Collagenase Activity of Cathepsin K Depends on Complex Formation with Chondroitin Sulfate
2002; Elsevier BV; Volume: 277; Issue: 32 Linguagem: Inglês
10.1074/jbc.m204004200
ISSN1083-351X
AutoresZhenqiang Li, Wu-Shiun Hou, Carlos R. Escalante-Torres, Bruce D. Gelb, Dieter Brömme,
Tópico(s)Connective tissue disorders research
ResumoBone resorption in balance with bone formation is vital for the maintenance of the skeleton and is mediated by osteoclasts. Cathepsin K is the predominant protease in osteoclasts that degrades the bulk of the major bone forming organic component, type I collagen. Although the potent collagenase activity of cathepsin K is well known, its mechanism of action remains elusive. Here, we report a cathepsin K-specific complex with chondroitin sulfate, which is essential for the collagenolytic activity of the enzyme. The complex is an oligomer consisting of five cathepsin K and five chondroitin sulfate molecules. Only the complex exhibits potent triple helical collagen-degrading activity, whereas monomeric cathepsin K has no collagenase activity. The primary substrate specificity of cathepsin K is not altered by complex formation, suggesting that the protease-chondroitin sulfate complex primarily facilitates the destabilization and/or the specific binding of the triple helical collagen structure. Inhibition of complex formation leads to the loss of collagenolytic activity but does not impair the proteolytic activity of cathepsin K toward noncollagenous substrates. The physiological relevance of cathepsin K complexes is supported by the findings that (i) the content of chondroitin sulfate present in bone and accessible to cathepsin K activity is sufficient for complex formation and (ii) Y212C, a cathepsin K mutant that causes pycnodysostosis (a bone sclerosing disorder) and that has no collagenase activity but remains potent as a gelatinase, is unable to form complexes. These findings reveal a novel mechanism of bone collagen degradation and suggest that targeting cathepsin K complex formation would be an effective and specific treatment for diseases with excessive bone resorption such as osteoporosis. Bone resorption in balance with bone formation is vital for the maintenance of the skeleton and is mediated by osteoclasts. Cathepsin K is the predominant protease in osteoclasts that degrades the bulk of the major bone forming organic component, type I collagen. Although the potent collagenase activity of cathepsin K is well known, its mechanism of action remains elusive. Here, we report a cathepsin K-specific complex with chondroitin sulfate, which is essential for the collagenolytic activity of the enzyme. The complex is an oligomer consisting of five cathepsin K and five chondroitin sulfate molecules. Only the complex exhibits potent triple helical collagen-degrading activity, whereas monomeric cathepsin K has no collagenase activity. The primary substrate specificity of cathepsin K is not altered by complex formation, suggesting that the protease-chondroitin sulfate complex primarily facilitates the destabilization and/or the specific binding of the triple helical collagen structure. Inhibition of complex formation leads to the loss of collagenolytic activity but does not impair the proteolytic activity of cathepsin K toward noncollagenous substrates. The physiological relevance of cathepsin K complexes is supported by the findings that (i) the content of chondroitin sulfate present in bone and accessible to cathepsin K activity is sufficient for complex formation and (ii) Y212C, a cathepsin K mutant that causes pycnodysostosis (a bone sclerosing disorder) and that has no collagenase activity but remains potent as a gelatinase, is unable to form complexes. These findings reveal a novel mechanism of bone collagen degradation and suggest that targeting cathepsin K complex formation would be an effective and specific treatment for diseases with excessive bone resorption such as osteoporosis. chondroitin 4-sulfate dithiothreitol benzyloxycarbonyl l-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidino)butane 7-amino-4-methylcoumarin 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid dynamic light scattering cathepsin K matrix metalloproteinase Bone is undergoing