Abstract
Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5-7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10-12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl "primer." Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.
Highlights
Important details of the structures of lipomannan and lipoarabinomannan remain unknown
Using an MSMEG_4241 mutant that lacks the ␣-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has [5,6,7] unbranched ␣-6-linked-mannosyl residues followed by two or three ␣-6-linked mannosyl residues branched with single ␣-mannopyranose residues at O-2
Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (⌬embC and ⌬MSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ϳ10 –12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl “primer.” these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM
Summary
Important details of the structures of lipomannan and lipoarabinomannan remain unknown. The innermost region consists of a linear ␣-(1,5)-linked Araf backbone of unknown and likely variable length that is attached at its reducing end to the mannan core This linear region is followed by a branched region the details of which are poorly understood, an interesting hypothesis suggesting elongation and further branching based on the structure of the more fully understood arabinan present in arabinogalactan has been put forth (9). Inactivation of the M. smegmatis orthologous gene (MSMEG_4247) by allelic exchange blocked the synthesis of mature, branched LM and resulted in the accumulation of a LAM-like product shown by MALDI-TOF/MS to be lower in molecular weight than WT LAM and devoid of 2,6-linked Manp and the corresponding terminal Manp residues This product had little alteration in the known arabinan motifs, suggesting that a linear ␣-(1,6) mannan devoid of any ␣-(1,2) Manp branching is capable of being arabinosylated in a normal fashion (16).
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