Characterization of mycobacterial protein glycosyltransferase activity using synthetic peptide acceptors in a cell-free assay.
Synthetic peptides derived from a 45-kDa glycoprotein antigen of Mycobacterium tuberculosis were shown to function as glycosyltransferase acceptors for mannose residues in a mannosyltransferase cell-free assay. The mannosyltransferase activity was localized within both isolated membranes and a P60 cell wall fraction prepared from the rapidly growing mycobacterial strain, Mycobacterium smegmatis. Incorporation of radiolabel from GDP-[(14)C]mannose was inhibited by the addition of amphomycin, indicating that the glycosyl donor for the peptide acceptors was a member of the mycobacterial polyprenol-P-mannose (PPM) family of activated glycosyl donors. Furthermore, a direct demonstration of transfer from the in situ generated PP[(14)C]Ms was also demonstrated. It was also found that the enzyme activity was sensitive to changes in overall peptide length and amino acid composition. Because glycoproteins are present on the mycobacterial cell surface and are available for interaction with host cells during infection, protein glycosyltransferases may provide novel drug targets. The development of a cell-free mannosyltransferase assay will now facilitate the cloning and biochemical characterisation of the relevant enzymes from M. tuberculosis.
Centre for Molecular Microbiology and Infection, Imperial College of Science, Technology and Medicine, South Kensington, London, SW7 2AZ, England.
Howard N
HN
Cooper
Sudagar S
SS
Gurcha
Jérôme
J
Nigou
Patrick J
PJ
Brennan
John T
JT
Belisle
Gurdyal S
GS
Besra
Douglas
D
Young
D000595:Amino Acid Sequence; D001345:Autoradiography; D002474:Cell-Free System; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D006031:Glycosylation; D016695:Glycosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology*
D016695:Glycosyltransferases; D008358:Mannose