Cathepsin K is a recently identified lysosomal cysteine proteinase. It is abundant in osteoclasts, where it is believed to play a vital role in the resorption and remodeling of bone. Pycnodysostosis is a rare inherited osteochondrodysplasia that is caused by mutations of the cathepsin-K gene, characterized by osteosclerosis, short stature, and acroosteolysis of the distal phalanges. With a view to delineating the role of cathepsin K in bone resorption, we generated mice with a targeted disruption of this proteinase. Cathepsin-K-deficient mice survive and are fertile, but display an osteopetrotic phenotype with excessive trabeculation of the bone-marrow space. Cathepsin-K-deficient osteoclasts manifested a modified ultrastructural appearance: their resorptive surface was poorly defined with a broad demineralized matrix fringe containing undigested fine collagen fibrils; their ruff led borders lacked crystal-like inclusions, and they were devoid of collagen-fibril-containing cytoplasmic vacuoles. Assaying the resorptive activity of cathepsin-K-deficient osteoclasts in vitro revealed this function to be severely impaired, which supports the contention that cathepsin K is of major importance in bone remodeling.

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The interaction of human recombinant full-length cathepsin S propeptide (amino acids 16-114) with mature cysteine proteinases was studied with respect to selectivity and pH dependence. The inhibitory capacity was tested towards mature human recombinant cathepsin S, purified cathepsin L from rat and Paramecium tetraurelia, rat cathepsin B, human cathepsin H, and papain. The propeptide of cathepsin S strongly inhibited cathepsin S (Ki = 0.27 nM) and the two cathepsin L species (Ki = 0.36 nM) at neutral pH. Papain, and to a minor extent cathepsin H, hydrolyzed the propeptide of cathepsin S, leading to competition with the hydrolysis of the fluorogenic substrates in the respective assays. Cathepsin B activity was nearly unaffected up to micromolar propeptide concentrations in the assay. The inhibition of cathepsin-L-like peptidases was diminished with decreasing pH, probably due to dramatic changes in the conformation of the propeptide. This assumption was supported by far-ultraviolet CD spectroscopy and by the finding of rapid hydrolysis of the cathepsin S propeptide by cathepsin L at pH values less than 5.5.

Cathepsin S, a lysosomal cysteine protease, is synthesized as inactive precursor. It is activated in the lysosomes by a proteolytic cleavage of the propeptide. HEK 293-cells which do not express cathepsin S were transfected with cDNA of either wild type human procathepsin S or a mutant procathepsin S in which Asn of the only glycosylation site in the proregion was replaced by Gln. The cells expressed glycosylated and non-glycosylated procathepsin S, respectively. Large amounts of the precursors were secreted into the culture media by both transfectants. Secreted wild type procathepsin S contained Man-6-phosphate in the oligosaccharide chain. Wild type procathepsin S was activated in the cells but no maturation occurred in the culture media. In vitro processing of glycosylated as well as of non-glycosylated procathepsin S gave fully active enzymes thus indicating that the oligosaccharide chain was not necessary for proper folding. A reuptake of the glycosylated and non-glycosylated procathepsin S by HEK 293-cells could be observed. Small amounts of mature cathepsin S were detected in the lysosomes of the mutant transfectants. Subcellular fractionation showed non-glycosylated procathepsin S in the membrane fraction. Non-glycosylated procathepsin S was bound to the plasma membrane at 2 degrees C, suggesting an additional sorting motif in the cathepsin S molecule besides the Man-6-phosphate residue.