Detection of femtomole quantities of mature cathepsin K with zymography

doi:10.1016/j.ab.2010.02.035

Analytical Biochemistry

Volume 401, Issue 1, 1 June 2010, Pages 91-98

https://doi.org/10.1016/j.ab.2010.02.035 Get rights and content

Abstract

Cathepsin K, the most potent mammalian collagenase, has been implicated in osteoporosis, cancer metastasis, atherosclerosis, and arthritis. Although procathepsin K is stable and readily detected, the active mature cathepsin K eludes detection by in vitro methods due to its shorter half-life and inactivation at neutral pH. We describe, for the first time, reliable detection, visualization, and quantification of mature cathepsin K to femtomole resolution using gelatin zymography. The specificity of the method was validated with cathepsin K knockdown using small interfering RNA (siRNA) transfection of human monocyte-derived macrophages, and enzymatic activity confirmed with benzyloxycarbonyl-glycine-proline-arginine-7-amino-4-methylcoumarin (Z-GPR-AMC) substrate hydrolysis was fit to a computational model of enzyme kinetics. Furthermore, cathepsin K zymography was used to show that murine osteoclasts secrete more cathepsin K than is stored intracellularly, and this was opposite to the behavior of the macrophages from which they were differentiated. In summary, this inexpensive, species-independent, antibody-free protocol describes a sensitive method with broad potential to elucidate previously undetectable cathepsin K activity.

Section snippets

Cell culture

Human Thp-1 acute monocytic leukemia cells (American Type Culture Collection [ATCC]) were cultured in RPMI medium 1640 (Mediatech) containing 10% fetal bovine serum (FBS), 0.05% β-mercaptoethanol, 1% l-glutamine, and 1% penicillin/streptomycin. Cells were maintained with 5% CO₂ at 37 °C. For macrophage differentiation, monocytes were incubated with 100 nM phorbol myristate acetate (PMA) for 24 h, followed by incubation for an additional 3 days in growth medium. RAW 264.7 murine macrophage cells

Cathepsin K activity detected by gelatin zymography is more sensitive than Western blot detection of mature cathepsin K

Procathepsin K was incubated in 0.1 M acetate buffer (pH 3.9), 10 mM DTT, and 5 mM EDTA for autocatalytic cleavage into the mature active form. Nonactivated procathepsin K (37.5 ng) and preactivated (cleaved) cathepsin K (37.5 ng) were then separately loaded for Western blots and cathepsin zymography (Fig. 1). The anti-cathepsin K antibody detects both the pro- and mature forms of cathepsin K, as seen in the 37-kDa bands of the nonactivated (0 min) sample and in the 29-kDa bands of mature cathepsin K

Discussion

Using recombinant cathepsin K and monocyte-derived macrophages, we have demonstrated a sensitive quantitative method of detecting femtomole amounts of mature cathepsin K activity by protecting the enzymes from irreversible denaturation with freshly added leupeptin and refolding them into active conformations after mild denaturation in 0.1% SDS. This simple electrophoresis method uses readily available and inexpensive reagents, and it visualizes the protein molecular mass to assert that the

Acknowledgments

Special thanks go to Randy Ankeny and Hannah Song for critical reading of this manuscript. Funding sources include the FACES/SURE program at Georgia Tech and Georgia Tech startup funds.

References (26)

P. Garnero et al.
The collagenolytic activity of cathepsin K is unique among mammalian proteinases
J. Biol. Chem.
(1998)
G.P. Shi et al.
Molecular cloning of human cathepsin O, a novel endoproteinase and homologue of rabbit OC2
FEBS Lett.
(1995)
J. Rantakokko et al.
Mouse cathepsin K: cDNA cloning and predominant expression of the gene in osteoclasts, and in some hypertrophying chondrocytes during mouse development
FEBS Lett.
(1996)
D. Greenbaum et al.
Chemical approaches for functionally probing the proteome
Mol. Cell. Proteomics
(2002)
B. Turk et al.
Lysosomal cysteine proteases: more than scavengers
Biochim. Biophys. Acta
(2000)
A. Klose et al.
Identification and discrimination of extracellularly active cathepsins B and L in high-invasive melanoma cells
Anal. Biochem.
(2006)
M.J. Bossard et al.
Proteolytic activity of human osteoclast cathepsin K: expression, purification, activation, and substrate identification
J. Biol. Chem.
(1996)
K.M. Bischoff et al.
The detection of enzyme activity following sodium dodecyl sulfate–polyacrylamide gel electrophoresis
Anal. Biochem.
(1998)
F. Lecaille et al.
Biochemical properties and regulation of cathepsin K activity
Biochimie
(2008)
S.A. Lacks et al.
Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate
J. Biol. Chem.
(1980)

M.E. McGrath

The lysosomal cysteine proteases

Annu. Rev. Biophys. Biomol. Struct.

(1999)

H.J. Salminen-Mankonen et al.

