Mirković Bojana, Premzl Ales, Hodnik Vesna, Doljak Bojan, Jevnikar Zala, Anderluh Gregor, Kos Janko
Faculty of Pharmacy, University of Ljubljana, Slovenia.
FEBS J. 2009 Sep;276(17):4739-51. doi: 10.1111/j.1742-4658.2009.07171.x. Epub 2009 Jul 27.
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.
组织蛋白酶B（EC 3.4.22.1）是一种具有内肽酶和外肽酶活性的溶酶体半胱氨酸蛋白酶。前者与细胞外基质蛋白的降解有关，这是肿瘤细胞侵袭和转移所必需的过程。在本研究中，我们表明我们小组制备的2A2单克隆抗体能够调节组织蛋白酶B的活性。使用斑点分析确定，位于组织蛋白酶B氨基酸残基133 - 138之间靠近封闭环的EPGYSP序列是2A2单克隆抗体的表位。通过表面等离子体共振，确定含有表位序列的九肽CIAEPGYSP与2A2单克隆抗体之间相互作用的平衡解离常数（Kd）为4.7 nM。2A2单克隆抗体增强了组织蛋白酶B的外肽酶活性，激活常数（Ka）为22.3 nM，尽管同时抑制了其内肽酶活性。抑制蛋白质底物BODIPY FL酪蛋白和DQ - 胶原蛋白IV水解的半数抑制浓度值分别为761和702 nM。如通过非变性凝胶电泳和凝胶过滤所观察到的，2A2单克隆抗体与组织蛋白酶B/胱抑素C复合物的结合导致胱抑素C从复合物中解离。本研究获得的结果表明，结合后，2A2单克隆抗体诱导组织蛋白酶B发生构象变化，稳定其外肽酶构象，从而消除与其内肽酶活性相关的有害作用。
