Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp

Abstract

Background: The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs). In the present study, nuclear magnetic resonance (NMR) was used to map the binding site(s) of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction.

Results: The capacity of a panel of 30 mer synthetic peptides covering the full length of Hlp to bind to heparin/heparan sulfate was analyzed by solid phase assays, NMR, and affinity chromatography. An additional active region between the residues Gly46 and Ala60 was defined at the N-terminal domain of Hlp, expanding the previously defined heparin-binding site between Thr31 and Phe50. Additionally, the C-terminus, rich in Lys residues, was confirmed as another heparan sulfate binding region. The amino acids in Hlp identified as mediators in the interaction with heparan sulfate were Arg, Val, Ile, Lys, Phe, and Thr.

Conclusion: Our data indicate that Hlp interacts with heparan sulfate through two distinct regions of the protein. Both heparan sulfate-binding regions here defined are preserved in all mycobacterial Hlp homologues that have been sequenced, suggesting important but possibly divergent roles for this surface-exposed protein in both pathogenic and saprophic species.

Figure 1

Hlp peptides corresponding to the C-terminal domain bind heparan sulfate. (A) Microtiter-plate wells coated with 50 μL of 0.1 μM Hlp or 50 μL of 0.65 μM individual 30-mer synthetic peptides covering the entire sequence of Hlp were incubated with biotinylated heparan sulfate in phosphate buffer 10 mM, pH 7.2. Heparan sulfate binding was measured by adding streptavidin peroxidase to the wells and expressed in absorbency units at 490 nm. The data are expressed as mean ± SD of a representative experiment from five independent ones performed in duplicate. (B) Microtiter-plate wells were coated with Hlp or peptides p31–60, p136–165, and p151–180 and biotinylated heparan sulfate was added in the presence of increasing concentrations of NaCl. Heparan sulfate binding was quantified as above.

Figure 2

Elution profiles of p31–60, p136–165, and p151–180 from a Heparin-Sepharose affinity colunm. Individual peptides bound to the column were eluted with a 0 – 2 M gradient of NaCl. Peptides p31–60, p136–165, and p151–180 were eluted, respectively, at 1.09, 1.31, and 1.29 M NaCl.

Figure 3

STD-TOCSY of p16–45 in presence of heparan sulfate. (A) Reference TOCSY spectrum of p16–45 (2 mM) in the presence of heparan sulfate (10 μM). (B) STD-TOCSY spectrum. Spectra were recorded in PBS-D₂O, pH 7.6, 25°C with mixing time of 66 ms, 32 scans per t1 increment. 200 increments were collected in an interlaced mode for on or off pre-saturation.

Figure 4

Association of peptides p31–60 and p46–75 with heparan sulfate. (A) STD-TOCSY of p31–60 (2 mM) in the presence of heparan sulfate (10 μM). (B) STD-TOCSY spectrum of the aromatic region. (C) STD-TOCSY of p46–75 (2 mM) in the presence of heparan sulfate (10 μM). Spectra were recorded as in Figure 3.

Figure 5

Association of peptides p136–165 and p151–180 with heparan sulfate. (A) STD-TOCSY of p136–165 (2 mM) in association with heparan sulfate (100 μg). (B) STD-TOCSY of p151–180 (2 mM) in the presence of heparan sulfate (10 μM). Spectra were recorded as in Figure 3.

Figure 6

Protein sequence alignment of Hlp from M. leprae, and MDP1 and HBHA from M. tuberculosis. The sequence of Hlp and MDP1 surrounded by the black straight-lined box corresponds to the heparin-interacting region (region I) proposed in this study. The sequence underlined by a red line represents the other heparin-interacting region (region II). The heparin-binding domain of M. tuberculosis HBHA is also shown on the bottom. The sequence alignment was prepared via the Jalview Java Alignment Editor version 2.2 [26].