a constant remodeling process that is balanced through the activities of bone-generating osteoblasts and bone-resorbing osteoclasts. Various bone diseases, such as osteoporosis, Paget's disease, certain forms of arthritis, and osseous metastases are characterized by excessive osteoclast-mediated bone resorption. The resorptive step can be divided into two basic processes: (i) mineral solubilization and (ii) focalized matrix degradation. Whereas mineral solubilization depends on the production and secretion of acid by the osteoclast, matrix degradation is mainly due to the activity of the cysteine protease, cathepsin K (1Bromme D. Okamoto K. Biol. Chem. Hoppe-Seyler. 1995; 376: 379-384Crossref PubMed Scopus (241) Google Scholar, 2Drake F.H. Dodds R.A. James I.E. Connor J.R. Debouck C. Richardson S. Lee-Rykaczewski E. Coleman L. Rieman D. Barthlow R. Hastings G. Gowen M. J. Biol. Chem. 1996; 271: 12511-12516Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). Type I collagen constitutes 90–95% of the organic bone mass (3Krane S.M. Simon L. Rubenstein E. Federman D.D. Scientific American Medicine. 3. Scientific American, Inc., New York1994: 1-26Google Scholar) and represents the major biological substrate for cathepsin K (4Brömme D. Okamoto K. Wang B.B. Biroc S. J. Biol. Chem. 1996; 271: 2126-2132Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Among all mammalian collagenases, cathepsin K is the only protease capable of cleaving interstitial collagens at multiple sites within their triple helical structures (5Garnero P. Borel O. Byrjalsen I. Ferreras M. Drake F.H. McQueney M.S. Foged N.T. Delmas P.D. Delaisse J.M. J. Biol. Chem. 1998; 273: 32347-32352Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 6Kafienah W. Bromme D. Buttle D.J. Croucher L.J. Hollander A.P. Biochem. J. 1998; 331: 727-732Crossref PubMed Scopus (297) Google Scholar). Deficiency in cathepsin K activity leads to an accumulation of undigested collagen fibrils in lysosomes within osteoclasts (7Everts V. Aronson D.C. Beertsen W. Calcif. Tissue Int. 1985; 37: 25-31Crossref PubMed Scopus (109) Google Scholar), as observed in patients with the autosomal recessive skeletal dysplasia pycnodysostosis (8Gelb B.D. Shi G.P. Chapman H.A. Desnick R.J. Science. 1996; 273: 1236-1238Crossref PubMed Scopus (859) Google Scholar). Analysis of cathepsin K mutants revealed that the collagen degradation defect is not necessarily coupled with the loss of proteolytic activity of cathepsin K. One disease causing mutation, Y212C, that is remote from the active site of the protease, only mildly affected the overall proteolytic activity of cathepsin K but completely eliminated its collagenase activity (9Hou W.-S. Brömme D. Zhao Y. Mehler E. Dushey C. Weinstein H. Miranda C.S. Fraga C. Greig F. Carey J. Rimoin D.L. Desnick R.J. Gelb B.D. J. Clin. Invest. 1999; 103: 731-738Crossref PubMed Scopus (141) Google Scholar). This observation indicated that in addition to the catalytic activity of cathepsin K, other features are required for the hydrolysis of collagens by cathepsin K. We have recently demonstrated that bone- and cartilage-resident glycosaminoglycans specifically enhance the degradation of interstitial collagens of types I and II by cathepsin K, an effect not observed with cathepsin L or matrix metalloproteinase I (10Li Z. Hou W.S. Bromme D. Biochemistry. 2000; 39: 529-536Crossref PubMed Scopus (140) Google Scholar). This finding suggested that the collagenase activity of cathepsin K requires specific interactions between cathepsin K protein and glycosaminoglycans. The mechanism by which cathepsin K degrades collagen, however, remained elusive. In this report, we demonstrate that the collagenolytic activity of cathepsin K depends on the formation of a novel oligomeric complex of cathepsin K protein with chondroitin sulfate. Wild-type human cathepsin K and its mutant protein, Y212C, were expressed in Pichia pastoris and purified as described previously (9Hou W.-S. Brömme D. Zhao Y. Mehler E. Dushey C. Weinstein H. Miranda C.S. Fraga C. Greig F. Carey J. Rimoin D.L. Desnick R.J. Gelb B.D. J. Clin. Invest. 