title: 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.
authors: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, 30332, USA.
authors: Catera L
authors: CL
authors: Wilder
authors: Keon-Young
authors: KY
authors: Park
authors: Philip M
authors: PM
authors: Keegan
authors: Manu O
authors: MO
authors: Platt
mesh_terms: D001671:Biochemistry / Q000379:methods*; D056657:Cathepsin K / Q000032:analysis / Q000378:metabolism; D056668:Cathepsin L / Q000032:analysis / Q000378:metabolism; D002403:Cathepsins / Q000032:analysis* / Q000378:metabolism*; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008175:Lung Neoplasms / Q000201:enzymology*; D008264:Macrophages / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C028217:cathepsin S; D056657:Cathepsin K; C117627:CTSV protein, human