MYCOBACTERIUM-SMEGMATIS PHOSPHOINOSITOLS-GLYCEROARABINOMANNANS - STRUCTURE AND LOCALIZATION OF ALKALI-LABILE AND ALKALI-STABLE PHOSPHOINOSITIDES

Lipoarabinomannans from fast growing Mycobacterium sp., namely AraLAMs , stimulate the early events of macrophage activation. The immunologic al activities of all of these AraLAMs drastically decrease with the lo ss of the mild alkali groups, which were believed to be restricted to the fatty acid residues from the phosphatidyl-myo-inositol anchor. Thi s report reveals the presence and the structure of mild alkali-labile phosphoinositide units linked via the phosphate to the C-5 of the beta -D-Araf in the AraLAMs of Mycobacterium smegmatis, a fast growing myco bacterial species. Their structure was unambiguously established with a strategy based on both one-dimensional P-31 and two-dimensional H-1- P-31 heteronuclear multiple quantum correlation spectroscopy (HMQC) an d HMQC-homonuclear Hartmann-Hahn spectroscopy NMR experiments applied to native AraLAMs and to AraLAMs treated in mild alkali conditions. Ne xt to these alkali-labile phosphoinositides estimated at three per mol ecule, two other mild alkali-stable phosphoinositide units were identi fied: the expected (myo-inositol-1)-phosphate-(3-glycerol) unit typify ing the well known glycosylphosphatidylinositol anchor of the mannan c ore and, more surprisingly, one (myo-inositol-1)-phosphate-(5-beta-D-A raf) unit having the same structure as the alkali-labile ones. Moreove r, these four phosphoinositide units were found capping the arabinan s ide chains. Thus, their different behavior toward mild alkaline hydrol ysis was explained according to their accessibility to the alkali reag ent. This novel class of LAMs, namely phosphoinositols-glyceroarabinom annans (PI-GAMs), are characterized by their phosphoinositide units bu t also by the absence of fatty acid residues. These PI-GAMs were found to elicit the secretion of tumor necrosis factor-alpha, suggesting th at phosphoinositides are the major PI-GAM epitope involved in this pro cess.