Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, 30332, USA.
Archives of Biochemistry and Biophysics (Impact Factor: 3.02). 12/2011; 516(1):52-7. DOI: 10.1016/
Source: PubMed


Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications. Here, we detail a method of multiplex cathepsin zymography to detect and distinguish the activity of mature cathepsins K, L, S, and V by exploiting differences in individual cathepsin substrate preferences, pH effects, and electrophoretic mobility under non-reducing conditions. Specific identification of cathepsins K, L, S, and V in one cell/tissue extract was obtained with cathepsin K (37 kDa), V (35 kDa), S (25 kDa), and L (20 kDa) under non-reducing conditions. Cathepsin K activity disappeared and V remained when incubated at pH 4 instead of 6. Application of this antibody free, species independent, and medium-throughput method was demonstrated with primary human monocyte-derived macrophages and osteoclasts, endothelial cells stimulated with inflammatory cytokines, and normal and cancer lung tissues, which identified elevated cathepsin V in lung cancer.

Download full-text


Available from: Philip Keegan, Jan 22, 2014
  • Source
    • "Excised suprarenal aortas or two pairs of carotids (4 arteries) were placed in 50 ul of lysis buffer (20 nM Tris–HCl [pH 7.5], 5 mM ethyleneglycoltetraacetic acid [EGTA], 150 mM NaCl, 20 mM β-glycerol phosphate, 10 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100, and 0.1% Tween 20) with 0.1 mM leupeptin freshly added to stabilize enzymes during electrophoresis, homogenized using disposable sample grinders (GE Healthcare), and protein concentrations were obtained by the bisinchoninic acid (BCA) assay (Pierce). Cathepsin zymography was performed as described previously (Hansen et al., 2012; Li et al., 2010; Wilder et al., 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: HIV positive patients on highly active antiretroviral therapy (HAART) have shown elevated incidence of a number of non-AIDS defining co-morbidities, including cardiovascular disease. Given that HAART regimens contain a combination of at least three drugs, that disease management often requires adjustment of these regimens, and HIV, independent of HAART, also plays a role in development of co-morbidities, determining the role of specific HAART drugs and HIV infection itself from clinical data remains challenging. To characterize specific mediators and underlying mechanisms of disease, in vitro and in vivo animal models are required, in parallel with clinical data. Given its low cost azidothymidine (AZT) contributes to the backbone of a large proportion of HAART treated patients in the developing world where much of the global burden of HIV resides. The goal of this study was to test the hypothesis that AZT can lead to proatherogenic changes including the subclinical markers of arterial stiffening and intima-media thickening in mice. AZT (100mg/kg) or vehicle was administered to wild-type FVB/N mice via oral gavage for 35 days. Cylindrical biaxial biomechanical tests on the common carotid arteries and suprarenal aortas exhibited arterial stiffening in AZT mice compared to controls. Multiphoton microscopy and histology demonstrated that AZT led to increased intima-media thickness. These data correlated with decreased elastin content and increased protease activity as measured by cathepsin zymography; no differences were observed in collagen content or organization, in vivo axial stretch, or opening angle. Thus, this study suggests the drug AZT has significant effects on the development of subclinical markers of atherosclerosis.
    Journal of Biomechanics 04/2013; 46(9). DOI:10.1016/j.jbiomech.2013.03.021 · 2.75 Impact Factor
  • Source
    • "Cathepsin zymography was performed as described previously [24]. Determination of cathepsin V band required incubation in acetate buffer, pH 4 [25]. Gels were imaged using an ImageQuant 4010 system (GE Healthcare). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Sickle cell disease is a genetic disease that increases systemic inflammation as well as the risk of pediatric strokes, but links between sickle-induced inflammation and arterial remodeling are not clear. Cathepsins are powerful elastases and collagenases secreted by endothelial cells and monocyte-derived macrophages in atherosclerosis, but their involvement in sickle cell disease has not been studied. Here, we investigated how tumor necrosis alpha (TNFα) and circulating mononuclear cell adhesion to human aortic endothelial cells (ECs) increase active cathepsins K and V as a model of inflammation occurring in the arterial wall. ECs were stimulated with TNFα and cultured with peripheral blood mononuclear cells (PBMCs) from persons homozygous for sickle (SS) or normal (AA) hemoglobin. TNFα was necessary to induce cathepsin K activity, but either PBMC binding or TNFα increased cathepsin V activity. SS PBMCs were unique; they induced cathepsin K in ECs without exogenous TNFα (n = 4, P < 0.05). Inhibition of c-Jun N-terminal kinase (JNK) significantly reduced cathepsins K and V activation by 60% and 51%, respectively. Together, the inflammation and activated circulating mononuclear cells upregulate cathepsin activity through JNK signaling, identifying new pharmaceutical targets to block the accelerated pathology observed in arteries of children with sickle cell disease.
    Anemia 04/2012; 2012:201781. DOI:10.1155/2012/201781
  • Source
    • "Zymography allows the detection of a 37 kDa (cathepsin K), 35 kDa (cathepsin V), 25 kDa (cathepsin S) and 20 kDa (cathepsin L). Cathepsin K activity disappeared and V remained when incubated at pH 4.0 instead of 6.0, allowing the visualization of each enzyme (Wilder et al. 2011). Kupai and colleagues also highlighted that substrate zymography is the method of choice, among several analyzed, to detect the activity of the different matrix metallopeptidase (MMP) isoenzymes from a wide range of biological samples (Kupai et al. 2010). "

    Gel Electrophoresis - Advanced Techniques, 04/2012; , ISBN: 978-953-51-0457-5
Show more