Cathepsin B (EC 3.4.22.1) is a lysosomal
cysteine protease with both
endopeptidase and
exopeptidase activity. The former is associated with the degradation of the
extracellular matrix proteins, which is a process required for tumour cell invasion and
metastasis. In the present study, we show that 2A2
monoclonal antibody, raised by our group, is able to regulate
cathepsin B activity. The EPGYSP sequence, located between
amino acid residues 133-138 of
cathepsin B in the proximity of the occluding loop, was determined to be the
epitope for 2A2
monoclonal antibody using SPOT analysis. By surface plasmon resonance, an equilibrium dissociation constant (Kd) of 4.7 nM was determined for the interaction between the nonapeptide CIAEPGYSP, containing the
epitope sequence, and 2A2
monoclonal antibody. 2A2
monoclonal antibody potentiated
cathepsin B exopeptidase activity with a activation constant (Ka) of 22.3 nM, although simultaneously inhibiting its
endopeptidase activity. The median inhibitory concentration values for the inhibition of hydrolysis of
protein substrates,
BODIPY FL
casein and DQ-
collagen IV were 761 and 702 nM, respectively. As observed by native gel electrophoresis and gel filtration, the binding of 2A2
monoclonal antibody to the
cathepsin B/
cystatin C complex caused the dissociation of
cystatin C from the complex. The results obtained in the present study suggest that, upon binding, the 2A2
monoclonal antibody induces a conformational change in
cathepsin B, stabilizing its
exopeptidase conformation and thus disabling its harmful action associated with its
endopeptidase activity.