Observing proteases in living cells

Abstract

The lysosomal cysteine protease cathepsin B has been implicated in tumor progression and metastasis in part due to its altered trafficking. In order to analyze the trafficking of cathepsin B in living cells, we utilized enhanced green fluorescent protein (EGFP) fused to various cathepsin B constructs for transfecting two cell lines: an invasive human breast adenocarcinoma cell line (BT20) and a cathepsin B deficient mouse embryonic fibroblast cell line (MEF T -/-). The cells were transiently transfected with four cathepsin B-EGFP fusion constructs: full-length preprocathepsin B-EGFP, cathepsin B pre region-EGFP, cathepsin B prepro region-EGFP, and cathepsin B prepro region-EGFP with a mutation of the glycosylation site in the pro region. The full length construct showed vesicular distribution throughout the cells in both cell lines. In both BT20 and MEF T -/- cells, preregion-EGFP was localized in a ring tightly associated with the cell nucleus, suggesting distribution to the endoplasmic reticulum. The distribution of the prepro region-EGFP construct was similar except that it also included some patchy areas adjacent to the nucleus. This suggested that the cathepsin B prepro region-EGFP might have entered the Golgi. Distribution of the mutated cathepsin B prepro region-EGFP was similar to that of wild-type prepro region-EGFP in the MEF T -/-. In the invasive BT20 cells, however, the mutated prepro region-EGFP showed a vesicular distribution throughout the cytoplasm and in cell processes. This distribution is similar to that of endogenous cathepsin B in these cells. Our results suggest that: 1) tumor cells have an alternative mechanism for trafficking of cathepsin B which is independent of the mannose-6-phosphate receptor pathway, and 2) the pro region of cathepsin B may contain the sorting sequence necessary for its trafficking via this pathway.