The genus Amycolatopsis is a member of the phylogenetic group nocardioform actinomycetes, which also includes the genus Mycobacterium. Members of this group have a characteristic cell envelope structure, dominated by various complex lipids and polysaccharides. Amongst these, lipoglycans are of particular interest since mycobacterial lipoarabinomannans are important immunomodulatory molecules. In this study we report the isolation and structural characterization of Amycolatopsis sulphurea lipoarabinomannan, designated AsuLAM. SDS/PAGE analysis revealed that AsuLAM was of an intermediate size between Mycobacterium tuberculosis lipoarabinomannan and lipomannan, confirmed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry that predicted an average molecular mass of 10 kDa. Using a range of chemical degradations, NMR experiments and capillary electrophoresis analysis, AsuLAM was revealed as an original structure. The mannosyl-phosphatidyl-myo-inositoI anchor exhibits a single acyl-form, characterized by a diacylated glycerol moiety, and contains, as one of the main fatty acids, 14-methyl-pentadecanoate, a characteristic fatty acid of the Amycolatopsis genus. AsuLAM also contains a short mannan domain; and is dominated by a multi-branched arabinan domain, composed of an (alpha1 --> 5)-Araf (arabinofuranose) chain substituted, predominately at the O-2 position, by a single beta-Araf. The arabinan domain is further elaborated by manno-oligosaccharide caps, with around one per molecule. This is the first description of manno-oligosaccharide caps found in a non-mycobacterial LAM. AsuLAM was unable to induce the production of the proinflammatory cytokine tumour necrosis factor alpha when tested with human or murine macrophage cell lines, reinforcing the paradigm that mannose-capped LAM are poor inducers of pro-inflammatory cytokines.
- capillary electrophoresis