title: Regulation of cathepsin B activity by 2A2 monoclonal antibody.
abstract: 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.
authors: Faculty of Pharmacy, University of Ljubljana, Slovenia.
authors: Bojana
authors: B
authors: Mirković
authors: Ales
authors: A
authors: Premzl
authors: Vesna
authors: V
authors: Hodnik
authors: Bojan
authors: B
authors: Doljak
authors: Zala
authors: Z
authors: Jevnikar
authors: Gregor
authors: G
authors: Anderluh
authors: Janko
authors: J
authors: Kos
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D002401:Cathepsin B / Q000737:chemistry*; D002460:Cell Line; D055316:Cystatin C / Q000737:chemistry; D000939:Epitopes; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D000911:Antibodies, Monoclonal; D055316:Cystatin C; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D002401:Cathepsin B