Proteolytic Activity of Human Osteoclast Cathepsin K

doi:10.1074/jbc.271.21.12517

Proteolytic Activity of Human Osteoclast Cathepsin K

EXPRESSION, PURIFICATION, ACTIVATION, AND SUBSTRATE IDENTIFICATION (*)

From the (1)Departments of Medicinal Chemistry,
(2) Gene Expression Sciences,
(3) Protein Biochemistry, and
(4) Cellular Biochemistry SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

^§To whom correspondence should be addressed:
Mail Code UW-2103, Dept. of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, P. O. Box 1539, King of Prussia, PA 19406.
Tel.: 610-270-6602; Fax: 610-270-4162.

Abstract

Human cathepsin K is a recently identified protein with high primary sequence homology to members of the papain cysteine protease superfamily including cathepsins S, L, and B and is selectively expressed in osteoclasts (Drake, F. H., Dodds, R., James, I., Connor, J., Debouck, C., Richardson, S., Lee, E., Rieman, D., Barthlow, R., Hastings, G., and Gowen, M.(1996) J. Biol. Chem. 271, 12511-12516). To characterize its catalytic properties, cathepsin K has been expressed in baculovirus-infected SF21 cells and the soluble recombinant protein isolated from growth media was purified. Purified protein includes an inhibitory pro-leader sequence common to this family of protease. Conditions for enzyme activation upon removal of the pro-sequence have been identified. Fluorogenic peptides have been identified as substrates for mature cathepsin K. In addition, two protein components of bone matrix, collagen and osteonectin, have been shown to be substrates of the activated protease. Cathepsin K is inhibited by E-64 and leupeptin, but not by pepstatin, EDTA, phenylmethylsulfonyl fluoride, or phenanthroline, consistent with its classification within the cysteine protease class. Leupeptin has been characterized as a slow binding inhibitor of cathepsin K (k/[I] = 273,000 M•s). Cathepsin K may represent the elusive protease implicated in degradation of protein matrix during bone resorption and represents a novel molecular target in treatment of disease states associated with excessive bone loss such as osteoporosis.

Footnotes

↵() The nomenclature of this protein sequence has taken several forms, including cathepsin K(6), cathepsin O(7), cathepsin X(8), and the rabbit homologue, OC2(9). A second, unrelated sequence also has been referred to as cathepsin O(31). Upon our inquiry, the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology has assigned the name cathepsin K (EC 3.4.22.38) to the protease described in this paper.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵() The abbreviations used are:

PAGE

polyacrylamide gel electrophoresis

Cbz

benzyloxycarbonyl

AMC

aminomethylcoumarin.
↵⁽⁾ During preparation of this manuscript, use of Cbz-Phe-Arg-AMC as a substrate for cathepsin K, referred to as cathepsin O2, was reported(32). Alternative methods for purification and activation as well as additional kinetic characterization of this protease has been described subsequent to the review of this paper (33).
- Received October 27, 1995.
- Revision received March 13, 1996.