Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin

The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains sev eral lysine-rich repeats at its carboxyl-terminal end. Using truncated reco mbinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these re peats are responsible for heparin binding. Immunofluorescence microscopy st udies revealed that their deletion abrogates binding of HBHA to human pneum ocytes. Conversely, when fused to MBP, the HBHA repeats confer pneumocyte a dherence properties to the hybrid protein. Treatment of pneumocytes with gl ycosaminoglycan-degrading enzymes showed that HBHA binding depends on the p resence of heparan sulfate chains on the cell surface. The epitope of a mon oclonal 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 anal yses showed that recombinant HBHA binds to immobilized heparin with fast as sociation 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)), yieldin g a K-D value of 26 nM, Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain t he entire heparin-binding site of HBHA. The molecular characterization of t he interactions of HBHA with epithelial glycosaminoglycans should help to b etter understand mycobacterial adherence within the lungs and may ultimatel y lead to new approaches for therapy or immunoprophylaxis.