Structural definition of the non-reducing termini of mannose-capped LAM from Mycobacterium tuberculosis through selective enzymatic degradation and fast atom bombardment-mass spectrometry

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Abstract
The application of extracellular arabinases from aCellulomonas sp. and fast atom bombardment-mass spectrometry (FAB-MS) provided new insight into the structure of lipoarabinomannan (LAM) of Mycobacterium tuberculosis, a key molecule in the pathogenesis and physiology of the tubercle bacillus. Previously, the non-reducing arabinan ends of LAM from the virulent (Erdman) strain of M.tuberculosis were shown to be ‘capped’ by short (a1→2)-linked mannopyranose (Manp)-containing oligosaccharides, a product called ManLAM. The structural relationship between these Manp units and the underlying arabinofuranose (Araf)-containing arabinan was examined by digesting ManLAM from M.tuberculosisErdman with the Cellulomonas enzyme, resolving fragments by various means and subjecting the derivatized oligoglycosylalditols to FAB-MS. The sequences Manp2Araf4, Manp3Araf4 and Manp1–6Araf6 were recognized as the major terminal motifs. Upon complete structural definition, all of the Ara6-containing products were shown to be based on a 3,5-linked branched Araf unit, whereas those containing Ara4 were linear. Minor non-mannosylated terminal arrangements containing Ara4–6, branched, linear and cyclical, were also recognized. In addition, the mannan ‘core’ of ManLAM was isolated from enzyme digests and shown to contain segments of the phosphatidylinositol anchor and a ‘stub’ of the arabinan side-chain in the form of a ‘linker’ a-Araf-(1→5)-Araf unit attached to C-2, apparently of the penultimate 2,6-linked Manp residue. The structural unravelling of this complex molecule further substantiates the case for structural and biological similarities to the enterobacterial lipopolysaccharides/lipoglycans and other important ‘capped’ lipooligomers such as the lipooligosac charides of Neisseriaspecies and the lipophosphoglycan of Leishmaniapromastigotes.

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