Structural determinants in a glucose-containing lipopolysaccharide from Mycobacterium tuberculosis critical for inducing a subset of protective T cells

Abstract

Mycobacteria synthesize intracellular, 6-O-methylglucose-containing lipopolysaccharides (mGLPs) proposed to modulate bacterial fatty acid metabolism. Recently, it has been shown that Mycobacterium tuberculosis mGLP specifically induces a specific subset of protective γ₉δ₂ T cells. Mild base treatment, which removes all the base-labile groups, reduces the specific activity of mGLP required for induction of these T cells, suggesting that acylation of the saccharide moieties is required for γ₉δ₂ T-cell activation. On the basis of this premise, we used analytical LC/MS and NMR methods to identify and locate the acyl functions on the mGLP saccharides. We found that mGLP is heterogeneous with respect to acyl functions and contains acetyl, isobutyryl, succinyl, and octanoyl groups and that all acylations in mGLP, except for succinyl and octanoyl residues, reside on the glucosyl residues immediately following the terminal 3-O-methylglucose. Our analyses also indicated that the octanoyl residue resides at position 2 of an internal glucose toward the reducing end. LC/MS analysis of the residual product obtained by digesting the mGLP with pancreatic α-amylase revealed that the product is an oligosaccharide terminated by α-(1→4)-linked 6-O-methyl-d-glucosyl residues. This oligosaccharide retained none of the acyl groups, except for the octanoyl group, and was unable to induce protective γ₉δ₂ T cells. This observation confirmed that mGLP induces γ₉δ₂ T cells and indicated that the acylated glucosyl residues at the nonreducing terminus of mGLP are required for this activity.

Keywords: analytical chemistry; carbohydrate chemistry; carbohydrate function; lipoglycan; structural analysis; tuberculosis; vaccine; γδ T cells.

Figure 1.

¹H NMR. A, mGP. The deacylation was achieved by mild base hydrolysis followed by desalting. The anomeric region (δ 4.7–5.5 ppm) revealed the type of glycosidic linkages present in mGLP backbone. B, mGLP. The aliphatic region (between δ 0.5 and 3.0 ppm) revealed the different acyl groups present in mGLP. The succinyl group was confirmed with HSQC NMR (in Fig. 2).

Figure 2.

HSQC NMR (H–C 2D correlation) spectrum of G-50–purified native mGLP. NMR was performed in D₂O at room temperature. Red contour peaks correspond to methylene (-CH₂-) groups, and blue contour peaks correspond to methyl (-CH₃) and methine (>CH-) groups. GA, glyceric acid.

Figure 3.

NOESY of native mGLP (D₂O; no spin; molecular weight, ∼3800; mixing time, 0.3 s). Shown is through-space correlation of all protons in the octanoyl residue with the Glcp ring protons, succinyl residue (inset, magnified δ 4.0–5.2 ppm), and acylated (possibly succinylated) methylene protons. α(1→4) and α(1→6) anomeric protons signify octanoyl as a ring substitution other than C-6 of Glcp.

Figure 4.

LC/MS (negative ionization) of native mGLP. A reverse-phase C₁₈ column with NH₄OAc:CH₃CN gradient was used to resolve the isoforms. Each ion cluster corresponds to one isoform of mGLP (altogether 14 isoforms; seven nonsuccinylated and seven succinylated). The upper panel represents [M − 2H]²⁻ ions, and the lower panel represents corresponding [M − 3H]³⁻ ions. isobut, isobutyryl.

Figure 5.

Tandem mass spectroscopic analysis of mGLP. ESI-collision-induced dissociation in negative ion mode shows 80- and 100-eV fragment ions [M − H]⁻ of LC-purified native mGLP isoform (molecular weight, 3795; m/z 1264.1 [M − 3H]³⁻). Z_i and X_i ions correspond to the number of glycosidic linkages from the reducing end; C_i, D_i, and A_i ions correspond to the number of glycosidic linkages from the nonreducing end. The fragment ions suggested the locations of isobutyryl, acetyl, octanoyl, and glyceric acid residues and β-d-Glcp-(1→3) branches on the mGLP skeleton.

Figure 6.

Representation of native mGLP and its enzymatic porcine α(1→4)-amylase) digestion product. The MS of the major product (Fig. S5) isolated corresponded to the above structure drawn of the reducing end of mGLP (with glyceric acid and octanoyl groups intact) arising after the enzymatic cleavage of three Glcp units plus one Glcp(3Me) unit from the nonreducing end carrying isobutyryl and acetyl residues.

Figure 7.

γ₉δ₂ T-cell expansion profile of mGLP derivatives with different human PBMC volunteers. A, concentration (0.01, 0.1, and 1.0 μg/ml)-wise γ₉δ₂ T-cell expansion profile of mGLP derivatives (absolute numbers of expanded T cells with three volunteers). ▵, medium-rested (MR) + interleukin 2 (IL-2) is the baseline control in the absence of any antigen. □, native mGLP + IL-2 showed the best expansion ability at 0.1 μg/ml; saturation of biological response may be responsible for a dip in expanded T-cell numbers at 1.0 μg/ml. The enzyme-digested product (○) mGLP E1 + IL-2, which lost four nonreducing-end hexoses, two to three acetyls, and one isobutyryl group, showed inability for T-cell expansion at 0.01 or 0.1 μg/ml but a very weak expansion at 1.0 μg/ml. B, concentration (0.01, 0.1, and 1.0 μg/ml)-wise γ₉δ₂ T-cell expansion profile of mGLP derivatives (absolute numbers of expanded T-cells with two volunteers). ▵, medium-rested (MR) + IL-2 is the baseline control in the absence of any antigen. □, native mGLP + IL-2 showed the best expansion ability at 0.1 μg/ml. ×, mGP + IL-2 showed inability for T-cell expansion at 0.01 or 0.1 μg/ml but a very weak expansion at 1.0 μg/ml. ♢, the Smith degraded product from mGLP + IL-2 showed inability for T-cell expansion at 0.01 or 0.1 μg/ml but a very weak expansion at 1.0 μg/ml.