Autoactivation and calpain-1-mediated shedding of hepsin in human hepatoma cells

Abstract

Hepsin is a transmembrane serine protease implicated in many biological processes, including hepatocyte growth, urinary protein secretion, auditory nerve development, and cancer metastasis. Zymogen activation is critical for hepsin function. To date, how hepsin is activated and regulated in cells remains an enigma. In this study, we conducted site-directed mutagenesis, cell expression, plasma membrane protein labeling, trypsin digestion, Western blotting, and flow cytometry experiments in human hepatoma HepG2 cells, where hepsin was originally discovered, and SMMC-7721 cells. Our results show that hepsin is activated by autocatalysis on the cell surface but not intracellularly. Moreover, we show that hepsin undergoes ectodomain shedding. In the conditioned medium from HepG2 and SMMC-7721 cells, we detected a soluble fragment comprising nearly the entire extracellular region of hepsin. By testing protease inhibitors, gene knockdown, and site-directed mutagenesis, we identified calpain-1 as a primary protease that acted extracellularly to cleave Tyr52 in the juxtamembrane space of hepsin. These results provide new insights into the biochemical and cellular mechanisms that regulate hepsin expression and activity.

Intracellular autoactivation of TMPRSS11A, an airway epithelial transmembrane serine protease

“…To date, several distinct mechanisms have been identified. For example, hepsin and matriptase 2 (hepatic TTSPs) are autoactivated on the cell surface but not inside the cell (38)(39)(40). Corin (a cardiac TTSP) is activated in the extracellular space by proprotein convertase subtilisin/kexin-6 (6,41).…”

A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells

“…The plasmids expressing human neprilysin, hepsin, and corin WT and mutants were described previously ( Dong et al, 2016 ; Knappe et al, 2004 ; Wang et al, 2019 ). Human full-length cDNAs encoding matriptase, TMPRSS3, TMPRSS4, and TMPRSS5 were amplified from human cell line-derived cDNA libraries and inserted into pcDNA 3.1/V5-His plasmid (Thermo Fisher, K4800-01), which encodes a C-terminal V5 tag.…”

Hepsin: a multifunctional transmembrane serine protease in pathobiology

“…It was unknown if the activation cleavage was mediated by hepsin autocatalysis or by unidentified protease(s) in tissue samples. Recently, hepsin activation was detected by western blotting in cultured human hepatoma cells [50]. The two-chain active form was found on the cell surface but not inside the cell.…”

“…Unexpectedly, calpain-1, a cysteine protease, has been identified as a hepsin sheddase [50]. Calpain-1 is a calcium-dependent cysteine protease that acts both intracellularly and extracellularly [66][67][68].…”

“…Calpain-1 is a calcium-dependent cysteine protease that acts both intracellularly and extracellularly [66][67][68]. In cultured human hepatoma cells, calpain-1 was detected in the conditioned medium [50]. The secreted calpain-1 cleaved hepsin at Tyr52-Pro53 in the external juxtamembrane region.…”