Premium
Regulation of cathepsin B activity by 2A2 monoclonal antibody
Author(s) -
Mirković Bojana,
Premzl Aleš,
Hodnik Vesna,
Doljak Bojan,
Jevnikar Zala,
Anderluh Gregor,
Kos Janko
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.07171.x
Subject(s) - chemistry , monoclonal antibody , cathepsin b , microbiology and biotechnology , epitope , biochemistry , cathepsin , antibody , biology , enzyme , immunology
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 ( K d ) of 4.7 n m 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 ( K a ) of 22.3 n m , 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 n m , 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.