An Active 32-kDa Cathepsin L Is Secreted Directly from HT 1080 Fibrosarcoma Cells and Not via Lysosomal Exocytosis

• Published in: PloS one Vol. 10; no. 12; p. e0145067
• Main Authors: Hashimoto, Yoko, Kondo, Chihiro, Katunuma, Nobuhiko
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 16.12.2015; Public Library of Science (PLoS)
• Subjects: Acid phosphatase; Alkaline phosphatase; Biochemistry; Breast cancer; Cancer; Cancer cells; Cathepsin L; Cathepsin L - metabolism; Cathepsins; Cell Line, Tumor; Concanavalin A; Core protein; Dentistry; Endocytosis; Endoglycosidase H; Enzymes; Exocytosis; Fibrosarcoma; Fibrosarcoma - metabolism; Gelatin; Gene expression; Genetic aspects; Glycoproteins; Glycosylation; Humans; Lectins; Leupeptin; Lysosomes; Lysosomes - metabolism; Metastasis; Phosphatase; Phosphorylation; Physiological aspects; Prostate cancer; Proteins; Ricinus communis; Secretory Pathway; Streptomyces griseus; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0145067

Cathepsin L [EC 3.4.22.15] is secreted via lysosomal exocytosis by several types of cancer cells, including prostate and breast cancer cells. We previously reported that human cultured fibrosarcoma (HT 1080) cells secrete cathepsin L into the medium; this secreted cathepsin is 10-times more active than intracellular cathepsin. This increased activity was attributed to the presence of a 32-kDa cathepsin L in the medium. The aim of this study was to examine how this active 32-kDa cathepsin L is secreted into the medium. To this end, we compared the secreted active 32-kDa cathepsin L with lysosomal cathepsin L by using a novel gelatin zymography technique that employs leupeptin. We also examined the glycosylation and phosphorylation status of the proteins by using the enzymes endoglycosidase H [EC 3.2.1.96] and alkaline phosphatase [EC 3.1.3.1]. Strong active bands corresponding to the 32-kDa and 34-kDa cathepsin L forms were detected in the medium and lysosomes, respectively. The cell extract exhibited strong active bands for both forms. Moreover, both forms were adsorbed onto a concanavalin A-agarose column. The core protein domain of both forms had the same molecular mass of 30 kDa. The 32-kDa cathepsin L was phosphorylated, while the 34-kDa lysosomal form was dephosphorylated, perhaps because of the lysosomal marker enzyme, acid phosphatase. These results suggest that the active 32-kDa form does not enter the lysosomes. In conclusion, our results indicate that the active 32-kDa cathepsin L is secreted directly from the HT 1080 cells and not via lysosomal exocytosis.