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 -
inositol 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)-Ara f (
arabinofuranose) chain substituted, predominately at the O -2 position, by a single beta-Ara f. 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 pro-inflammatory
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.