Identification and Cloning of the Membrane-associated Serine Protease, Hepsin, from Mouse Preimplantation Embryos

Vu, Thien Khai H; Liu, Rose W.; Haaksma, Carol J.; Tomasek, James J.; Howard, Eric W.

doi:10.1074/jbc.272.50.31315

Cited by 65 publications

(81 citation statements)

References 38 publications

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“…The K i values were calculated according to the relationship K i ϭ IC 50 /(1 ϩ [S]/K m ) (23) using the experimentally determined IC 50 and K m values for each enzyme-substrate pair. For HAI-2, the apparent K i values (K i *) were determined by fitting the data to the equation for tight binding inhibition (24,25), 2 , 0.05 mg/ml BSA, 5 mM glucose), and 0.8 ml of 100 nM pro-uPA in prewarmed HBSA-glucose buffer was added to the cell layers. The inhibitors KD1 or HI-10331 were added to give final concentrations of 1 and 10 M, respectively.…”

Section: Real-time Reverse Transcription-pcr-totalmentioning

confidence: 99%

“…The physiologic function of hepsin remains unknown. Despite its expression in the very early stages of embryogenesis (2), hepsin-deficient mice were viable and developed normally (3,4). The studies further showed that hepsin was not essential for liver regeneration and for coagulationrelated physiological functions (3,4).…”

mentioning

confidence: 96%

“…Intriguingly, hepsin overexpression was associated with basement membrane disruption (16) pointing toward the possibility that hepsin activity is somehow linked to the degradation of basement membrane components. In vitro, hepsin was able to convert the latent growth factor pro-hepatocyte growth factor (pro-HGF) 2 into its active two-chain form (HGF), which induced Met receptor signaling (17,18). Because the HGF/Met pathway has been implicated in invasive tumor growth and metastasis, it is possible that overexpression of hepsin activates the HGF/Met axis in prostate cancer.…”

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confidence: 99%

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Pro-urokinase-type Plasminogen Activator Is a Substrate for Hepsin

Moran¹,

Li²,

Fan³

et al. 2006

Journal of Biological Chemistry

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Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys 158 -Ile 159 (P 1 -P 1 ) peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (k cat /K m 4.8 ؋ 10 5 M ؊1 s ؊1 ) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 ؎ 1.9 nM uPA h ؊1 , which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression.Hepsin is a type II transmembrane serine protease expressed on the surface of epithelial cells. The 417-amino acid protein is composed of a short N-terminal cytoplasmic domain, a transmembrane domain, and a single scavenger receptor cysteinerich domain that packs tightly against the C-terminal protease domain (1). The physiologic function of hepsin remains unknown. Despite its expression in the very early stages of embryogenesis (2), hepsin-deficient mice were viable and developed normally (3, 4). The studies further showed that hepsin was not essential for liver regeneration and for coagulationrelated physiological functions (3, 4). However, hepsin has been implicated in ovarian cancer (5) and prostate cancer (6 -11), where several gene expression studies have identified it as one of the most highly induced genes. Hepsin RNA levels were found to be low in normal prostate and benign hyperplasia but strongly increased in prostate carcinoma, particularly in advanced stages (8 -10). Hepsin protein staining with a monoclonal anti-hepsin antibody showed that hepsin expression was highest at sites of bone metastasis and in late stage primary tumors (12), which is consistent with the finding that increased hepsin RNA levels correlated with higher Gleason grades...

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Section: Real-time Reverse Transcription-pcr-totalmentioning

confidence: 99%

mentioning

confidence: 96%

mentioning

confidence: 99%

See 1 more Smart Citation

Pro-urokinase-type Plasminogen Activator Is a Substrate for Hepsin

Moran¹,

Li²,

Fan³

et al. 2006

Journal of Biological Chemistry

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“…For example, the extracellular matrix degrading peptidases such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) promote blastocyst invasion (Salamonsen, 1999;Eliahu et al, 2004;Aflalo et al, 2005). Likewise, an implantation specific serine proteinase (ISP1) (O'Sullivan et al, 2001(O'Sullivan et al, , 2002 and Hepsin, a membrane associated serine protease, are thought to be involved in hatching of the embryo (Vu et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

Acid peptidase activity released from in vitro produced porcine embryos: A candidate marker to predict developmental competence

Telugu

Spate

Prather

et al. 2008

Molecular Reproduction Devel

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SUMMARYThe ability to efficiently create high quality embryos, competent to produce normal viable offspring in vitro, facilitates diverse technological advancements in animal agriculture and assisted reproduction. Current methods for evaluation of embryos are predominantly based on morphological characteristics which are prone to potential bias of the scorer. Metabolic and genetic markers have also been explored for quality assessment, but they are cost prohibitive or require longer periods of time for evaluation. We hypothesized that secreted enzymes could provide another means of embryo quality assessment. In this report, we provide evidence that medium conditioned by porcine embryos often has proteolytic activity that operates in acidic conditions (acid peptidase activity or APA). The APA could be inhibited by pepstatin A, suggesting that the activity is derived from one or more aspartic peptidases. We also provide evidence that single embryos, incubated for as few as 24 hr, released enough APA that it was possible to measure it accurately at day 5 of culture. We also observed that such activity on day 6 could be positively correlated with advanced developmental stage and embryo quality. In addition, those embryos that were graded identically by morphological evaluations often differed in the amount of APA-with some being significantly higher than the experimental threshold value. Therefore, the APA of embryos might serve as an additional marker for evaluation of embryos.

