title: 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.
authors.affiliation: Department of Biology, Amgen San Francisco, 1120 Veterans Boulevard, South San Francisco, CA 94080, USA.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Herter
authors.forename: Derek E
authors.initials: DE
authors.lastname: Piper
authors.forename: Wade
authors.initials: W
authors.lastname: Aaron
authors.forename: Timothy
authors.initials: T
authors.lastname: Gabriele
authors.forename: Gene
authors.initials: G
authors.lastname: Cutler
authors.forename: Ping
authors.initials: P
authors.lastname: Cao
authors.forename: Ami S
authors.initials: AS
authors.lastname: Bhatt
authors.forename: Youngchool
authors.initials: Y
authors.lastname: Choe
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.forename: Nigel
authors.initials: N
authors.lastname: Walker
authors.forename: David
authors.initials: D
authors.lastname: Meininger
authors.forename: Timothy
authors.initials: T
authors.lastname: Hoey
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Austin
mesh_terms: D001665:Binding Sites; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D005260:Female; D015870:Gene Expression; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D008297:Male; D010051:Ovarian Neoplasms / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000378:metabolism*; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D015854:Up-Regulation
chemical_list: D017228:Hepatocyte Growth Factor; D012697:Serine Endopeptidases; C055645:hepsin