1999; 103: 731-738Crossref PubMed Scopus (141) Google Scholar, 11Linnevers C.J. McGrath M.E. Armstrong R. Mistry F.R. Barnes M. Klaus J.L. Palmer J.T. Katz B.A. Brömme D. Protein Sci. 1997; 6: 919-921Crossref PubMed Scopus (107) Google Scholar). Chondroitin 4-sulfate (C4-S)1 was purchased from Sigma and fractionated on a Sephadex G-75 Superfine column. The molecular masses of the individual fractions were determined by dynamic light scattering (see below). Cathepsin K complexes were obtained by mixing purified cathepsin K and excess C4-S in acetate buffer, pH 5.0 containing 1 mm dithiothreitol (DTT) and 1 mm EDTA. 40 μg of purified recombinant human cathepsin K were preincubated with 0.1% C4-S (with 16-, 25-, and 29.8-kDa fractions, respectively, as determined by dynamic light scattering, see below) in the elution buffer for 20 min and then applied to a Superdex200 column and eluted either in the presence or absence of 300 mm NaCl with 100 mmsodium acetate buffer, pH 5.5, containing 1 mm EDTA/DTT. Protein elution was monitored at 280 nm, and cathepsin K activity was assayed for the hydrolysis of the cathepsin K substrate Z-Leu-Arg-MCA as described previously (4Brömme D. Okamoto K. Wang B.B. Biroc S. J. Biol. Chem. 1996; 271: 2126-2132Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Steady state kinetics were performed with fluorogenic dipeptide substrates as described previously (4Brömme D. Okamoto K. Wang B.B. Biroc S. J. Biol. Chem. 1996; 271: 2126-2132Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). The enzymatic activity was followed by monitoring the release of the fluorogenic leaving group, MCA, at an excitation wavelength of 380 nm and an emission wavelength of 450 nm using the Molecular Devices SpectraMax Gemini spectro-microfluorimeter. The k cat and K m values were determined using nonlinear regression analysis. The cathepsin K activities were assayed at 25 °C in the absence or presence of 0.1% C4-S at a fixed enzyme concentration (0.5 nm) and variable substrate concentrations (1–100 μm) in 100 mm sodium acetate buffer, pH 5.5, containing 2.5 mm DTT and 2.5 mm EDTA. In addition, Z-Leu-Arg-MCA hydrolysis was assayed in the presence or absence of 300 mm NaCl. The active site concentration of cathepsin K and the mutant protein were determined by titration with E64 (12Barrett A.J. Kembhavi A.A. Brown M.A. Kirschke H. Knight C.G. Tamai M. Hanada K. Biochem. J. 1982; 201: 189-198Crossref PubMed Scopus (922) Google Scholar). DCG-04, an epoxide inhibitor derivative of E64 containing a tyrosine residue for iodination and a biotin moiety (13Bogyo M. Verhelst S. Bellingard-Dubouchaud V. Toba S. Greenbaum D. Chem. Biol. 2000; 7: 27-38Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), was kindly provided by Dr. M. Bogyo, (Celera Corp., South San Francisco, CA). The compound was iodinated as described in Ref. 14Greenbaum D. Medzihradszky K.F. Burlingame A. Bogyo M. Chem. Biol. 2000; 7: 569-581Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar. An aqueous solution of 15 μm (0.1 μCi/ml) of [125I]DCG-04 was incubated with recombinant human cathepsin K (or recombinant Y212C mutant; both at 2.5 μm) in 100 mm acetate buffer, pH 5.0, containing 2.5 mm DTT and 2.5 mm EDTA for 4 h. Free inhibitor was removed by gel filtration using a PD-10 column (Amersham Biosciences). 50 ng of [125I]DCG-04-labeled cathepsin K were preincubated in 100 mm sodium acetate buffer, pH 5.0, containing 1 mm EDTA/DTT in the presence or absence of 0.1% of C4-S, respectively, for 20 min and then mixed at 37 °C with nonreducing protein loading buffer and preheated agarose gel. The sample was loaded into the well of a 0.5% agarose gel and separated at room temperature at 55 V, 250 mA for 30 min in a running buffer containing 125 mm sodium acetate, pH 5.0, 0.5 mm EDTA, 1 mm DTT, 40 mm NaCl, and 0.1% Chaps. The gel was dried and subsequently exposed to an x-ray film. To determine the effect of NaCl concentration on the formation of cathepsin K·C4-S complexes, labeled cathepsin K was incubated as described above in the presence of increasing NaCl concentrations (100, 200, and 300 mm). Equal salt concentrations were added to the agarose gel and the electrophoresis running buffer. To determine the complex ratio between cathepsin K protein and C4-S, a constant amount of C4-S (292 pmol) was incubated with variable amounts of [125I]DCG-04-labeled