K. Pethe et al., Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin, J BIOL CHEM, 275(19), 2000, pp. 14273-14280
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.