Mycobacterium smegmatis phosphoinositols-glyceroarabinomannans. Structure and localization of alkali-labile and alkali-stable phosphoinositides.

M. Gilleron (1) , N. Himoudi (1) , O. Adam (1) , P. Constant (1) , A. Venisse (1) , M. Rivière (1) , G. Puzo (1)

1 IPBS - Institut de pharmacologie et de biologie structurale

Lipoarabinomannans from fast growing Mycobacterium sp., namely AraLAMs, stimulate the early events of macrophage activation. The immunological activities of all of these AraLAMs drastically decrease with the loss of the mild alkali groups, which were believed to be restricted to the fatty acid residues from the phosphatidyl-myo-inositol anchor. This 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 mycobacterial species. Their structure was unambiguously established with a strategy based on both one-dimensional 31P and two-dimensional 1H-31P heteronuclear multiple quantum correlation spectroscopy (HMQC) and HMQC-homonuclear Hartmann-Hahn spectroscopy NMR experiments applied to native AraLAMs and to AraLAMs treated in mild alkali conditions. Next to these alkali-labile phosphoinositides estimated at three per molecule, two other mild alkali-stable phosphoinositide units were identified: the expected (myo-inositol-1)-phosphate-(3-glycerol) unit typifying the well known glycosylphosphatidylinositol anchor of the mannan core and, more surprisingly, one (myo-inositol-1)-phosphate-(5-beta-D-Araf) unit having the same structure as the alkali-labile ones. Moreover, these four phosphoinositide units were found capping the arabinan side chains. Thus, their different behavior toward mild alkaline hydrolysis was explained according to their accessibility to the alkali reagent. This novel class of LAMs, namely phosphoinositols-glyceroarabinomannans (PI-GAMs), are characterized by their phosphoinositide units but also by the absence of fatty acid residues. These PI-GAMs were found to elicit the secretion of tumor necrosis factor-alpha, suggesting that phosphoinositides are the major PI-GAM epitope involved in this process.

Mots clés: TNF; tumor necrosis factor; IL; interleukin; LPS; lipopolysaccharide; BCG; bacillus Calmette Guérin; DC-SIGN; dendritic cellspecific intracellular adhesion molecule-3 grabbing nonintegrin; KC; keratinocyte-derived chemokine; MR; mannose receptor; LAM; lipoarabinomannan; LM; lipomannans; Man; mannosyl unit; ManLAM; mannose-capped LAM; MyD88; myeloid differentiation protein 88; PI; phosphatidyl-myo-inositol; PIM; PI mannosides; TLR; Toll-like receptor; MTT; 3-[4; 5-dimethylthiazol-2-yl]-2; 5diphenyltetrazolium bromide; GPI; glycosylphosphatidylinositol; TRIF; TIR domain-containing adaptor protein inducing interferon ␤; KO; knock out

Domaines: Biologie structurale [q-bio.BM]

M. Gilleron, N. Himoudi, O. Adam, P. Constant, A. Venisse, et al.. Mycobacterium smegmatis phosphoinositols-glyceroarabinomannans. Structure and localization of alkali-labile and alkali-stable phosphoinositides.. Journal of Biological Chemistry, 1997, 272 (1), pp.117-24. ⟨10.1074/jbc.m109.037846⟩. ⟨hal-00177659⟩