title: An Active 32-kDa Cathepsin L Is Secreted Directly from HT 1080 Fibrosarcoma Cells and Not via Lysosomal Exocytosis.
abstract: 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.
authors.affiliation: Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Hashimoto
authors.affiliation: Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Kondo
authors.affiliation: Institute for Health Sciences, Tokushima Bunri University, Tokushima, Japan.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Katunuma
mesh_terms: D056668:Cathepsin L / Q000378:metabolism*; D045744:Cell Line, Tumor; D005089:Exocytosis; D005354:Fibrosarcoma / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D055571:Secretory Pathway
chemical_list: D056668:Cathepsin L