cathepsin K and then applied to 0.5% agarose gel electrophoresis. Electrophoretically separated bands of free cathepsin K represented the exact amount of cathepsin K in its free form in the equilibrium. The free form of labeled cathepsin K was detected by autoradiography and quantified by densitometry using a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA). The free cathepsin K concentrations were determined using a calibration curve derived from known concentrations of cathepsin K. Plotting the quotient cathepsin Kc/C4-S ([cathepsin K in complex] divided by [C4-S]; complex cathepsin K = total cathepsin K-free cathepsin K) versus total cathepsin K concentration, the ratio between cathepsin K and C4-S in the complex could be determined. With increasing cathepsin K concentrations, the value for cathepsin K·C4-S increased until reaching saturation. The data could be fitted to the following equation, which was developed using the equilibrium equation and the assumption of a 1:1 complex, [CatKc]/[C]t=(Kd+[CatK]t+[C]t−((Kd+[CatK]t+[C]t)2−4[C]t[CatK]t)0.5)/2[C]tEquation 1 where [CatK]t is the total cathepsin K concentration, [C]t is total C-4S concentration, and [CatKc] is the cathepsin K concentration in the complex form. The saturation line of the curve represents the complex ratio of cathepsin K to C4-S. The molecular masses of monomeric cathepsin K, C4-S, and the CatK·C4-S complex were determined using a DynaPro-801 TC (ProteinSolutions, Inc., Charlottesville, VA) with helium/neon laser light scattering. The samples were dissolved in 100 mm sodium acetate buffer, pH 5.5, containing 80 mm NaCl. CatK·C4-S complexes were generated by mixing cathepsin K and C4-S (29.8-kDa fraction) at a ratio of 1:1. The data were analyzed using Dynamics 4.0 software (ProteinSolutions), and the molecular masses were calculated from experimental hydrodynamic radii using the Pullunan standard curve for C4-S and a globular protein standard for monomeric cathepsin K and its complex form. The experiment was performed in the Optima XL-I Analytical Ultracentrifuge using a Beckman An50-Ti 8 hole rotor equipped with a six-sector charcoal-filled Epon centerpiece and quartz windows. The absorbance data were acquired at 280 nm as an average of 10 measurements at each radial position in 0.003-cm increments. A total of six data sets were collected corresponding to three different concentrations (10, 20, and 50 μm of cathepsin K and C4-S in 100 mm sodium acetate buffer, pH 5.5, containing 1 mm DTT, 1 mm EDTA, and 80 mm NaCl) and two speeds (6000 and 8000 rpm). Data editing and evaluation of the equilibrium were performed using the programs REEDIT and MATCH, respectively (provided by the National Analytical Ultracentrifugation facility of the University of Connecticut). The data were analyzed using the program NONLIN (15Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar) allowing a global fit to s, where s = M(1 −vD)w2/RT is the reduced apparent molecular mass. Calculation of the molecular mass was performed using molar masses (24.8 kDa for cathepsin K and 29.8 kDa for C4-S) and partial specific volumes (0.714 for protein and 0.58 for glycosaminoglycan) (16Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992Google Scholar). 0.4 mg/ml type I collagen (calf skin, Calbiochem) were incubated with human cathepsin K (800 nm) or its mutant protein (800 nm) in 100 mm sodium acetate buffer, pH 5.0, containing 2.5 mm DTT and EDTA. Collagen digestion was performed at 28 and 37 °C in the absence and the presence of 0.15% (w/v) C4-S. Heat-denatured type I collagen (gelatin) was incubated with cathepsin K (0.5 nm) or its mutant (1 nm). The digestion reaction was stopped by the addition of 10 μm E64 after 4 and 8 h. The samples were subjected to SDS-polyacrylamide electrophoresis using 4–20% Tris/glycine gels that subsequently were stained with Coomassie Blue. The glycosaminoglycans present in commercial type I collagen were removed by treatment with chondroitinase ABC. 100 μl of 8 mg/ml collagen (U.S. Biochemical Corp.) in 100 mm sodium acetate buffer, pH 5.5, were incubated with 50 milliunits of chondroitinase ABC (Sigma) at 25 °C for 4 h. The residual glycosaminoglycan content was determined as follows: 8 mg/ml