Elastin, a novel extracellular matrix protein adhering to mycobacterial antigen 85 complex

Abstract

The antigen 85 complex (Ag85) consists of three predominantly secreted proteins (Ag85A, Ag85B, and Ag85C), which play a key role in the mycobacterial pathogenesis and also possess enzymatic mycolyltransferase activity involved in cell wall synthesis. Ag85 is not only considered to be a virulence factor because its expression is essential for intracellular survival within macrophages, but also because it contributes to adherence, invasion, and dissemination of mycobacteria in host cells. In this study, we report that the extracellular matrix components, elastin and its precursor (tropoelastin) derived from human aorta, lung, and skin, serve as binding partners of Ag85 from Mycobacterium tuberculosis. The binding affinity of M. tuberculosis Ag85 to human tropoelastin was characterized (K(D) = 0.13 ± 0.006 μm), and a novel Ag85-binding motif, AAAKAA(K/Q)(Y/F), on multiple tropoelastin modules was identified. In addition, the negatively charged Glu-258 of Ag85 was demonstrated to participate in an electrostatic interaction with human tropoelastin. Moreover, binding of Ag85 on elastin siRNA-transfected Caco-2 cells was significantly reduced (34.3%), implying that elastin acts as an important ligand contributing to mycobacterial invasion.

FIGURE 1.

Binding of M. tuberculosis Ag85 to elastin and tropoelastin. A, comparison of the elastin-binding affinities of M. tuberculosis Ag85A, Ag85B, and Ag85C. His-tagged M. tuberculosis Ag85 proteins (0.2 μm) were added to microtiter plate wells coated with 1 μg of elastin (extracted from human aorta). Bound proteins were detected by ELISA. B, binding of M. tuberculosis Ag85B to native Fn, elastin, or tropoelastin. M. tuberculosis Ag85B (0.2 μm) was added to wells coated with 1 μg of Fn (extracted from human plasma, serving as a positive control), elastins (extracted from human aorta, lung, and skin, respectively), tropoelastin (extracted from chicken aorta), or BSA (negative control). C, SPR analysis of recombinant GST-tagged human tropoelastin (full-length) interacting with immobilized M. tuberculosis Ag85B. The measured k_on, k_off, and K_D values were 5.8 × 10⁴ ± 0.7 m⁻¹ s⁻¹, 7.7 × 10⁻³ ± 0.5 s⁻¹, and 0.13 ± 0.006 μm, respectively. RU, response units.

FIGURE 2.

Mapping the binding fragments of MTB Ag85B on HTE. A, schematic diagram showing the HTE and truncated HTE fragments (including HTE(1–18), HTE(17–27), and HTE(27–36)) used in this study. All repeating module types are indicated on the full-length HTE, although only KA-cross-linking domains are shown on the truncated HTE fragments. B, binding of truncated M. tuberculosis Ag85B to immobilized recombinant GST-tagged truncated HTE fragments. Various concentrations (0, 0.2, 0.4, 1, 2, and 3.2 μm) of truncated HTE fragments were coated on microtiter plate wells, incubated with His-tagged M. tuberculosis Ag85B (0.2 μm), and detected by ELISA. The measured K_D values were 0.44 ± 0.08 μm (HTE(1–18)) and 0.49 ± 0.15 μm (HTE (17–27)), and 0.53 ± 0.18 μm (HTE (27–36)). Each value represents the mean ± S.D. of three trials performed in triplicate samples. C, eight synthetic peptides (2 μm) based on the sequence of HTE(27–36) fragments were immobilized on microtiter plate wells, incubated with His-tagged M. tuberculosis Ag85B (0.2 μm), and detected by ELISA. D, sequence alignment of the M. tuberculosis Ag85B-binding peptides and other HTE KA-cross-linking domains reveals a conserved motif, AAAKAAKY. The conserved residues are highlighted (identical is black; and similar is gray).

FIGURE 3.

Identification of the key residues of HTE involved in MTB Ag85B binding. A, schematic representation of mutants (V1A, P2A, G3A, L5A, K9A, K12A, Y13A, K9R, K12R, and Y13F) derived from GST-HTE(27–28) by single residue scanning substitution. B, K_D determination of recombinant GST-HTE(27–28) to M. tuberculosis Ag85B by SPR sensorgrams analysis. The measured k_on, k_off, and K_D values were 3.1 × 10³ ± 0.7 m⁻¹ s⁻¹, 1.5 × 10⁻³ ± 0.3 s⁻¹, and 0.48 ± 0.04 μm, respectively. C, immobilized GST-tagged HTE (27–28) mutants or wild-type (0.2 μm) was incubated with M. tuberculosis Ag85B (0.5 μg) on microtiter plate wells and detected using ELISA. D, NMR structure of pHTE 27 (previously determined by Tamburro and Bochicchio (35)) was colored by residue using the relative decrease in binding affinity for the corresponding alanine mutation. Alanine residues (black) were not included in the scan and analysis.

