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

Yoko Hashimoto,Chihiro Kondo,Nobuhiko Katunuma,Hong Wanjin

doi:10.1371/journal.pone.0145067

Yoko Hashimoto, Chihiro Kondo + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0145067

Copy DOI

Journal: PloS one	Publication Date: Dec 16, 2015
Citations: 17	License type: CC BY 4.0

Affiliation: Aichi Gakuin University, Tokushima Bunri University

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.

Highlights

Lysosomes are cellular organelles that play crucial roles in intracellular protein degradation and recycling [1]
Our results demonstrate that the major active form of cathepsin L in the lysosomes of the HT 1080 cells is 34-kDa cathepsin L, which is glycosylated and dephosphorylated
We hypothesize that a nonsignal constitutive secretory pathway [33] transports the active 32-kDa single-chain cathepsin L directly to the cell surface from the trans Golgi network (TGN) in the HT 1080 cells [34]

Summary

Introduction

Lysosomes are cellular organelles that play crucial roles in intracellular protein degradation and recycling [1]. Lysosomes are translocated to the vicinity of the cell surface; this process occurs independent of the intracellular calcium [Ca2+] concentration [2]. Secretion of Active 32-kDa Cathepsin L by HT 1080 Cells membrane; this process depends on an increase in the intracellular [Ca2+] concentration [2,3,4,5,6,7,8,9]. Increased [Ca2+] triggers the fusion of the lysosomes with the plasma membrane. This lysosomal docking and fusion are regulated by a transcription factor EB (TFEB) for lysosome biogenesis and autophagy [8,9]. The fusion of the lysosomes with the plasma membrane is called lysosomal exocytosis. Lysosomal exocytosis leads to the extracellular release of lysosomal enzymes [8,9]

Objectives

Methods

Results

Conclusion