Structural characterization of a partially arabinosylated lipoarabinomannan variant isolated from a Corynebacterium glutamicum ubiA mutant

Abstract

Arabinan polysaccharide side-chains are present in both Mycobacterium tuberculosis and Corynebacterium glutamicum in the heteropolysaccharide arabinogalactan (AG), and in M. tuberculosis in the lipoglycan lipoarabinomannan (LAM). This study shows by quantitative sugar and glycosyl linkage analysis that C. glutamicum possesses a much smaller LAM version, Cg-LAM, characterized by single t-Araf residues linked to the alpha(1-->6)-linked mannan backbone. MALDI-TOF MS showed an average molecular mass of 13,800-15 400 Da for Cg-LAM. The biosynthetic origin of Araf residues found in the extracytoplasmic arabinan domain of AG and LAM is well known to be provided by decaprenyl-monophosphoryl-D-arabinose (DPA). However, the characterization of LAM in a C. glutamicum : : ubiA mutant devoid of prenyltransferase activity and devoid of DPA-dependent arabinan deposition into AG revealed partial formation of LAM, albeit with a slightly altered molecular mass. These data suggest that in addition to DPA utilization as an Araf donor, alternative pathways exist in Corynebacterianeae for Araf delivery, possibly via an unknown sugar nucleotide.

Fig. 1.

Lipoglycan profiles of wild-type C. glutamicum (a) and C. glutamicum : : ubiA (b). Lipoglycans were analysed using SDS-PAGE and visualized using the Pro-Q emerald glycoprotein stain (Invitrogen) specific for carbohydrates. Individual lipoglycans (1 and 2) were purified as described in Methods. C, crude lipoglycan; M, molecular mass markers (kDa).

Fig. 2.

Glycosyl compositional analysis of lipoglycan-2 from wild-type C. glutamicum (a) and C. glutamicum : : ubiA (b). Samples of individually purified lipoglycans (2) were hydrolysed with 2 M TFA, reduced and per-O-acetylated. Alditol acetates were subjected to GC analysis. Peak area integration shows that the arabinose content is 23 % in wild-type C. glutamicum (a) and 4.5 % in the C. glutamicum : : ubiA mutant (b). Ara, arabinose; Man, mannose.

Fig. 3.

Glycosyl linkage analysis of per-O-methylated lipoglycan-2 from C. glutamicum (a) and C. glutamicum : : ubiA (b). Lipoglycan 2 from C. glutamicum and C. glutamicum : : ubiA was per-O-methylated, hydrolysed using 2 M TFA, reduced, and per-O-acetylated. The resulting partially per-O-methylated and per-O-acetylated glycosyl derivatives were analysed by GC/MS as described previously (Alderwick et al., 2005).

Fig. 4.

(a–d) NMR characterization of wild-type Cg-LAM (a, b) and Cg-LAM from the UbiA mutant (c, d). 1D ¹H (a, c) and 2D ¹H-¹³C HMQC (b, d) NMR spectra of Cg-LAMs in D₂O at 313 K. Expanded regions (δ ¹H: 4.85–5.30) (a, c) and (δ ¹H: 4.85–5.30, δ ¹³C: 100–114) (b, d) are shown. Glycosyl residues are labelled in Roman numerals and their carbons and protons in Arabic. I, II, t-α-Araf; III, t-α-Manp; IV, V, VI, 2,6-α-Manp; VII, 6-α-Manp; VIII, 2-α-Manp. (e) Structural representation of Cg-LAM (Tatituri et al., 2007). Cg-LAM contains a (1→6)-Manp backbone almost completely substituted by t-Araf, t-Manp, t-Manp(1→2)-Manp and t-Araf(1→2)-Manp units. X, either a t-Araf or a t-Manp unit.

Fig. 5.

MALDI-TOF MS spectra of Cg-LAM from wild-type C. glutamicum (a) and C. glutamicum : : ubiA (b). MALDI spectra were acquired in the linear negative mode with delayed extraction using 2,5-dihydrobenzoic acid as matrix.

Fig. 6.

Proposed biosynthetic pathway of Cg-LAM. The addition of the first Araf unit is thought to occur on the cytoplasmic side of the membrane by a GT-A/B glycosyltransferase (Liu & Mushegian, 2003). After transportation across the membrane by an unknown ‘flippase’ enzyme, further elaboration of the lipoglycan then occurs through the addition of mannose and arabinose units catalysed by several GT-C glycosyltransferases utilizing DPM and DPA, respectively (Berg et al., 2007; Seidel et al., 2007; Tatituri et al., 2007).