Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin.
The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains several lysine-rich repeats at its carboxyl-terminal end. Using truncated recombinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these repeats are responsible for heparin binding. Immunofluorescence microscopy studies revealed that their deletion abrogates binding of HBHA to human pneumocytes. Conversely, when fused to MBP, the HBHA repeats confer pneumocyte adherence properties to the hybrid protein. Treatment of pneumocytes with glycosaminoglycan-degrading enzymes showed that HBHA binding depends on the presence of heparan sulfate chains on the cell surface. The epitope of a monoclonal antibody that inhibits mycobacterial adherence to epithelial cells was mapped within the lysine-rich repeats, confirming their involvement in mycobacterial adherence to epithelial cells. Surface plasmon resonance analyses showed that recombinant HBHA binds to immobilized heparin with fast association kinetics (k(a) = 5.62 (+/- 0.10) x 10(5) m(-1) s(-1)), whereas the dissociation kinetics were slower (k(d) = 0.015 (+/- 0.002) s(-1)), yielding a K(D) value of 26 nm. Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain the entire heparin-binding site of HBHA. The molecular characterization of the interactions of HBHA with epithelial glycosaminoglycans should help to better understand mycobacterial adherence within the lungs and may ultimately lead to new approaches for therapy or immunoprophylaxis.
Pethe
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INSERM U447, Mécanismes Moléculaires de la Pathogénie Microbienne, Institut Pasteur de Lille, Institut de Biologie de Lille, 1 rue A. Calmette, 59019 Lille Cedex, France.
Aumercier
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M
Fort
E
E
Gatot
C
C
Locht
C
C
Menozzi
F D
FD
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D016264:Dextran Sulfate / Q000737:chemistry; D006388:Hemagglutinins / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D037102:Lectins; D008168:Lung / Q000166:cytology / Q000378:metabolism; D009161:Mycobacterium / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D017384:Sequence Deletion; D020349:Surface Plasmon Resonance
D017931:DNA Primers; D016264:Dextran Sulfate; D006388:Hemagglutinins; D006493:Heparin; D006497:Heparan Sulfate; D037102:Lectins; D011994:Recombinant Proteins; C104578:heparin-binding hemagglutinin