Characterization of novel cathepsin K mutations in the pro and mature polypeptide regions causing pycnodysostosis

Cathepsin K, a lysosomal cysteine protease critical for bone remodeling by osteoclasts, was recently identified as the deficient enzyme causing pycnod ysostosis, an autosomal recessive osteosclerotic skeletal dysplasia. To inv estigate the nature of molecular lesions causing this disease, mutations in the cathepsin K gene from eight families were determined, identifying seve n novel mutations (K52X, G79E, Q190X, Y212C, A277E, A277V, and R312G). Expr ession of the first pro region missense mutation in a cysteine protease, G7 9E, in Pichia pastoris resulted in an unstable precursor protein, consisten t with misfolding of the proenzyme. Expression of five mature region missen se defects revealed that G146R, A277E, A277V, and R312G precursors were uns table, and no mature proteins or protease activity were detected. The Y212C precursor was activated to its mature form in a manner similar to that of the wild-type cathepsin K. The mature Y212C enzyme retained its dipeptide s ubstrate specificity and gelatinolytic activity, but it had markedly decrea sed activity toward type I collagen and a cathepsin K-specific tripeptide s ubstrate, indicating that it was unable to bind collagen triple helix. Thes e studies demonstrated the molecular heterogeneity of mutations causing pyc nodysostosis, indicated that pro region conformation directs proper folding of the proenzyme, and suggested that the cathepsin K active site contains a critical collagen-binding domain.