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Differential contribution of the repeats to heparin binding of HBHA, a major adhesin of Mycobacterium tuberculosis.

Lebrun P, Raze D, Fritzinger B, Wieruszeski JM, Biet F, Dose A, Carpentier M, Schwarzer D, Allain F, Lippens G, Locht C - PLoS ONE (2012)

Bottom Line: The NMR studies show significant shifts of all residues in the HBD upon interaction with heparin, with stronger shifts in the last repeats compared to the upstream repeats, and indicated that the HS fragments with 14 sugar units cover the entire C-terminal lysine-rich domain of HBHA.Mycobacterial HBHA undergoes structural adaptation upon interaction with GAGs, which is likely involved in binding specificities of the adhesin, and mycobacterial pathogens may use HBD polymorphisms for host or organ specificity.Further studies will aim at decoding the complementarity between HBD repeats and HS sequence.

View Article: PubMed Central - PubMed

Affiliation: INSERM U1019, Lille, France.

ABSTRACT

Background: Tuberculosis remains one of the most important causes of global mortality and morbidity, and the molecular mechanisms of the pathogenesis are still incompletely understood. Only few virulence factors of the causative agent Mycobacterium tuberculosis are known. One of them is the heparin-binding haemagglutinin (HBHA), an important adhesin for epithelial cells and an extrapulmonary dissemination factor. HBHA mediates mycobacterial adherence to epithelial cells via the interactions of its C-terminal, lysine rich repeat domain with sulfated glycoconjugates on the surface of epithelial cells.

Methodology/principal findings: Using defined heparin sulfate (HS) analogs, we determined the minimal heparin fragment length for HBHA binding and structural adaptations of the HBHA heparin-binding domain (HBD) upon binding to heparin. The NMR studies show significant shifts of all residues in the HBD upon interaction with heparin, with stronger shifts in the last repeats compared to the upstream repeats, and indicated that the HS fragments with 14 sugar units cover the entire C-terminal lysine-rich domain of HBHA. The differential implication of the repeats is determined by the relative position of prolines and lysines within each repeat, and may contribute to binding specificity. GAG binding induces a non-homogeneous structural rearrangement in the HBD, with stabilization of a nascent α-helix only in the last penta-repeats.

Conclusion/significance: Mycobacterial HBHA undergoes structural adaptation upon interaction with GAGs, which is likely involved in binding specificities of the adhesin, and mycobacterial pathogens may use HBD polymorphisms for host or organ specificity. Further studies will aim at decoding the complementarity between HBD repeats and HS sequence.

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Related in: MedlinePlus

Analysis of the oligosaccharide minimal unit length required for HBHA binding.Oligosaccharides from dp4 to dp14 (5 nmol of each dp oligosaccharide per lane) were incubated with 0.2 nmol of HBHA (A & B) or MalE-Ct (C & D) and subjected to mobility shift assays. The migration of the oligosaccharides was visualized by coloration with azur blue (A & C) and that of the proteins by Coomassie blue staining (B & D). Cyclophilin B (CycB) and free HBHA or MalE-Ct were used in the positive and negative control lanes, respectively.
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pone-0032421-g001: Analysis of the oligosaccharide minimal unit length required for HBHA binding.Oligosaccharides from dp4 to dp14 (5 nmol of each dp oligosaccharide per lane) were incubated with 0.2 nmol of HBHA (A & B) or MalE-Ct (C & D) and subjected to mobility shift assays. The migration of the oligosaccharides was visualized by coloration with azur blue (A & C) and that of the proteins by Coomassie blue staining (B & D). Cyclophilin B (CycB) and free HBHA or MalE-Ct were used in the positive and negative control lanes, respectively.

Mentions: To determine the minimal length of the heparin fragment required for HBHA binding, heparin-derived oligosaccharides ranging in size from dp4 to dp14 were used in a mobility shift assay. The oligosaccharides (5 nmol per sample) were incubated with 0.2 nmol of HBHA and subjected to non-denaturant electrophoresis (Figure 1A and B). In these conditions, HBHA is positively charged and cannot shift to the anode. However, charge compensation by complex formation with negatively charged heparin fragments allows the migration of the complex, and can thereby be visualized as a shifted band at the top of the gel. The unbound oligosaccharides in excess are visible in the middle region of the gel. Tetra- and hexa-saccharides were not significantly shifted in the presence of HBHA, indicating that they were inefficient in forming complexes with HBHA. In contrast, significant amounts of octasaccharides and larger oligosaccharides were shifted in the presence of HBHA. Cyclophilin B (CycB) and dp10 were used as positive controls for interaction, as the heparin:CycB interaction was previously described and HBHA and Cyclophilin B have similar values for pI and molecular weight [21]. These results indicate that the minimal length of heparin capable of interacting with HBHA is between 8 and 10 sugar units. For subsequent studies, we used the dp14 fragment.


