[7] Hepsin

Kurachi, Kotoku; Torres-Rosado, Adrian; Tsuji, Akihiko

doi:10.1016/0076-6879(94)44009-3

Cited by 33 publications

(26 citation statements)

References 7 publications

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“…2). The results are consistent with reports of other type II transmembrane serine proteases including EK (32), matriptases (11,33,34), hepsin (35,36), spinesin (16), human airway trpsin-like protease (17), and polyserase-I (20), showing that soluble enzymes lacking the transmembrane domain can be catalytically active. It appears, therefore, that the main func- 98 is indicated by an arrow (top).…”

Section: Discussionsupporting

confidence: 81%

Functional Analysis of the Transmembrane Domain and Activation Cleavage of Human Corin

Knappe

Masikat

et al. 2003

Journal of Biological Chemistry

111

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Corin is a cardiac transmembrane serine protease. In cell-based studies, corin converted pro-atrial natriuretic peptide (pro-ANP) to mature ANP, suggesting that corin is potentially the pro-ANP convertase. In this study, we evaluated the importance of the transmembrane domain and activation cleavage in human corin. We showed that a soluble corin that consists of only the extracellular domain was capable of processing recombinant human pro-ANP in cell-based assays. In contrast, a mutation at the conserved activation cleavage site, R801A, abolished the function of corin, demonstrating that the activation cleavage is essential for corin activity. These results allowed us to design, express, and purify a mutant soluble corin, EKsolCorin, that contains an enterokinase recognition sequence at the activation cleavage site. Purified EKsolCorin was activated by enterokinase in a dose-dependent manner. Activated EKsolCorin had hydrolytic activity toward peptide substrates with a preference for Arg and Lys residues in the P-1 position. This activity of EKsolCorin was inhibited by trypsin-like serine protease inhibitors but not inhibitors of chymotrypsin-like, cysteine-, or metallo-proteases. In pro-ANP processing assays, purified active EKsolCorin converted recombinant human pro-ANP to biologically active ANP in a highly sequence-specific manner. The pro-ANP processing activity of EKsolCorin was not inhibited by human plasma. Together, our data indicate that the transmembrane domain is not necessary for the biological activity of corin but may be a mechanism to localize corin at specific sites, whereas the proteolytic cleavage at the activation site is an essential step in controlling the activity of corin.Corin is a mosaic serine protease that was recently identified from the human heart (1, 2). It consists of 1,042 amino acids and contains an integral transmembrane domain near the N terminus. In the extracellular region of corin, there are two frizzled-like cysteine-rich domains, eight low density lipoprotein receptor type A repeats, a scavenger receptor-like cysteinerich domain, and a C-terminal trypsin-like protease domain. Topologically, corin belongs to the newly defined type II transmembrane serine protease family (3-6), which includes enterokinase (EK) 1 (7), hepsin (8), matriptases (9 -12), TMPRSS2-5 (13-16), human airway trypsin-like protease (17), MSPL (18), DESC1 (19), and polyserase-I (20). The combination of domains present in corin, however, is unique among the trypsin-like serine proteases, because corin is the only serine protease identified so far that contains frizzled-like cysteinerich domains. Corin mRNA and protein are abundantly expressed in the heart (1, 2), suggesting that corin might have a role in the cardiovascular system. In cell-based experiments, we showed that recombinant human corin mediated the conversion of proatrial natriuretic peptide (pro-ANP) and pro-brain natriuretic peptide to mature ANP and brain natriuretic peptide (21), both of which are cardiac hormones important in maintaining ...

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Section: Discussionsupporting

confidence: 81%

Functional Analysis of the Transmembrane Domain and Activation Cleavage of Human Corin

Knappe

Masikat

et al. 2003

Journal of Biological Chemistry

111

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“…At its NH 2 terminus, hepsin contains a cytoplasmic domain and an integral transmembrane domain. In the extracellular region of hepsin, there is a macrophage scavenger receptor-like domain and a serine protease domain at the COOH terminus (9)(10)(11)(12). Biochemical studies indicate that hepsin is an enzyme with trypsin-like substrate specificity that cleaves peptide bonds after basic residues such as arginine and lysine (11,12).…”

Section: Introductionmentioning

confidence: 99%

“…In the extracellular region of hepsin, there is a macrophage scavenger receptor-like domain and a serine protease domain at the COOH terminus (9)(10)(11)(12). Biochemical studies indicate that hepsin is an enzyme with trypsin-like substrate specificity that cleaves peptide bonds after basic residues such as arginine and lysine (11,12). The overall topology of hepsin is similar to those of other type II transmembrane serine proteases of the trypsin superfamily (13), which include corin (14,15), enterokinase (16), MT-SP1/matriptase (17,18), human airway trypsin-like protease (19), TMPRSS2 (20), and Stubble-stubbloid (21).…”

Section: Introductionmentioning

confidence: 99%

Antibodies Neutralizing Hepsin Protease Activity Do Not Impact Cell Growth but Inhibit Invasion of Prostate and Ovarian Tumor Cells in Culture