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“…13,14) Hepsin zymogen is activated autocatalytically by cleavage at 14) forming a heterodimeric enzyme. Many lines of evidence have shown that hepsin contributes to activation of coagulation factor VII 15) and growth promoting and suppressing activities [16][17][18] and is increased in ovarian 19) and prostate cancer.…”

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confidence: 99%

Activation of Rat Liver Microsomal Glutathione Transferase by Hepsin

Nakama

Oshiro

Aniya

2010

Biological & Pharmaceutical Bulletin

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Glutathione transferases (GST) catalyze the conjugation of various electrophiles including antitumor drugs with reduced glutathione and distribute in cytosol, microsomes, and mitochondria.1-3) Rat liver microsomal glutathione transferase (MGST1) is a homotrimer that contains a sole thiol (Cys49) per each subunit protein and is activated through a modification of the thiol such as alkylation, thiol/disulfide exchange, or oxidation to sulfenic acid. [4][5][6][7][8][9] MGST1 is also activated by limited proteolysis at lysine 41 with trypsin 10) and oxidatively modified MGST1 becomes sensitive to the proteolytic activation.11) It is of great interest whether protease is capable of activating MGST1 in the liver.Recently we purified a serine protease, hepsin, from rat liver microsomes and found that when purified MGST1 was incubated with hepsin for 3-6 d at 4°C, MGST1 activity was increased and a disulfide-linked dimer of MGST1 was observed, 12) suggesting that hepsin stimulates MGST1 dimer formation. However, the activation mechanism of MGST1 by hepsin has not been clarified.Hepsin is a cell surface-expressed type II transmembrane serine protease found in plasma membrane and microsomes. 13,14) Hepsin zymogen is activated autocatalytically by cleavage at 14) forming a heterodimeric enzyme. Many lines of evidence have shown that hepsin contributes to activation of coagulation factor VII 15) and growth promoting and suppressing activities [16][17][18] and is increased in ovarian 19) and prostate cancer. 20,21) Hepsin is a 417 amino acid polypeptide containing a short N-terminal cytoplasmic tail, a transmembrane region, and an extracellular domain (Arg45-Leu417) composed of the scavenger receptor cysteine-rich (SRCR) domain and protease domain. 22) Since intra-or inter-domain disulfide bonds are involved in hepsin, it is suggested that hepsin may activate MGST1 via thiol/disulfide exchange. To confirm the activation mechanism of MGST1 by hepsin, we investigated the possibility of thiol/disulfide exchange between MGST1 and hepsin molecules. MATERIALS AND METHODSChemicals Reduced glutathione (GSH), Triton X-100, and CM Sepharose CL 6B were purchased from Sigma Chemicals (St. Louis, MO, U.S.A.). Lubrol PX, benzamidine hydrochloride, and sodium cholate were from Nacalai Tesque (Kyoto, Japan). 1-Chloro-2,4-dinitrobenzene (CDNB) and glycerol were obtained from Wako Pure Chemicals (Osaka, Japan). Hydroxyapatite, silver stain kit, gout anti-rabbit immunoglobulin G (IgG) (HϩL), and horseradish peroxidase conjugate substrate kit were from Bio-Rad Laboratories (Richmond, CA, U.S.A.). Benzamidine-Sepharose 6B and synthetic protease substrate t-butyloxycarbonyl (Boc)-GlnArg-Arg-4-methylcoumaryl-7-amide (MCA) were from Pharmacia-LKB Biotechnology Inc. (Tokyo, Japan) and Peptide Institute Inc. (Osaka, Japan), respectively. Anti-hepsin antibody was prepared against C-terminal 17 peptide as described previously.12) Anti-MGST1 antibody was prepared in our laboratory as described previously.23) All other reagents were of analytical grade.P...

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Identification and Cloning of the Membrane-associated Serine Protease, Hepsin, from Mouse Preimplantation Embryos

Cited by 65 publications

References 38 publications

Pro-urokinase-type Plasminogen Activator Is a Substrate for Hepsin

Pro-urokinase-type Plasminogen Activator Is a Substrate for Hepsin

Acid peptidase activity released from in vitro produced porcine embryos: A candidate marker to predict developmental competence

Activation of Rat Liver Microsomal Glutathione Transferase by Hepsin

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