Role of cathepsin K in normal joints and in the development of arthritis

Curr. Drug Targets

(2007)

M.O. Platt et al.

Expression of cathepsin K is regulated by shear stress in cultured endothelial cells and is increased in endothelium in human atherosclerosis

Am. J. Physiol. Heart Circ. Physiol.

(2007)

Cited by (37)

Mechanical strain modulates extracellular matrix degradation and byproducts in an isoform-specific manner
2023, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
Cathepsins are a superfamily of cysteine proteases, and some members can proteolyze ECM. Cathepsin K is the most potent mammalian collagenase, and it can cleave type I collagen both in the native triple helix and in the telopeptide regions [13,14]. Previous studies have established that mechanical strain can modulate the enzymatic degradation of collagen fibers by altering the ‘mechanochemistry’ of collagen-protease binding [7,10,15].
Many studies have shown that mechanical forces can alter collagen degradation by proteases, and this mechanochemical effect may potentially serve an important role in determining extracellular matrix content and organization in load-bearing tissues. However, it is not yet known whether mechano-sensitive degradation depends on particular protease isoforms, nor is it yet known whether particular degradation byproducts can be altered by mechanical loading. In this study, we tested the hypothesis that different types of proteases exhibit different sensitivities to mechanical loading both in degradation rates and byproducts. Decellularized porcine pericardium samples were treated with human recombinant matrix metalloproteinases-1, −8, −9, cathepsin K, or a protease-free control while subjected to different levels of strain in a planar, biaxial mechanical tester. Tissue degradation was monitored by tracking the decay in mechanical stresses during displacement control tests, and byproducts were assessed by mass spectrometry analysis of the sample supernatant after degradation. Our key finding shows that cathepsin K-mediated degradation of collagenous tissue was enhanced with increasing strain, while MMP1-, MMP8-, and MMP9-mediated degradation were first decreased and then increased by strain. Degradation induced changes in tissue mechanical properties, and proteomic analysis revealed strain-sensitive degradome signatures with different ECM byproducts released at low vs. high strains. This evidence suggests a potentially new type of mechanobiology wherein mechanical forces alter the degradation products that can provide important signaling feedback functions during tissue remodeling.
Design, synthesis and biological evaluation of inhibitors of cathepsin K on dedifferentiated chondrocytes
2019, Bioorganic and Medicinal Chemistry
Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC₅₀ values and inhibition constants (K_i) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with K_i amounting to 1.14 and 7.21 nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5 µM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1 µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.
Direct detection of cysteine peptidases for MALDI-TOF MS analysis using fluorogenic substrates
2019, Analytical Biochemistry
Citation Excerpt :
The alignment of T. molitor peptidases with those from mammals indicates that the sequences of the identified peptidases contain conserved motifs characteristic of C1 family peptidases. Previous studies have described detection of various peptidases using protein (gelatin) substrates, either individual [18], or in different biological systems including plant tissue extracts [19] or supernatants of cells and tissues [31–36]. Some techniques apply in-gel detection after electrophoresis in mild denaturing conditions, using SDS or lithium dodecyl sulfate (LDS) gels copolymerized with gelatin.
A method is described for the direct detection of unstable cysteine peptidase activity in polyacrylamide gels after native electrophoresis using new selective fluorogenic peptide substrates, pyroglutamyl-phenylalanyl-alanyl-4-amino-7-methylcoumaride (Glp-Phe-Ala-AMC) and pyroglutamyl-phenylalanyl-alanyl-4-amino-7-trifluoromethyl-coumaride (Glp-Phe-Ala-AFC). The detection limit of the model enzyme papain was 17 pmol (0.29 μg) for Glp-Phe-Ala-AMC and 43 pmol (0.74 μg) for Glp-Phe-Ala-AFC, with increased sensitivity and selectivity compared to the traditional method of protein determination with Coomassie G-250 staining or detection of activity using chromogenic substrates. Using this method, we easily identified the target digestive peptidases of Tenebrio molitor larvae by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. The method offers simplicity, high sensitivity, and selectivity compared to traditional methods for improved identification of unstable cysteine peptidases in multi-component biological samples.
Differential cathepsin responses to inhibitor-induced feedback: E-64 and cystatin C elevate active cathepsin S and suppress active cathepsin L in breast cancer cells
2016, International Journal of Biochemistry and Cell Biology