collagen were heated at 70 °C for 20 min and then incubated with 0.1 mg/ml pepsin at 25 °C, pH 3.7, for 2 h. Glycosaminoglycan concentration was measured using the Blyscan glycosaminoglycan assay kit (Biocolor Ltd., Newtown Abbey, Northern Ireland). A human femoral head bone specimen obtained after hip joint replacement surgery was washed several time with ice-cold water and was milled under liquid nitrogen. The bone powder was defatted with acetone at 4 °C for 2 h, washed with ice-cold water, and eventually demineralized in 10% formic acid for 3 days at 4 °C with daily renewals of formic acid. Subsequently, the washed demineralized bone powder was freeze-dried. 4 mg of bone powder were degraded for 4 h with 800 nm cathepsin K at 25 °C in 0.3 ml of 100 mm sodium acetate buffer, pH 5.5, containing 2.5 mm DTT and EDTA. The soluble glycosaminoglycan content was determined using the Blyscan glycosaminoglycan assay kit. For experiments to identify complex formation, glycosaminoglycan-containing supernatants were heat-inactivated at 70 °C for 10 min after cathepsin K digestion. Heat-inactivated supernatants were then incubated with [125I]DCG-04-labeled cathepsin K at room temperature. The samples were analyzed by the electrophoretic mobility shift assay as described above. As a control, supernatants of undigested bone powder were used. Size exclusion chromatography of purified recombinant cathepsin K and C4-S mixtures revealed proteolytically active, high molecular mass complexes. The molecular masses varied between 180 and 310 kDa depending on the molecular mass fraction of C4-S used (16, 25, and 29.8 kDa determined by dynamic light scattering) (Fig.1 A). When cathepsin K·C4-S mixtures were eluted in the presence of 300 mm NaCl, cathepsin K activity corresponded to a peak of 25 kDa, which was identical to the elution peak obtained when cathepsin K was applied to the gel filtration column without C4-S. The complex form was also detected in an electrophoretic mobility shift assay using [125I]DCG-04-labeled cathepsin K in the presence of C4-S. Cathepsin K in the presence of C4-S migrated further to the anode than the enzyme without C4-S. In the presence of 0.3 m NaCl, the complex dissociated and cathepsin K migrated identically to the protease sample in the absence of C4-S (Fig. 1 B). For the dissociation of the complexes, NaCl concentrations of >0.2m are required (Fig. 1 B). The existence of high molecular mass cathepsin K·C4-S complexes was confirmed by DLS and analytical ultracentrifugation. Measurements by DLS yielded a molecular mass of 274 ± 12 kDa, and the value obtained by ultracentrifugation was 285 kDa using the 29.8-kDa fraction of C4-S. Mobility shift assays allowed us to determine the ratio of [125I]DCG-04-labeled cathepsin K to C4-S in the complex. The plot of the amounts of cathepsin Kc·C4-Sversus cathepsin K resulted in a saturation curve that reached a plateau at a 1:1 ratio of cathepsin K to C4-S (Fig.2). Based on the molecular mass of the complex determined by DLS and analytical ultracentrifugation and the ratio of 1:1 of cathepsin K to C4-S, we suggest the following stoichiometry for the complex: (CatK)5 (C4-S)5. The calculated molecular mass of the complex is 275 kDa (based on the masses of its components: cathepsin K, 25.2 ± 1.2 kDa, and C4-S, 29.8 ± 3.2 kDa as determined by DLS) and is in close proximity to the experimental values obtained by DLS and ultracentrifugation. The hydrodynamic radius of the complex was determined by DLS to be 65 ± 1.4 Å. To determine whether chondroitin sulfate and sodium chloride affect the catalytic activity of cathepsin K, we determined the kinetic parameters for the cathepsin K-catalyzed hydrolysis of the synthetic fluorogenic substrate, Z-Leu-Arg-MCA, in the absence and presence of C4-S and sodium chloride, respectively (TableI). C4-S increased the specificity constant, k cat/K m, 2-fold, which was equally reflected in slight decreases in theK m value and increases in thek cat value. NaCl had no or only a very weak effect on the appropriate K m andk cat values in the presence or the absence of C4-S. This suggests that the observed activating effect of C4-S on cathepsin K activity toward Z-Leu-Arg-MCA is not associated with the formation of high