HEK 293 cells were transfected with LGMN-plasmid encoding rat legumain, to establish a cell line transiently and/or stably expressing legumain. In addition, HEK 293 cells and a number of melanoma cell lines (from Rikshospitalet University Hospital HF) were successfully transfected with CST6-plasmid encoding human cystatin M, a potent secreted endogenous inhibitor of legumain and other cysteine proteases. Successful CST6-transfection was evaluated as increase in total inhibitory activity (IU/ml) against papain as the target enzyme in media from transfected cells versus control cells (using empty vector; pTracer). The increase in total inhibitory activity was much higher in media from the CST6-transfected HEK 293 cells than the CST6-transfected melanoma cells. In addition, legumain activity was measured in cell lysates from CST6-transfected cells, showing decreased activity compared to control cells. The legumain activity was nearly abolished in cell lysates from CST6-transfected HEK 293 cells and moderately decreased (10-41 %) in CST6-transfected melanoma cells. Interestingly, decrease in legumain activity was strongly correlated to the potency of total inhibitory activity in media from the same cells. Despite many adjustments made to optimize transfection efficacy of HEK 293 cells with the LGMN-plasmid, legumain could not be over-expressed.
Cystatin M-conditioned media from CST6-transfected cells were used to treat living HEK 293, THP-1 and PC12 cells to investigate how this affected legumain activity in the cells. Legumain activity was only moderately decreased in cell lysates of HEK 293 cells treated with cystatin M-conditioned compared to control cell media. However, no regulation of legumain activity was observed in either THP-1 or PC12 cells after treatment with cystatin M-conditioned media.
Cell lysates from HEK 293, THP-1 and PC12 cells were subjected to size exclusion chromatography in an attempt to characterize the molecular weight of active legumain in these cells. Surprisingly, different molecular weights were found to be responsible for legumain activity in the different cell lines. Addition of cystatin M-conditioned media to HEK 293, THP-1 and PC12 cell fractions of highest legumain activity resulted in inhibition of legumain activity. Legumain activity in HEK 293 and PC12 cells was almost completely suppressed, but surprisingly only partially inhibited in THP-1 cells. Adding of the cathepsin-inhibitors E64 and CA074 to the highest legumain activity fractions from the same cell lines resulted in no inhibition of legumain in HEK 293 and PC12 cell lysate fractions. On the contrary, the legumain activity was greatly decreased in the THP-1 cell fraction by both E64 and CA074. This is the first observation of legumain inhibition by E64 and CA074 in any cell type. This might indicate an uncharacterized form of legumain or a legumain-like enzyme in monocytes/macrophages.