title: Chemical composition of antigen 60 from Mycobacterium bovis BCG.
abstract: Antigen 60 (A60), the main thermostable immunogen of tuberculin and PPD, has been purified from Mycobacterium bovis BCG cytoplasm, and identified by crossed immunoelectrophoresis with anti-BCG polyclonal antiserum. Two A60 fractions, free lipids and lipid-conjugated compounds, have been recognized. The free lipids represented about 30% (dry weight), and consisted essentially of C16-C18 fatty acids, and of phosphatidyl-inositol-mannosides. Lipoconjugates, upon DEAE-cellulose chromatography and gel filtration, yielded two main fractions of neutral and polar components. Chromatography of delipidated and deproteinized A60 on Sephadex G-100 yielded: a high molecular weight fraction (Al, 18%, a lipoglucan of congruent to 10(6)), and a low molecular weight fraction (B, 10%, a lipopeptidoglycan of congruent to 10(4)) containing mannose, glucose, and small amounts of arabinose. The polysaccharide moieties of fractions Al and B were submitted to acetylation, methylation, and acid hydrolysis, and the structure of the hydrolysed polymer was deduced by combined gas chromatography/mass spectrometry analysis. The results indicated a branched structure involving 1,4-, 1,6-, and 1,4,6-linked D-gluco- or D-manno-pyranosyl residues. Glucan- and peptidoglycan-bound fatty acids were identified as saturated (C16-C18) and monounsaturated linear acids (C12-C18). Immunodiffusion on agarose gel indicated that delipidation and proteolysis did not suppress the ability of A60 to yield immunoprecipitates with anti-A60 antiserum. The high polymer fractions obtained by chromatography on DEAE cellulose and Sephadex G-100 were also reactive. It is concluded that A60 is made of free lipids and of lipopeptidoglycans of high molecular weights (10(6)-10(7)) endowed with immunogenic properties.
mesh_terms: D000942:Antigens, Bacterial / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000032:analysis; D005227:Fatty Acids / Q000032:analysis; D008055:Lipids / Q000032:analysis; D009163:Mycobacterium bovis / Q000276:immunology*; D011135:Polysaccharides, Bacterial / Q000032:analysis
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D005227:Fatty Acids; D008055:Lipids; D011135:Polysaccharides, Bacterial