molecular cathepsin K complexes. A potential effect of complex formation on the substrate specificity of cathepsin K was excluded by the kinetic analysis of second order rate constants for the hydrolysis of dipeptide substrates (Z-Xaa-Arg-MCA, where Xaa = Leu, Phe, Val, or Arg) in the presence or absence of C4-S. The formation of cathepsin K complexes had no effect on the substrate specificity of the S2 subsite pocket (Fig.3). The order of preference for P2 amino acid residues in the tested substrate series was identical in both assay conditions (Leu > Phe > Val > Arg), suggesting that the binding and cleavage preferences were identical in the free and the complex forms of cathepsin K.Table IKinetic parameters for the cleavage of Z-Leu-Arg-MCA by cathepsin K in the presence or absence of C4-S (1.5 mg/ml) and NaCl (0.3m)Conditionk catK mk cat/K ms−1μm×106m−1 s−17.957 ± 0.3036.632 ± 0.4681.1997NaCl8.420 ± 0.4058.570 ± 0.6980.9825C4-S9.730 ± 0.1094.047 ± 0.1022.4042C4-S/NaCl11.375 ± 0.2117.045 ± 0.2541.6146 Open table in a new tab Type I collagen was incubated with free cathepsin K or with cathepsin K·C4-S complexes at 28 and 37 °C in the presence or absence of 300 mm NaCl. In the absence of NaCl, type I collagen was completely degraded by the protease·C4-S complex, whereas in the presence of NaCl cathepsin K alone or in a mixture with C4-S showed no or only a minimal degradation of the extracellular matrix protein even at 37 °C (Fig.4 A). Because the degradation of native collagen requires longer incubation times, the question was raised whether this activity is facilitated by an increased stability of the enzyme in the presence of C4-S. The residual activities listed in Table II indicate that both C4-S and NaCl stabilize cathepsin K activity. However, the stabilizing effects were nonadditive. Of note, there was no difference in the measured residual activities when the enzyme was incubated with C4-S alone or together with NaCl. Because the collagenolytic activity of cathepsin K·C4-S complexes was abrogated in the presence of NaCl, we conclude that the observed collagenolytic activity depends solely on the complex formation and not on the stabilization of the protease. In contrast, neither C4-S nor NaCl had any significant effect on the degradation of denatured type I collagen (gelatin) (Fig. 4 B). This indicates that complex formation is not required for the cathepsin K-catalyzed degradation of noncollagenous protein substrates.Table IIStability of cathepsin K in collagen I degradation assays in the presence or absence of C4-S (1.5 mg/ml) and NaCl (0.3 m) at 28 °CConditionsResidual activities4 h8 h%7.8 ± 1.82.8 ± 1.0NaCl26.7 ± 7.69.3 ± 3.1C4-S57.1 ± 6.434.1 ± 5.5C4-S/NaCl50.8 ± 8.130.6 ± 3.8Residual activities were measured using Z-Leu-Arg-MCA as fluorogenic substrate. Open table in a new tab Residual activities were measured using Z-Leu-Arg-MCA as fluorogenic substrate. Contrary to this report, previous studies have shown a collagenolytic activity of cathepsin K in the apparent absence of chondroitin sulfate (4Brömme D. Okamoto K. Wang B.B. Biroc S. J. Biol. Chem. 1996; 271: 2126-2132Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 5Garnero P. Borel O. Byrjalsen I. Ferreras M. Drake F.H. McQueney M.S. Foged N.T. Delmas P.D. Delaisse J.M. J. Biol. Chem. 1998; 273: 32347-32352Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 6Kafienah W. Bromme D. Buttle D.J. Croucher L.J. Hollander A.P. Biochem. J. 1998; 331: 727-732Crossref PubMed Scopus (297) Google Scholar). This activity is probably caused by glycosaminoglycan contaminations in commercially available collagen preparations (approximately 0.2 μg/ml in soluble type I calf skin collagen fromCalbiochem after pepsin degradation). At this concentration, C4-S was sufficient to partially form cathepsin K·C4-S complexes and to exhibit a relatively minor but detectable collagenase activity as shown in Fig. 5. A reduction of the glycosaminoglycan content in the collagen preparations by pretreatment with chondroitinase ABC resulted in a significant reduction of the collagenolytic activity of cathepsin K toward type I collagen (Fig.5 B), whereas chondroitinase ABC had no effect on the activity of cathepsin K toward synthetic peptide