Cathepsins are powerful proteases, once referred to as the lysosomal cysteine proteases, that have been implicated in breast cancer invasion and metastasis, but pharmaceutical inhibitors have suffered failures in clinical trials due to adverse side effects. Scientific advancement from lysosomotropic to cell impermeable cathepsin inhibitors have improved efficacy in treating disease, but off-target effects have still been problematic, motivating a need to better understand cellular feedback and responses to treatment with cathepsin inhibitors. To address this need, we investigated effects of E-64 and cystatin C, two broad spectrum cathepsin inhibitors, on cathepsin levels intra- and extracellularly in MDA-MB-231 breast cancer cells. Cathepsins S and L had opposing responses to both E-64 and cystatin C inhibitor treatments with paradoxically elevated amounts of active cathepsin S, but decreased amounts of active cathepsin L, as determined by multiplex cathepsin zymography. This indicated cellular feedback to selectively sustain the amounts of active cathepsin S even in the presence of inhibitors with subnanomolar inhibitory constant values. These differences were identified in cellular locations of cathepsins L and S, trafficking for secretion, co-localization with endocytosed inhibitors, and longer protein turnover time for cathepsin S compared to cathepsin L. Together, this work demonstrates that previously underappreciated cellular compensation and compartmentalization mechanisms may sustain elevated amounts of some active cathepsins while diminishing others after inhibitor treatment. This can confound predictions based solely on inhibitor kinetics, and must be better understood to effectively deploy therapies and dosing strategies that target cathepsins to prevent cancer progression.
Early cathepsin K degradation of type II collagen in vitro and in vivo in articular cartilage
2016, Osteoarthritis and Cartilage
To characterize the initial events in the cleavage of type II collagen mediated by cathepsin K and demonstrate the presence of the resulting products in human and equine articular osteoarthritic cartilage.
Equine type II collagen was digested with cathepsin K and the cleavage products characterized by mass spectrometry. Anti-neoepitope antibodies were raised against the most N-terminal cleavage products and used to investigate the progress of collagen cleavage, in vitro, and the presence of cathepsin K-derived products in equine and human osteoarthritic cartilage.
Six cathepsin K cleavage sites distributed throughout the triple helical region were identified in equine type II collagen. Most of the cleavages occurred following a hydroxyproline residue. The most N-terminal site was within three residues of the previously identified site in bovine type II collagen. Western blotting using anti-neoepitope antibodies showed that the initial cleavages occurred at the N-terminal sites and this was followed by more extensive degradation resulting in products too small to be resolved by SDS gel electrophoresis. Immunohistochemical staining of cartilage sections from equine or human osteoarthritic joints showed staining in lesional areas which was not observed in non-arthritic sites.
Cathepsin K cleaves triple helical collagen by erosion from the N-terminus and with subsequent progressive cleavages. The liberated fragments can be detected in osteoarthritic cartilage and may represent useful biomarkers for disease activity.
Purification and characterization of a fish granzymeA involved in cell-mediated immunity
2016, Developmental and Comparative Immunology
Granzymes are serine proteases involved in the induction of cell death against non-self cells. The enzymes differ in their primary substrate specificity and have one of four hydrolysis activities: tryptase, Asp-ase, Met-ase and chymase. Although granzyme genes have been isolated from several fishes, evidence for their involvement in cytotoxicity has not yet been reported. In the present study, we attempted to purify and characterize a fish granzyme involved in cytotoxicity using ginbuna crucian carp. The cytotoxicity of leukocytes was significantly inhibited by the serine protease inhibitor ‘‘3, 4-dichloroisocoumarin’’. In addition, we found that granzymeA-like activity (hydrolysis of Z-GPR-MCA) was inhibited by the same inhibitor and significantly enhanced by allo-antigen stimulation in vivo. Proteins from leukocyte extracts were subjected to two steps of chromatographic purification using benzamidine-Sepharose and SP-Sepharose. The molecular weight of the purified enzyme was estimated to be 26,900 Da by SDS-PAGE analysis. The purified enzyme displayed a K_m of 220 μM, a K_cat of 21.7 sec⁻¹ and a K_cat/K_m of 98,796 sec⁻¹ M⁻¹ with an optimal pH of 9.5 for the Z-GPR-MCA substrate. The protease was totally inhibited by serine protease inhibitors and showed granzymeA-like substrate specificity. Therefore, we conclude that the purified enzyme belongs to the mammalian granzymeA (EC 3.4.21.78) and appears to be involved in cytotoxicity in fish.

View all citing articles on Scopus

View full text

Detection of femtomole quantities of mature cathepsin K with zymography

Abstract

Section snippets

Cell culture

Cathepsin K activity detected by gelatin zymography is more sensitive than Western blot detection of mature cathepsin K

Discussion

Acknowledgments

J. Biol. Chem.

FEBS Lett.

FEBS Lett.

Mol. Cell. Proteomics

Biochim. Biophys. Acta

Anal. Biochem.

J. Biol. Chem.

Anal. Biochem.

Biochimie

J. Biol. Chem.

The lysosomal cysteine proteases

Annu. Rev. Biophys. Biomol. Struct.

Role of cathepsin K in normal joints and in the development of arthritis

Curr. Drug Targets

Expression of cathepsin K is regulated by shear stress in cultured endothelial cells and is increased in endothelium in human atherosclerosis

Am. J. Physiol. Heart Circ. Physiol.