. Cory.Wenzel@nrc-cnrc.gc.ca
Cory Q
CQ
Wenzel
Frank
F
St Michael
Jacek
J
Stupak
Jianjun
J
Li
Andrew D
AD
Cox
James C
JC
Richards
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004982:Ethanolaminephosphotransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009345:Neisseria meningitidis / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D008565:Membrane Proteins; C023023:lipid-linked oligosaccharides; D004982:Ethanolaminephosphotransferase