Molecular cloning and expression of an alpha-mannosidase gene in Mycobacterium tuberculosis.
Mannose is a major component of glycolipids and glycoproteins of the cell envelope of M. tuberculosis (Mtb). However, the enzymes involved in the biosynthesis and catabolism of mannosylated glycans are largely unknown. We demonstrate alpha-mannosidase activity towards the fluorescent substrate 4-methylumberlliferyl-alpha-D-mannopyranoside (4MU-Man) in cell lysates of attenuated and virulent Mtb bacilli, with two-fold higher activity in the virulent strain Erdman. Mannosidase activity was optimal at pH 6.5, was not inhibited by deoxymannojirimycin (dMNJ), was mildly inhibited by swainsonine (SW) and stimulated two-fold by EDTA. GenBank BLAST analysis for sequences homologous to eukaryotic alpha-mannosidases revealed a 3.6 kb putative gene (Rv0648) in Mtb cosmid SCY20H10 (Acc# z92772), with strong homology (48%) to the rat ER/cytosolic alpha-mannosidase and containing signature sequences of class 2 mannosidases. By RT-PCR, gene Rv0648 was found differentially expressed, with lower expression during growth in A549 pneumocyte cultures. Gene Rv0648 was cloned, expressed in E. coli, and alpha-mannosidase activity in cell lysates determined. Expression of alphaMan-pET in E. coli cells resulted in an eight-fold increase in mannosidase activity toward 4-MU-Man, upon IPTG induction. Partial purification of the histidine-tagged Mtb mannosidase by metal chelation affinity chromatography, and analysis by SDS-PAGE, showed a protein with the predicted m.w. of 137.5 kDa. Enzyme assays of the column fractions showed alpha-mannosidase activity toward synthetic aryl-mannose substrates, in fractions enriched in the recombinant Mtb mannosidase. These results demonstrate that gene Rv0648 encodes an active alpha-mannosidase in Mtb.
Rivera-Marrero
C A
CA
Department of Medicine, Emory University School of Medicine, Decatur, GA 30033, USA. crivera@emory.edu
Ritzenthaler
J D
JD
Roman
J
J
Moremen
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D008361:Mannosidases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity; D051381:Rats; D017386:Sequence Homology, Amino Acid; D043323:alpha-Mannosidase
C008512:methylumbelliferyl-alpha-D-mannopyranoside; D006923:Hymecromone; D008361:Mannosidases; D043323:alpha-Mannosidase