Differential contribution of the repeats to heparin binding of HBHA, a major adhesin of Mycobacterium tuberculosis.

Lebrun P, Raze D, Fritzinger B, Wieruszeski JM, Biet F, Dose A, Carpentier M, Schwarzer D, Allain F, Lippens G, Locht C - PLoS ONE (2012)

Analysis of the oligosaccharide minimal unit length required for HBHA binding.Oligosaccharides from dp4 to dp14 (5 nmol of each dp oligosaccharide per lane) were incubated with 0.2 nmol of HBHA (A & B) or MalE-Ct (C & D) and subjected to mobility shift assays. The migration of the oligosaccharides was visualized by coloration with azur blue (A & C) and that of the proteins by Coomassie blue staining (B & D). Cyclophilin B (CycB) and free HBHA or MalE-Ct were used in the positive and negative control lanes, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293801&req=5

pone-0032421-g001: Analysis of the oligosaccharide minimal unit length required for HBHA binding.Oligosaccharides from dp4 to dp14 (5 nmol of each dp oligosaccharide per lane) were incubated with 0.2 nmol of HBHA (A & B) or MalE-Ct (C & D) and subjected to mobility shift assays. The migration of the oligosaccharides was visualized by coloration with azur blue (A & C) and that of the proteins by Coomassie blue staining (B & D). Cyclophilin B (CycB) and free HBHA or MalE-Ct were used in the positive and negative control lanes, respectively.
Mentions: To determine the minimal length of the heparin fragment required for HBHA binding, heparin-derived oligosaccharides ranging in size from dp4 to dp14 were used in a mobility shift assay. The oligosaccharides (5 nmol per sample) were incubated with 0.2 nmol of HBHA and subjected to non-denaturant electrophoresis (Figure 1A and B). In these conditions, HBHA is positively charged and cannot shift to the anode. However, charge compensation by complex formation with negatively charged heparin fragments allows the migration of the complex, and can thereby be visualized as a shifted band at the top of the gel. The unbound oligosaccharides in excess are visible in the middle region of the gel. Tetra- and hexa-saccharides were not significantly shifted in the presence of HBHA, indicating that they were inefficient in forming complexes with HBHA. In contrast, significant amounts of octasaccharides and larger oligosaccharides were shifted in the presence of HBHA. Cyclophilin B (CycB) and dp10 were used as positive controls for interaction, as the heparin:CycB interaction was previously described and HBHA and Cyclophilin B have similar values for pI and molecular weight [21]. These results indicate that the minimal length of heparin capable of interacting with HBHA is between 8 and 10 sugar units. For subsequent studies, we used the dp14 fragment.

Bottom Line: The NMR studies show significant shifts of all residues in the HBD upon interaction with heparin, with stronger shifts in the last repeats compared to the upstream repeats, and indicated that the HS fragments with 14 sugar units cover the entire C-terminal lysine-rich domain of HBHA.Mycobacterial HBHA undergoes structural adaptation upon interaction with GAGs, which is likely involved in binding specificities of the adhesin, and mycobacterial pathogens may use HBD polymorphisms for host or organ specificity.Further studies will aim at decoding the complementarity between HBD repeats and HS sequence.

View Article: PubMed Central - PubMed

Affiliation: INSERM U1019, Lille, France.

ABSTRACT

Background: Tuberculosis remains one of the most important causes of global mortality and morbidity, and the molecular mechanisms of the pathogenesis are still incompletely understood. Only few virulence factors of the causative agent Mycobacterium tuberculosis are known. One of them is the heparin-binding haemagglutinin (HBHA), an important adhesin for epithelial cells and an extrapulmonary dissemination factor. HBHA mediates mycobacterial adherence to epithelial cells via the interactions of its C-terminal, lysine rich repeat domain with sulfated glycoconjugates on the surface of epithelial cells.

Methodology/principal findings: Using defined heparin sulfate (HS) analogs, we determined the minimal heparin fragment length for HBHA binding and structural adaptations of the HBHA heparin-binding domain (HBD) upon binding to heparin. The NMR studies show significant shifts of all residues in the HBD upon interaction with heparin, with stronger shifts in the last repeats compared to the upstream repeats, and indicated that the HS fragments with 14 sugar units cover the entire C-terminal lysine-rich domain of HBHA. The differential implication of the repeats is determined by the relative position of prolines and lysines within each repeat, and may contribute to binding specificity. GAG binding induces a non-homogeneous structural rearrangement in the HBD, with stabilization of a nascent α-helix only in the last penta-repeats.

Conclusion/significance: Mycobacterial HBHA undergoes structural adaptation upon interaction with GAGs, which is likely involved in binding specificities of the adhesin, and mycobacterial pathogens may use HBD polymorphisms for host or organ specificity. Further studies will aim at decoding the complementarity between HBD repeats and HS sequence.

Show MeSH
Related in: MedlinePlus