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Investigation of the collagenolytic activity of cathepsin K complexes by site-directed mutagenesis Takimoto, Shinako


Cathepsin K (catK) is a lysosomal cysteine protease predominantly expressed in osteoclasts. It is the most potent collagenase and elastase in human and involved in a variety of physiological functions including bone degradation, wound healing, maturation of hormones, and a range of important proteolytic activities required for normal cellular function. The main organic constituent of bone, type I collagen, has a highly organized and tightly packed structure and is resistant to proteolysis by most proteases. CatK is the only human protease that efficiently cleaves triple helical collagen, leading to the complete degradation of bone organic matter. This enzyme is the main protease expressed by osteoclasts and is responsible for bone degradation during bone resorption. In recent years, it has been shown that catK forms a complex with bone associated glycosaminoglycans (GAGs) to gain this collagenase activity. However, precise mechanism remains unclear. Due to the major role in bone resorption, catK has been a pharmaceutical target for osteoporosis treatment. Several catK inhibitors have been developed, yet adverse side effects remain a concern. A major issue of the active site inhibitors is its interference to other functions of this enzyme. Gaining the insight of mechanical details of collagenolytic activity of catK can lead to substrate-targeting specific inhibitors that can treat osteoporosis with minimum side effects. There are two catK-GAG complex models based on x-ray crystallography developed in our laboratory; the dimer and the tetramer models. In this study, mutant proteases were made to assess the role of specific protein interaction sites in these proposed models. Mutation at one residue in particular, N99, exhibited 40~50% reduction of degradation activity toward soluble and insoluble collagen without affecting regular proteolytic activity. The atomic force microscopy analysis revealed that the complex formation, observed in the wild type enzyme, was compromised in this mutant protease. This indicates that N99 contributes to the protein interaction necessary for the collagenolytic catK complex formation. Additionally, two mutant proteases showed facilitated collagenase activity against insoluble fibers. These finding contribute to gain better understanding of catK’s collagenolytic activity and will lead development of exosite inhibitors to treat osteoporosis in the future.

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