STRUCTURAL DEFINITION OF THE NONREDUCING TERMINI OF MANNOSE-CAPPED LAM FROM MYCOBACTERIUM-TUBERCULOSIS THROUGH SELECTIVE ENZYMATIC DEGRADATION AND FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY

The application of extracellular arabinases from a Cellulomonas 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 (alpha1-->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.tuberculosis Erdman 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' alpha-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 lipooligosaccharides of Neisseria species and the lipophosphoglycan of Leishmania promastigotes.