Abstract
The lipopolysaccharides of mycobacteria, lipoarabinomannan (LAM) and lipomannan (LM), of key importance in host-pathogen interaction, were recently shown to contain a phosphatidylinositol "anchoring domain." We now have established that LAM and LM are based on the phosphatidylinositol mannosides, the characteristic glycophospholipids of mycobacteria. Digestion of the arabinose-free LM with an endo-alpha 1----6-mannosidase yielded evidence for the presence of the 1-(sn-glycerol-3-phospho)-D-myo-inositol-2,6-bis-alpha-D-mannopyranoside unit, indistinguishable from that derived from phosphatidylinositol dimannoside. This same inositol substitution pattern was shown to be present in LAM by methylation analysis before and after dephosphorylation. Positions C-2 and C-6 of the inositol unit of LAM are occupied by mannosyl residues and C-1 by a phosphoryl group. Partial acid hydrolysis of per-O-methylated LAM and comparison by gas chromatography-mass spectrometry of the resulting derivatized oligosaccharides with like products from phosphatidylinositol hexamannoside demonstrated that the C-6 of inositol is the point of attachment of the mannan core of LAM, which consists of an alpha 1----6-linked backbone with considerable alpha-1----2 side chains. Thus, a structural and presumably biosynthetic relationship is established between some of the membranous mannosylphosphatidylinositols described some 25 years ago and the newly emerging, biologically active lipopolysaccharides of mycobacteria.
Highlights
The lipopolysaccharides of mycobacteria, lipoarabi- of mycobacterial protein antigensby antigen-presenting cells nomannan(LAM) and lipomannan(LM), of key impor- (4) and may abrogate T-cell activation, further ensuring pertanceinhost-pathogeninteraction, were recently sistence of pathogen within the host
LAM induces the showntocontain a phosphatidylinositol“anchoring production of tumor necrosis factor (5, 6) and may be domain.”We have established thatLAM and LM responsible for mediating granuloma formation, nerve damare based on the phosphatidylinositol mannosides, theage, and tissue necrosis
PositionsC-2 and C-6 of the report that LAM and LM are based on the phosphatidylinoinositol uniotf LAM are occupied by mannosyl residuessitol mannosides (PIM), a group of distinct mycobacterial and C-1 by a phosphoryl groPuapr.tial acid hydrolysis glycophospholipids,known since the 1940sand fully described of per-0-methylatedLAM and comparison bgyaschro- by Ballou and colleagues (12,13)in the1960s
Summary
The HFwas removed by continuous evacuation over KOH and the products applied to columns of Bio-Gel (P-2 for PIM and P-100 for LAM and LM). PIM2 and the deacylated product (dPIMz; Gro-P-Ins-Manz)were readily purified (Fig. 1)and characterized (20); the structure described by Lee and Ballou (20) for the M. tuberculosis dPIM2 was shown to apply to the product from M. leprae (results not shown). Initial digestion of [3H]Ins-dLM with an exoa-D-mannosidase and purification of the product on Bio-Gel in 0.1 N CH3COOH (10)resulted inan exomannosidaseresistant [3H]Ins-containing product (Fig. 3A), analysis of which had previously suggested a linear a-1-6-linked nonaor decamannoside with some single t-a-D-Manp side chains of Sephacryl S-200 in detergent; the presence of deoxycholate attached to C-2 (10).This product proved susceptible to endoin buffers apparently led to dissociation of aggregates of LAM, a-(1+6)-mannosidase, yielding a [3H]Ins-containingsmall molecular weight product after ion-exchange chromatography.
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