Cathepsin K is a lysosomal cysteine peptidase, which is crucial in bone resorption, but also important in other processes. Cathepsin K is released from the lysosomes of activated osteoclasts in the sealing zone, where mineral components of the bone are decomposed in the environment with low pH, which allows peptidases access to the organic matrix. Cathepsin K efficiently degrades collagen, producing NTX and CTX fragments, which are biomarkers important in bone diseases such as osteoporosis or pycnodysostosis.
In the experimental work, we studied the impact of the point mutation Y283C of human cathepsin K, which causes pycnodysostosis. The wild type and the mutant cathepsin K cDNA were inserted into the plasmid vector pET-28b(+) and expressed in E. coli. We compared their expression, activation, and proteolytic activity. The activation of the wild type form of procathepsin K was more than a hundred times faster than that of the mutated protein. Proteolytic activity was determined using the fluorescent substrate Z-Phe-Arg-AMC, and collagenolytic activity using collagen. We determined low proteolytic activity of the mutant on the substrate Z-Phe-Arg-AMC, which is probably the result of a poor zymogen activation. The cathepsin K Y283C mutant was also a much poorer collagenase and gelatinase compared to wild type cathepsin K.
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