A NEW INTERPRETATION OF THE STRUCTURE OF THE MYCOLYL ARABINOGALACTAN COMPLEX OF MYCOBACTERIUM-TUBERCULOSIS AS REVEALED THROUGH CHARACTERIZATION OF OLIGOGLYCOSYLALDITOL FRAGMENTS BY FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY AND H-1 NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Previous structural analysis of small oligosaccharide fragments had al lowed the recognition of several small structural motifs within arabin ogalactan, the dominant cell wall structural polysaccharide of Mycobac terium tuberculosis. To determine how these motifs are connected to on e another to form the complete polymer, oligosaccharide fragments cont aining up to 26 glycosyl residues were released by gentle acid hydroly sis of the per-O-methylated arabinogalactan, converted to fully per-O- alkylated oligoglycosylalditols, and purified by high-performance liqu id chromatography, and the molecular weights and alkylation patterns o f the resultant oligoglycosyl fragments were determined by fast atom b ombardment mass spectrometry. The results, combined with previous stud ies, allowed further understanding of the intricate structural feature s of the nonreducing ends of arabinogalactan. Thus, the extended nonre ducing ends of the arabinan were shown to consist of a tricosaarabinos ide (23-mer). We reason that three such arabinan motifs are attached t o the homogalactan component of arabinogalactan, which was previously shown to consist of alternating 5- and 6-linked beta-D-galactofuranosy l residues. Using the same approach as applied to the arabinan branche s, an extended stretch of the galactan was isolated that consisted of at least 23 alternating beta-1,6 and beta-1,5 D-Galf residues, devoid of any branching, demonstrating that the points of attachment of the a rabinan chains to galactan are close to the reducing end of galactan, which itself is linked to peptidoglycan via the linker disaccharide ph osphate L-Rhap-(1-->3)-alpha-D-GlcNAc-P. By nuclear magnetic resonance analysis, the L-Rhap was shown to be in the a configuration, The long -chain alpha-alkyl-beta-hydroxy mycolic acids, known to occupy the 5-p ositions of both the terminal beta-D-Araf and internal 2-alpha-D-Araf residues of the terminal branched pentaarabinosyl motif, are now shown to be nonacylated at the beta-hydroxy function, Lack of acylation poi nts to intramolecular hydrogen bonding between the beta-hydroxyl and c arbonyl functions of the mycolic acid, providing a highly ordered arra ngement of mycolic acids in accord with evolving models of the orienta tion of the cell wall polymers in mycobacterial cell walls, A revised model is proposed for the composition and orientation of the mycolyl-a rabinogalactan in the cell walls of M. tuberculosis, which should incr ease our understanding of cell wall hydrophobicity, impermeability, an d role in disease pathogenesis.