Hepatocyte growth factor is a preferred in vitro substrate for human hepsin, a membrane-anchored serine protease implicated in prostate and ovarian cancers

Abstract

Hepsin is a membrane-anchored, trypsin-like serine protease with prominent expression in the human liver and tumours of the prostate and ovaries. To better understand the biological functions of hepsin, we identified macromolecular substrates employing a tetrapeptide PS-SCL (positional scanning-synthetic combinatorial library) screen that rapidly determines the P1-P4 substrate specificity. Hepsin exhibited strong preference at the P1 position for arginine over lysine, and favoured threonine, leucine or asparagine at the P2, glutamine or lysine at the P3, and proline or lysine at the P4 position. The relative activity of hepsin toward individual AMC (7-amino-4-methylcoumarin)-tetrapeptides was generally consistent with the overall peptide profiling results derived from the PC-SCL screen. The most active tetrapeptide substrate Ac (acetyl)-KQLR-AMC matched with the activation cleavage site of the hepatocyte growth factor precursor sc-HGF (single-chain HGF), KQLR downward arrowVVNG (where downward arrow denotes the cleavage site), as identified by a database analysis of trypsin-like precursors. X-ray crystallographic studies with KQLR chloromethylketone showed that the KQLR peptide fits well into the substrate-binding cleft of hepsin. This hepsin-processed HGF induced c-Met receptor tyrosine phosphorylation in SKOV-3 ovarian cancer cells, indicating that the hepsin-cleaved HGF is biologically active. Activation cleavage site mutants of sc-HGF with predicted non-preferred sequences, DPGR downward arrowVVNG or KQLQ downward arrowVVNG, were not processed, illustrating that the P4-P1 residues can be important determinants for substrate specificity. In addition to finding macromolecular hepsin substrates, the extracellular inhibitors of the HGF activator, HAI-1 and HAI-2, were potent inhibitors of hepsin activity (IC50 4+/-0.2 nM and 12+/-0.5 nM respectively). Together, our findings suggest that the HGF precursor is a potential in vivo substrate for hepsin in tumours, where hepsin expression is dysregulated and may influence tumorigenesis through inappropriate activation and/or regulation of HGF receptor (c-Met) functions.

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1

“…Mouse studies also support a tight regulatory relationship between matriptase HAI-1 and prostasin (11). In vitro inhibition of additional membrane-associated serine proteases hepsin (12), TMPRSS13 (13), HAT (14), and HATL5 (15) as well as the soluble proteases trypsin, plasmin, and plasma kallikrein have also been reported (16).…”

Matriptase inhibition by hepatocyte growth factor activator inhibitor-1 is essential for placental development

“…Kunitz-type domains are serine protease inhibitor domains that bind to the active site of trypsin-like serine proteases to form stable inhibitor complexes to restrict the proteolytic activity of their target proteases (Ascenzi et al, 2003). Although originally isolated as an inhibitor of hepatocyte growth factor activator, HAI-1 also has been shown to display potent inhibitory activity towards the membrane-bound trypsin-like serine proteases, hepsin, prostasin, and matriptase (Lin et al, 1999;Fan et al, 2005;Herter et al, 2005).…”

Serine protease hepsin regulates hepatocyte size and hemodynamic retention of tumor cells by hepatocyte growth factor signaling in mice

“…4 Antisenseoligonucleotide blockade of hepsin affects cell growth and enlarges and flattens hepatoma cells. 5 Several in vitro studies have identified substrates for hepsin, including coagulation factor VII, 6 prohepatocyte growth factor (pro-HGF), 7 and prourokinase-type plasminogen activator. 8 In addition, hepsin colocalizes with desmoplakin at the sites of desmosomal junctions.…”