substrates (data not shown). Thus, we conclude that the presence of traces of glycosaminoglycans in collagen preparations concealed the fact that monomeric cathepsin K does not have collagenolytic activity. With increasing C4-S concentrations, the collagenolytic activity dramatically increased and reached a maximum of activity at ∼10 μg/ml C4-S (∼500 nm), which was close to the protease concentration used in the assay. This again is suggestive for a cathepsin K to C4-S ratio of 1:1. The main sources of chondroitin sulfates in human bone are the proteoglycans, decorin and biglycan. Following incubation of human bone powder with cathepsin K, glycosaminoglycans were found in the supernatant at a concentration of 0.1 mg/ml (0.75% of dry weight of bone powder), which probably only represents the amount of glycosaminoglycans released from the surface of the bone powder particles. Because no soluble glycosaminoglycans were detected prior to the treatment with cathepsin K, we suggest that the protease degrades proteoglycans and releases the glycosaminoglycans. When [125I]DCG-04-labeled cathepsin K was incubated with supernatants of cathepsin K-predigested bone, we observed a shift in the mobility in agarose gel electrophoresis typical for cathepsin K·C4-S complexes. Preincubation of labeled cathepsin K with supernatants of undigested bone did not show the shift, and the enzyme migrated identically to the free enzyme (Fig.6). These data clearly indicate that surface digestion of bone particles by cathepsin K releases sufficient amounts of glycosaminoglycans required for the generation of cathepsin K complexes. Moreover, they explain the collagenolytic activity of cathepsin K using bone powder as substrate as described previously by Garnero et al. (5Garnero P. Borel O. Byrjalsen I. Ferreras M. Drake F.H. McQueney M.S. Foged N.T. Delmas P.D. Delaisse J.M. J. Biol. Chem. 1998; 273: 32347-32352Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). The data presented are derived from in vitro experiments and do not indicate whether complex formation is an in vitro artifact or a physiological feature of the collagenolytic activity of cathepsin K in vivo. To address this question, cathepsin K mutations that cause pycnodysostosis were reviewed. Nine mutations were either nonsense defects that obliterated the protein or missense mutations that resulted in unstable proteins (17Gelb B.D. Brömme D. Desnick R.J. Sriver C.R. Beaudet A.L. Valle D. Sly W.CS. The Metabolic and Molecular Bases of Inherited Diseases. III. McGraw-Hill, Inc., New York2001: 3453-3468Google Scholar). A single mutation, Y212C, produced a cathepsin K polypeptide that lacked collagenase activity but retained its gelatinase activity (9Hou W.-S. Brömme D. Zhao Y. Mehler E. Dushey C. Weinstein H. Miranda C.S. Fraga C. Greig F. Carey J. Rimoin D.L. Desnick R.J. Gelb B.D. J. Clin. Invest. 1999; 103: 731-738Crossref PubMed Scopus (141) Google Scholar). When tested, no complex formation between Y212C and C4-S was observed (Fig. 7 A). Similar to cathepsin K in the absence of glycosaminoglycans, the Y212C protein did not have collagenase activity in the presence nor in the absence of C4-S, even at 37 °C (Fig. 7 B). Thus, the lack of complex formation for the Y212C variant provides an explanation about the inability of this mutant to hydrolyze native collagen. In contrast, the gelatinase activities of wild-type and mutant Y212C cathepsin K in the presence of C4-S were comparable (Fig. 7 C), supporting the hypothesis that complex formation is required for the in vivo collagenase activity of cathepsin K but not for its action toward noncollagenous substrates. Type I collagen constitutes the major organic component in bone and is subjected to a constant turnover during the bone remodeling process. Only a few proteases are capable of degrading native triple helical collagens. Besides cathepsin K, several members of the matrix metalloproteinase (MMP) family are classified as mammalian collagenases. MMPs cleave interstitial collagens at a distinct Gly-(Ile/Leu) bond to generate ¾ and ¼ fragments (18Welgus H.G. Jeffrey J.J. Eisen A.Z. J. Biol. Chem. 1981; 256: 9511-9515Abstract Full Text PDF PubMed Googl
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