Xuan¹,

Schneider²,

Toy³

et al. 2006

Cancer Research

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Hepsin is a type II transmembrane serine protease that is expressed in normal liver, and at lower levels in kidney, pancreas, and testis. Several studies have shown that hepsin mRNA is significantly elevated in most prostate tumors, as well as a significant fraction of ovarian and renal cell carcinomas and hepatomas. Although the overexpression of mRNA in these tumors has been extensively documented, there has been conflicting literature on whether hepsin plays a role in tumor cell growth and progression. Early literature implied a role for hepsin in human tumor cell proliferation, whereas recent studies with a transgenic mouse model for prostate cancer support a role for hepsin in tumor progression and metastases. To evaluate this issue further, we have expressed an activatable form of hepsin, and have generated a set of monoclonal antibodies that neutralize enzyme activity. The neutralizing antibodies inhibit hepsin enzymatic activity in biochemical and cell-based assays. Selected neutralizing and nonneutralizing antibodies were used in cell-based assays with tumor cells to evaluate the effect of antibodies on tumor cell growth and invasion. Neutralizing antibodies failed to inhibit the growth of prostate, ovarian, and hepatoma cell lines in culture. However, potent inhibitory effects of the antibodies were seen on invasion of ovarian and prostate cells in transwell-based invasion assays. These results support a role for hepsin in tumor cell progression but not in primary tumor growth. Consistent with this, immunohistochemical experiments with a mouse monoclonal antibody reveal progressively increased staining of prostate tumors with advanced disease, and in particular, extensive staining of bone metastatic lesions.

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“…This pattern on SDS polyacrylamide gel electrophoresis is similar to that of hepsin. 31,34,35) Furthermore the BZ-II (M.W. of 31 kDa) reacted with the antihepsin antibody as seen by Western blotting and the peptide analyses of the BZ-II by MALDI-TOF MS showed a 99% similarity to that of hepsin.…”

Section: Discussionmentioning

confidence: 99%

“…Preparation of Antihepsin Antibody Hepsin antibodies prepared by immunization of two rabbits with a synthetic peptide corresponding to amino acid 397-416 (carboxy terminus) of rat hepsin 31,32) were obtained from Peptide Institute Inc. (Osaka, Japan).…”

Section: S-transferasementioning

confidence: 99%

Purification of Liver Serine Protease Which Activates Microsomal Glutathione S-Transferase: Possible Involvement of Hepsin

Kunii

Shimoji

Nakama

et al. 2006

Biological & Pharmaceutical Bulletin

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Glutathione S-transferases (GST, EC 2.5.1.18) are phase II drug metabolizing enzymes which catalyze the glutathione conjugation of a number of xenobiotics such as carcinogens, therapeutic drugs or toxic metabolites.3,4) Mammalian GSTs involve cytosolic and membrane-bound forms which are encoded by different gene families. 4,5) Rat liver microsomal GST (MGST1), distinct from cytosolic transferases, is a homotrimer which contains one SH group per subunit and is activated by covalent binding of the SH group with N-ethylmaleimide 6) or reactive metabolites formed during drug metabolism via cytochrome P450 system. 7,8) MGST1 is also activated by reactive oxygen species through disulfide bondlinked dimer formation.9-13) Furthermore, it has been evidenced that MGST1 is activated by limited proteolysis at Lys-41 with trypsin resulting in formation of the fragment of a molecular weight (M.W.) of 12 kDa. 14) We previously reported that MGST1 activity is increased 4-7 fold by trypsin treatment and the oxidized MGST1 with a mixed disulfide bond is more activated by the trypsin treatment and MGST1 dimer is also degraded to the fragment with a M.W. of 24 kDa. 15)Such a marked activation of MGST1 by trypsin prompts us to examine whether MGST1 is regulated by some protease(s) in the liver. In deed it is known that the oxidatively modified proteins become more sensitive to a proteolytic attack than native proteins and the proteolysis is regarded as a mechanism of protein metabolism or modulation of the enzyme activity. [16][17][18][19] As preliminary experiments, we observed that MGST1 is oxidized gradually and degraded when liver microsomes were stored at a cold room. It was therefore assumed that MGST1-activating/degrading protease may be involved in liver microsomes.In the present study, we purified the protease which can activate MGST1 and identified that the protease is hepsin. Hepsin is a type II transmembrane serine protease which is found in plasma/endoplasmic membranes 20) and may plays a role in cell growth or coagulation. 21,22) There are also considerable data showing a link between this protease and some types of cancer. 23,24) However the function of the microsomal hepsin has not been clarified yet. In this study we firstly demonstrated the function of hepsin as an activating/degrading enzyme of MGST1. MATERIALS AND METHODS ChemicalsReduced glutathione (GSH), Triton X-100, CM Sepharose CL 6B, and calpastatin were purchased from Sigma Chemicals (St. Louis, MO, U.S.A.). Lubrol PX and sodium cholate were from Nacalai Tesque (Tokyo, Japan). 1-Chloro-2,4-dinitrobenzene (CDNB), diisopropylfluorophosphate (DFP), and leupeptin were obtained from Wako Pure Chemicals (Osaka, Japan). Soybean trypsin inhibitor and Hydroxyapatite were from Mils Laboratories (Stoke Poges, Slough, U.K.) and Bio-Rad Laboratories (Richmond, CA, U.S.A.), respectively. Benzamidine-Sepharose 6B and synthetic protease substrates including t-butyloxycarbonyl (Boc)-Gln-Arg-Arg-4-methylcoumaryl-7-amide (MCA) were from Pharmacia-LKB Biotechnology Inc. (Tok...

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[7] Hepsin

Cited by 33 publications

References 7 publications

Functional Analysis of the Transmembrane Domain and Activation Cleavage of Human Corin

Functional Analysis of the Transmembrane Domain and Activation Cleavage of Human Corin

Antibodies Neutralizing Hepsin Protease Activity Do Not Impact Cell Growth but Inhibit Invasion of Prostate and Ovarian Tumor Cells in Culture

Purification of Liver Serine Protease Which Activates Microsomal Glutathione S-Transferase: Possible Involvement of Hepsin

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