Structural study of the LipoMannans from Mycobacterium bovis BCG:: Characterisation of multiacylated forms of the phosphatidyl-myo-inositol anchor

A biosynthetic filiation is postulated between the mycobacterial phosphatidyl-myo-inositol mannosides (PIMs), the lipomannans (LMs) and the lipoarabinomannans (LAMs), the major antigens of the envelopes. Moreover, as the PI anchor is thought to play a role in the biological functions of the LAMs, we characterized the lipid moiety of the PI anchor from Mycobacterium bovis BCG cellular LMs. Their structure was investigated along with that of a purified tetra-acylated form of PLM, (Ac4PIM2). A two-dimensional H-1-P-31 heteronuclear multiple quantum correlation homonuclear Hartmann-Hahn spectroscopy study of Ac4PIM2 unambiguously localised a fourth fatty acid on the C3 of the myo-Ins beside the fatty acids already described on the C1 and C2 position of the glycerol and on the C6 position of the mannose. This analytical strategy was extended to the structural study of the cellular LM anchor. Using an appropriate solvent system, the one dimensional P-31 NMR spectrum exhibited four major resonances typifying the LM populations. These populations differed in number and location of the fatty acids. For one of these populations, we established the presence of an extra fatty acid on the C3 of the myo-Ins of the LM anchor. The fact that both types of mol ecules have an elaborated anchor in common, indicates that cellular LMs are multimannosylated forms of PIMs. In addition, the LM mannan core structure was analysed by two-dimensional NMR, pointing to a high level of branching by single alpha 1 --> 2 Manp side-chains. (C) 1999 Academic Press.