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Laminin-332 is a substrate for hepsin, a protease associated with prostate cancer progression.

ABSTRACT: Hepsin, a cell surface protease, is widely reported to be overexpressed in more than 90% of human prostate tumors. Hepsin expression correlates with tumor progression, making it a significant marker and target for prostate cancer. Recently, it was reported that in a prostate cancer mouse model, hepsin up-regulation in tumor tissue promotes progression and metastasis. The underlying mechanisms, however, remain largely uncharacterized. Hepsin transgenic mice displayed reduced laminin-332 (Ln-332) expression in prostate tumors. This is an intriguing cue, since proteolytic processing of extracellular matrix macromolecules, such as Ln-332, is believed to be involved in cancer progression, and Ln-332 expression is lost during human prostate cancer progression. In this study, we provide the first direct evidence that hepsin cleaves Ln-332. Cleavage is specific, since it is both inhibited in a dose-dependent manner by a hepsin inhibitor (Kunitz domain-1) and does not occur when catalytically inactive hepsin is used. By Western blotting and mass spectrometry, we determined that hepsin cleaves the beta3 chain of Ln-332. N-terminal sequencing identified the cleavage site at beta3 Arg(245), in a sequence context (SQLR(245) LQGSCFC) conserved among species and in remarkable agreement with reported consensus target sequences for hepsin activity. In vitro cell migration assays showed that hepsin-cleaved Ln-332 enhanced motility of DU145 prostate cancer cells, which was inhibited by Kunitz domain-1. Further, hepsin-overexpressing LNCaP prostate cancer cells also exhibited increased migration on Ln-332. Direct cleavage of Ln-332 may be one mechanism by which hepsin promotes prostate tumor progression and metastasis, possibly by up-regulating prostate cancer cell motility.

SUBMITTER: Tripathi M

PROVIDER: S-EPMC2576550 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Laminin-332 is a substrate for hepsin, a protease associated with prostate cancer progression.

Tripathi Manisha M Nandana Srinivas S Yamashita Hironobu H Ganesan Rajkumar R Kirchhofer Daniel D Quaranta Vito V

The Journal of biological chemistry 20080909 45

Hepsin, a cell surface protease, is widely reported to be overexpressed in more than 90% of human prostate tumors. Hepsin expression correlates with tumor progression, making it a significant marker and target for prostate cancer. Recently, it was reported that in a prostate cancer mouse model, hepsin up-regulation in tumor tissue promotes progression and metastasis. The underlying mechanisms, however, remain largely uncharacterized. Hepsin transgenic mice displayed reduced laminin-332 (Ln-332) ...[more]

PMID: 18784072

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Project description: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.

Project description:BackgroundMucous membrane pemphigoid is a group of chronic subepithelial autoimmune blistering diseases that mainly affect mucous membranes. Laminin 332-specific autoantibodies are present in approximately 1/3 of the patients, being associated with an increased risk of malignancy. Because of the severe complications, an early recognition of the disease allowing a timely therapy is essential. The gold standard methods for detection of laminin 332-specific autoantibodies, including the immunoprecipitation and immunoblotting are non-quantitative, laborious and restricted to a few specialized laboratories worldwide. In addition, the use of radioimmunoassays, although highly sensitive and specific, are laborious, expensive and tightly regulated. Therefore, there is a stringent need for a quantitative immunoassay for the routine detection of laminin 332-specific autoantibodies more broadly available to diagnostic laboratories. The aim of this study was to compare different antigenic substrates, including native, recombinant laminin 332 and laminin 332-rich keratinocyte extracellular matrix, for development of an ELISA to detect autoantibodies in mucous membrane pemphigoid.ResultsUsing a relatively large number of sera from MMP patients with well-characterized autoantibody reactivity we show the suitability of ELISA systems using laminin 332 preparations as adjunct diagnostic tools in MMP. While glycosylation of laminin 332 does not appear to influence its recognition by MMP autoantibodies, ELISA systems using both purified, native and recombinant laminin 332 demonstrated a high sensitivity and good correlation with the detection of autoantibodies by immunoblotting. ELISA systems using different laminin 332 preparations represent a feasible and more accessible alternative for a broad range of laboratories.ConclusionsOur findings qualify the use of immunoassays with the laminin 332-rich preparations as an ancillary diagnostic tool in mucous membrane pemphigoid.

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Laminin-332 is a substrate for hepsin, a protease associated with prostate cancer progression.

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Laminin-332 is a substrate for hepsin, a protease associated with prostate cancer progression.

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