title: Molecular cloning and expression of a novel glycolipid sulfotransferase in Mycobacterium tuberculosis.
abstract: Sulfated trehalose glycolipids are among the most characteristic cell wall molecules of virulent strains of Mycobacterium tuberculosis. They comprise a family of trehalose-2-sulfate esters with an array of acyl fatty acids at various positions of the trehalose moiety. Although their structure has been well characterized, most of the enzymes involved in their biosynthesis, such as sulfotransferases, are unknown. It is demonstrated here by metabolic labelling with 35S abundant incorporation into sulfolipids of M. tuberculosis strains, in comparison to Mycobacterium avium, Mycobacterium bovis BCG and Mycobacterium smegmatis. The most abundant sulfolipid, sulfolipid I, is present in virulent strains H37Rv and Erdman, but absent in attenuated H37Ra. Sulfotransferase assays with the donor substrate 3'-phosphoadenosine-5'-[35S]phosphosulfonate and whole cell lysates of H37Ra resulted in the synthesis of four major sulfolipids (I, II, IV and VI). A search for sulfotransferase gene sequences in M. tuberculosis yielded gene Rv1373, a 981 bp gene slightly homologous (24% identity) to eukaryotic aryl-sulfotransferases. Rv1373 was cloned by PCR and expressed as a 39 kDa recombinant his-tagged protein. The recombinant M. tuberculosis aryl-sulfotransferase exhibited activity towards the cerebroside glycolipids glucosyl- and galactosylceramide. No activity was detected with sulfatide (3'-sulfated galactosylceramide), suggesting that sulfation of galactosylceramide may occur at C-3 of the galactose. Treatment of sulfated products with ceramide glycanase resulted in the release of 35S-labelled material showing that sulfation was at the saccharide moiety (galactose or glucose) of the ceramide. Assays with the M. tuberculosis aryl-sulfotransferase and total H37Ra glycolipids showed one major product corresponding to sulfolipid IV. These results demonstrate that Rv1373 encodes a novel glycolipid sulfotransferase with activity towards typical ceramide glycolipids and mycobacterial trehalose glycolipids.
authors.affiliation: Department of Medicine, Pulmonary and Critical Care Division, Atlanta VA Medical Center, Emory University School of Medicine, Room 12C 106, 1670 Clairmont Rd, Decatur, GA 30033, USA1.
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Rivera-Marrero
authors.affiliation: Department of Medicine, Pulmonary and Critical Care Division, Atlanta VA Medical Center, Emory University School of Medicine, Room 12C 106, 1670 Clairmont Rd, Decatur, GA 30033, USA1.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Ritzenthaler
authors.affiliation: Department of Medicine, Pulmonary and Critical Care Division, Atlanta VA Medical Center, Emory University School of Medicine, Room 12C 106, 1670 Clairmont Rd, Decatur, GA 30033, USA1.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Newburn
authors.affiliation: Department of Medicine, Pulmonary and Critical Care Division, Atlanta VA Medical Center, Emory University School of Medicine, Room 12C 106, 1670 Clairmont Rd, Decatur, GA 30033, USA1.
authors.forename: Jesse
authors.initials: J
authors.lastname: Roman
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA2.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005699:Galactosylceramides / Q000378:metabolism; D005963:Glucosylceramides / Q000378:metabolism; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017422:Sequence Analysis, DNA; D015238:Sulfotransferases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D005699:Galactosylceramides; D005963:Glucosylceramides; D015238:Sulfotransferases; C020556:galactosylceramide sulfotransferase