FIGURE 4.

Mapping the HTE-binding site on MTB Ag85. A, schematic representation of the truncated M. tuberculosis Ag85B constructs used in this study, including M. tuberculosis Ag85B-N (residues 41–133), M. tuberculosis Ag85B-Ctr1 (residues 134–198), M. tuberculosis Ag85B-Ctr2 (residues 199–253), and M. tuberculosis Ag85B-C (residues 254–325). B, comparison of HTE binding affinity of truncated M. tuberculosis Ag85B proteins. Full-length and truncated M. tuberculosis Ag85B (0.2 μm) was added to microtiter plate wells containing recombinant GST-tagged HTE or BSA (0.2 μg/well). Bound proteins were detected by ELISA.

FIGURE 5.

Identification of the key positively charged residues of MTB Ag85 involved in HTE binding. A, sequence alignment of the C termini of M. tuberculosis Ag85, subtypes Ag85A, Ag85B, and Ag85C. The arrows indicated the conserved negatively charged residues. B, four His-tagged mutants (E258A, E270A, D282A, and D317A) derived from M. tuberculosis Ag85B were added to microtiter plate wells coated with 1 μg of recombinant GST-tagged HTE. Bound proteins were detected by ELISA. Wild-type M. tuberculosis Ag85B (WT) served as a positive control. C, structure of M. tuberculosis Ag85B (Protein Data Bank code 1f0n) (36) was used to depict the M. tuberculosis Ag85B-C (residues 254–325) (cyan) region and the conserved negatively charged residues in this region. The HTE-interacting residue, Glu-258, is shown in red. D, Ag85B-pHTE 27 docking model. ClusPro 2.0 (34) was used to dock pHTE 27 (green) (35) onto the Ag85B structure (white) (36). The lowest energy structure after refinement with FireDock (37) includes the proposed electrostatic interaction. E, detailed view of the Ag85B-pHTE 27-binding site. pHTE 27 is positioned within a groove on the Ag85B surface.

FIGURE 6.

Binding of MTB Ag85B and MTB H37Ra to elastin siRNA-transfected Caco-2 cells. A, quantification of elastin. Various amounts (1, 2, 5, 10, and 20 μg) of human skin elastin were detected by Western blotting using elastin antibody. Intensity of blot was analyzed and quantified by ImageJ (upper panel). In lower panel, quantification data were processed to obtain a linear standard curve equation (y = 7.75x + 12). B, detection of the expression of elastin and β-actin in Caco-2 cells after 72 h of transfection with elastin or negative siRNA. Elastin and β-actin were detected by Western blotting using elastin and β-actin antibodies. Blot intensity was calculated by ImageJ and quantified by interpolating from the standard curve equation described above. The expression amounts of endogenous elastin were estimated as 5.43 μg in negative siRNA-transfected Caco-2 cells and 0.88 μg in elastin siRNA-transfected Caco-2 cells (that equaled a 16.2% knockdown of endogenous elastin). C, binding of M. tuberculosis Ag85B was reduced by 34.3% in elastin siRNA-transfected Caco-2 cells. His-tagged M. tuberculosis Ag85B (0.5 μm) was added to elastin siRNA- or negative siRNA-transfected Caco-2 cell (5 × 10⁶)-coated wells and incubated at 37 °C for 1 h. Bound M. tuberculosis Ag85B was detected by anti-His antibody (solid bar). A total of 2 × 10⁸ M. tuberculosis H37Ra cells was incubated with immobilized Caco-2 cells (5 × 10⁶) transfected with negative siRNA or elastin siRNA on microtiter plate wells. The wells incubated without Caco-2 cells served as the negative control. The number of M. tuberculosis cells adhering to negative siRNA-transfected Caco-2 cells and elastin siRNA transfected Caco-2 cells were counted as (8.1 ± 0.8) × 10⁶ and (6.6 ± 0.5) × 10³, respectively. Binding of M. tuberculosis H37Ra to elastin siRNA-transfected Caco-2 cells was slightly abolished (12.3% reduction) (open bar). Each datum is the mean ± S.D. of quadruplicate wells (n = 4). Statistically significant (p < 0.05) differences compared with the negative reference are indicated by an asterisk.