tailieunhanh - Báo cáo khoa học: Regulation of cathepsin B activity by 2A2 monoclonal antibody

Cathepsin B (EC ) is a lysosomal cysteine protease with both endo-peptidase and exopeptidase activity. The former is associated with the deg-radation 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 cathep-sin B activity. | ỊFEBS Journal Regulation of cathepsin B activity by 2A2 monoclonal antibody Bojana Mirkovic1 Ales Premzl2 Vesna Hodnik3 Bojan Doljak1 Zala Jevnikar1 Gregor Anderluh3 and Janko Kos1 2 1 Faculty of Pharmacy University of Ljubljana Slovenia 2 Department of Biotechnology Jozef Stefan Institute Ljubljana Slovenia 3 Department of Biology BiotechnicalFaculty University of Ljubljana Slovenia Keywords cathepsin B cystatin C endopeptidase inhibition monoclonalantibody Correspondence J. Kos University of Ljubljana Faculty of Pharmacy Askerceva 7 SI-1000 Ljubljana Slovenia Tel 386 1 4769 604 Fax 386 1 4258 031 E-mail Received 5 October 2008 revised 10 June 2009 accepted 25 June 2009 doi Cathepsin B EC 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 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 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