Chemical Composition of Antigen 60 from Mycobacterium bovis BCG

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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, fatly acids, and of phosphatidyl-inositol-mannosides. Lipocon-jugates, upon DEAE-cellulose chromatography and gel filtration, yielded two main fractions of neutral and polar components. Chromatography of delipidated and deproieinizd A60 on Sephadex G-100yielded: a high molecular weight fraction (Al, 18%, a lipoglucan of ± 106) and a low molecular weight fraction (B, 10%, a lipopeptidoglycan of ± 104) containing mannose, glucose, and small amount of arabinose. The polysaccharide moieties of fractions Al and B were submitted to aeetylalion, methylation, and acid hydrolysis, and ihe structure of the hydrolysed polymer was deduced by combined gas chromalography/mass spectrometry analysis. The results indicated a branched structure involving 1. 4-, 1, 6-, and 1, 4, 6-linked D-gluco- or n-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 delipidalion and proteolysis did not suppress the ability of A60 to yield immunoprrecipitates with anti -A60 antiserum. The high polymer fractions obtained by chromatography on DEAF cellulose and Sephadex G-100 were also reactive. It is concluded that A60 is made of tree lipids and of hpopcptidoglycans of high molecular weights (106-107) endowed with immunogenic properties.