Observing proteases in living cells.
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 preregion-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.
Moin
K
K
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Demchik
L
L
Mai
J
J
Duessing
J
J
Peters
C
C
Sloane
B F
BF
D000230:Adenocarcinoma / Q000201:enzymology / Q000473:pathology; D000818:Animals; D001692:Biological Transport; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology; D002401:Cathepsin B / Q000032:analysis* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology / Q000648:ultrastructure; D002477:Cells / Q000201:enzymology* / Q000648:ultrastructure; D003587:Cytomegalovirus / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000648:ultrastructure; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000201:enzymology / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000032:analysis; D006056:Golgi Apparatus / Q000201:enzymology / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis; D008247:Lysosomes / Q000201:enzymology; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000201:enzymology / Q000648:ultrastructure
D004792:Enzyme Precursors; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002403:Cathepsins; C057831:preprocathepsin B; D002401:Cathepsin B