Cleavage efficiency of cysteine cathepsins at distinct subcellular conditions. Single cathepsins or combinations thereof were incubated with thyroglobulin (Tg) in vitro in buffers representative of extracellular conditions (A), endosomes (B) or lysosomes (C) for 2 hours at 40°C. As a control, purified Tg was incubated in the respective buffers without proteases (A-C, last lanes). Samples were PAGE-separated and Tg fragments were visualized by immunoblotting. Efficiency of cleavage at neutral and oxidizing conditions was indicated by the appearance of a 250 kDa and a 200 kDa fragment (A, arrowheads). Similar fragments were observed under endosome-representative conditions (B, arrowheads). Most effective substrate degradation, discernible by numerous low molecular weight fragments, was observed under reducing, acidic conditions mimicking lysosomes (C). Cathepsin L can be considered most effective in Tg-processing within the lysosomal compartment, because samples containing this protease (C, asterisks) featured numerous low molecular weight fragments (arrows) and almost lacked intact, full-length Tg (dashed arrow). Note, that under neutral and oxidizing conditions, cathepsin S qualified to be most effective in Tg-processing. All samples including this protease featured a specific 70 kDa fragment (A, arrow) additionally to higher molecular mass products. In contrast to extracellular or lysosomal conditions, all peptidases tested performed equally well at endosome-representative conditions.
Jordans et al. BMC Biochemistry 2009 10:23 doi:10.1186/1471-2091-10-23