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

Abstract

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.

Cathepsin K-mediated notch1 activation contributes to neovascularization in response to hypoxia

“…Equal amounts of total protein (40 mg) were mixed with SDS sample buffer without a reducing agent and loaded onto a 12.5% SDS polyacrylamide gel containing 0.2% gelatin at 4°C (ref. 20). Gels were then incubated in activity buffer (0.1 M sodium phosphate buffer, pH 6.0, 1 mM EDTA and 2 mM DTT freshly added) overnight at 37°C.…”

“…CatK is one of the most potent collagenases 14,15 . Acidic gelatin zymography has been used to detect Cat-specific activity 20 . As compared with the control, VEGF with CoCl 2 stimulation exhibited increased collagen degradation; both the CatK-specific inhibitor CatK-II and the nonspecific Cat inhibitor E64 inhibited this activity (Fig.…”

Proteolytic Characterization and Lysosomal Localization of Echinoderm Cathepsin D

“…Total proteins were separated using 80% ammonium sulfate precipitation and cathepsin D was purified according to [10]. A zymogram assay was conducted to qualitatively determine the activity of the enzyme in gel using hemoglobin (Hb) as substrate [19]. A native PAGE was polymerized with 0.2% Hb and purified cathepsin D was loaded onto the gel.…”

Applications of Zymography (Substrate-SDS-PAGE) for Peptidase Screening in a Post-Genomic Era

“…Wilder and colleagues, for instance, report that zymography can selectively distinguish cathepsins K, L, S and V in cells and tissues by its electrophoretic mobility and by simply manipulating substrate and pH. The sequence homology among these cathepsins leads to a substrate promiscuity, which precludes desired specificity for in solution assays with specific chromogenic or fluorogenic peptide substrate (Wilder et al 2011). Zymography allows the detection of a 37 kDa (cathepsin K), 35 kDa (cathepsin V), 25 kDa (cathepsin S) and 20 kDa (cathepsin L).…”

“…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).…”