Lipoarabinomannans:: characterization of the multiacylated forms of the phosphatidyl-myo-inositol anchor by NMR spectroscopy

Lipoarabinomannans. which exhibit a large spectrum of immunological activities, emerge as the major antigens of mycobacterial envelopes. The lipoarabinomannan structure is based on a phosphatidyl-myo-inositol anchor whose integrity has been shown to be crucial for lipoarabinomannan biological activity and particularly for presentation to CD4/CD8 double-negative alpha beta T cells by CD1 molecules. In this report, an analytical approach was developed for high-resolution P-31-NMR analysis of native, i.e, multiacylated, lipoarabinomannans. The one-dimensional P-31 spectrum of cellular lipoarabinomannans, from Mycobacterium bovis Bacillus Calmette-Guerin, exhibited four P-31 resonances typifying four types of lipoarabinomannans. Two-dimensional H-1-P-31 heteronuclear multiple-quantum-correlation/homonuclear Hartmann-Hahn analysis of the native molecules showed that these four types of lipoarabinomannan differed in the number and localization of fatty acids (from 1 to 4) esterifying the anchor. Besides the three acylation sites previously described, i.e. positions 1 and 2 of glycerol and 6 of the mannosyl unit linked to the C-2 of myo-inositol, we demonstrate the existence of a fourth acylation position at the C-3 of myo-inositol. We report here the first structural study of native multiacylated lipoarabinomannans, establishing the structure of the intact phosphatidyl-myo-inositol anchor. Our findings would help gain more understanding of the molecular basis of lipoarabinomannan discrimination in the binding process to CD1 molecules.