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

Arch Biochem Biophys. 2011 Dec 1;516(1):52-7. doi: 10.1016/j.abb.2011.09.009. Epub 2011 Sep 29.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biochemistry / methods*
  • Cathepsin K / analysis
  • Cathepsin K / metabolism
  • Cathepsin L / analysis
  • Cathepsin L / metabolism
  • Cathepsins / analysis*
  • Cathepsins / metabolism*
  • Cells, Cultured
  • Cysteine Endopeptidases / analysis
  • Cysteine Endopeptidases / metabolism
  • Humans
  • Hydrogen-Ion Concentration
  • Lung Neoplasms / enzymology*
  • Macrophages / enzymology*
  • Substrate Specificity

Substances

  • Cathepsins
  • Cysteine Endopeptidases
  • Cathepsin L
  • cathepsin S
  • Cathepsin K
  • CTSV protein, human