Use of Synthetic Glycolipids to Probe the Number and Position of Arabinan Chains on Mycobacterial Arabinogalactan

The arabinogalactan of Corynebacterianeae is a critical heteropolysaccharide that tethers outer membrane mycolic acids to peptidoglycan thus forming the characteristic cell wall core of these prokaryotes. An essential α-(1→5)-arabinosyltransferase, AftA, is responsible for the transfer of the first arabinofuranosyl (Ara f ) unit of the arabinan domain to the galactan backbone of arabinogalactan, but the number and precise position at which Ara f residue(s) is/are added in mycobacteria remain ill-defined. Using membrane preparations from Mycobacterium smegmatis overexpressing aftA , farnesyl-phospho-arabinose as an Ara f donor, and a series of synthetic galactan acceptors of various lengths, we here show that a single priming arabinosyl residue substitutes the C-5 position of a precisely positioned internal 6-linked galactofuranosyl residue of the galactan acceptors, irrespective of their length. This unexpected result suggests that, like the structurally related mycobacterial lipoarabinomannans, the arabinogalactan of mycobacteria may in fact harbor a single arabinan chain.