Characterization of novel cathepsin K mutations in the pro and mature polypeptide regions causing pycnodysostosis
Wu-Shiun Hou, Dieter Brömme, Yingming Zhao, Ernest Mehler, Craig Dushey, Harel Weinstein, Clara Sa Miranda, Claudia Fraga, Fenella Greig, John Carey, David L. Rimoin, Robert J. Desnick, Bruce D. Gelb
Wu-Shiun Hou, Dieter Brömme, Yingming Zhao, Ernest Mehler, Craig Dushey, Harel Weinstein, Clara Sa Miranda, Claudia Fraga, Fenella Greig, John Carey, David L. Rimoin, Robert J. Desnick, Bruce D. Gelb
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Characterization of novel cathepsin K mutations in the pro and mature polypeptide regions causing pycnodysostosis

Abstract

Cathepsin K, a lysosomal cysteine protease critical for bone remodeling by osteoclasts, was recently identified as the deficient enzyme causing pycnodysostosis, an autosomal recessive osteosclerotic skeletal dysplasia. To investigate the nature of molecular lesions causing this disease, mutations in the cathepsin K gene from eight families were determined, identifying seven novel mutations (K52X, G79E, Q190X, Y212C, A277E, A277V, and R312G). Expression of the first pro region missense mutation in a cysteine protease, G79E, in Pichia pastoris resulted in an unstable precursor protein, consistent with misfolding of the proenzyme. Expression of five mature region missense defects revealed that G146R, A277E, A277V, and R312G precursors were unstable, 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 substrate specificity and gelatinolytic activity, but it had markedly decreased activity toward type I collagen and a cathepsin K–specific tripeptide substrate, indicating that it was unable to bind collagen triple helix. These studies demonstrated the molecular heterogeneity of mutations causing pycnodysostosis, 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.

Authors

Wu-Shiun Hou, Dieter Brömme, Yingming Zhao, Ernest Mehler, Craig Dushey, Harel Weinstein, Clara Sa Miranda, Claudia Fraga, Fenella Greig, John Carey, David L. Rimoin, Robert J. Desnick, Bruce D. Gelb

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Novel pycnodysostosis mutations in the cathepsin K gene. DNA changes to ...
Novel pycnodysostosis mutations in the cathepsin K gene. DNA changes to codons and their predicted effects on the cathepsin K protein are indicated above and positioned on the cathepsin K gene, which is divided into eight exons. The initiation and stop codons are indicated in exons 2 and 8, respectively. The effects of these mutations on the cathepsin K prepropeptide are shown below. The positions of the cysteine, histidine, and asparagine residues that form the active triad in the active cleft are indicated on the normalpolypeptide. The families in which individual mutations were found are indicated on the right.