Hepsin, a type II transmembrane serine protease, is highly upregulated in prostate cancer and promotes tumor progression and metastasis. We generated a soluble form of hepsin comprising the entire extracellular domain to show that it efficiently converts single-chain hepatocyte growth factor (pro-HGF) into biologically active two-chain HGF. Hepsin activity was potently inhibited by soluble forms of the bi-Kunitz domain inhibitors HAI-1B (IC 50 21.1 ± 2.7 nM) and HAI-2 (IC 50 1.3 ± 0.3 nM). Enzymatic assays with HAI-1B Kunitz domain mutants (R260A and K401A) further demonstrated that inhibition was due to Kunitz domain-1. The results suggest a functional link between hepsin and the HGF/Met pathway, which may contribute to tumor progression.
Hepatocyte growth factor activator inhibitor-1 (HAI-1) is an integral membrane protein expressed on epithelial cells and contains two extracellular Kunitz domains (N-terminal KD1 and C-terminal KD2) known to inhibit trypsin-like serine proteases. In tumorigenesis and tissue regeneration, HAI-1 regulates the hepatocyte growth factor (HGF)/c-Met pathway by inhibiting the activity of HGF activator (HGFA) and matriptase, two serine proteases that convert pro-HGF into its biologically active form. By screening a placental cDNA library, we discovered a new splice variant of HAI-1 designated HAI-1B that contains an extra 16 amino acids adjacent to the C terminus of KD1. To investigate possible consequences on Kunitz domain function, a soluble form of HAI-1B (sHAI-1B) comprising the entire extracellular domain was produced. First, we found that sHAI-1B displayed remarkable enzyme specificity by potently inhibiting only HGFA (IC 50 ؍ 30.5 nM), matriptase (IC 50 ؍ 16.5 nM), and trypsin (IC 50 ؍ 2.4 nM) among 16 serine proteases examined, including plasminogen activators (urokinase-and tissue-type plasminogen activators), coagulation enzymes thrombin, factors VIIa, Xa, XIa, and XIIa, and activated protein C. Relatively weak inhibition was found for plasmin (IC 50 ؍ 399 nM) and plasma kallikrein (IC 50 ؍ 686 nM). Second, the functions of the KD1 and KD2 domains in sHAI-1B were investigated using P 1 residue-directed mutagenesis to show that inhibition of HGFA, matriptase, trypsin, and plasmin was due to KD1 and not KD2. Furthermore, analysis by reverse transcription-PCR demonstrated that HAI-1B and HAI-1 were co-expressed in normal tissues and various epithelial-derived cancer cell lines. Both isoforms were up-regulated in eight examined ovarian carcinoma specimens, three of which had higher levels of HAI-1B RNA than of HAI-1 RNA. Therefore, previously demonstrated roles of HAI-1 in various physiological and pathological processes likely involve both HAI-1B and HAI-1.Hepatocyte growth factor activator inhibitor-1 (HAI-1)1 is an integral cell surface protein of 66 kDa expressed on epithelial cells (1-3). HAI-1 is known to inhibit the enzymatic activity of HGF activator (HGFA) (1, 4) and matriptase (5-9), two trypsinlike serine proteases capable of converting the inactive singlechain form of hepatocyte growth factor (pro-HGF) (10 -14) into its biologically active two-chain form (HGF). When activated HGF binds to its receptor c-Met, it promotes phospho-transfer activity of the intracellular tyrosine kinase domain leading to activation of multiple intracellular signaling pathways. Therefore, as an inhibitor of HGFA and matriptase, HAI-1 may control the local generation of HGF and thus modulate the activity of the HGF/c-Met receptor system, which is involved in such biological processes as tissue regeneration, morphogenesis, and tumorigenesis (reviewed in Refs. 15-18).The activation of the HGF-converting enzymes represents yet another level of HGF/c-Met pathway regulation. Similar to the coagulation factors, HGFA is ma...
Hepatocyte growth factor (HGF), a plasminogen-related growth factor, is the ligand for Met, a receptor tyrosine kinase implicated in development, tissue regeneration, and invasive tumor growth. HGF acquires signaling activity only upon proteolytic cleavage of single-chain HGF into its ␣/ heterodimer, similar to zymogen activation of structurally related serine proteases.
Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase c-Met, is composed of an ␣-chain containing four Kringle domains (K1-K4) and a serine protease domain-like -chain.
Tissue factor is the cell membrane-anchored cofactor for factor VIIa and triggers the coagulation reactions. The initial step is the conversion of factor VII to factor VIIa which, in vitro, is efficiently catalyzed by low concentrations of factor Xa. To identify the tissue factor region that interacts with the activator factor Xa during this process, we evaluated a panel of soluble tissue factor (1-219) mutants for their ability to support factor Xa-mediated activation of factor VII. The tissue factor residues identified as most important for this interaction (Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, and Tyr185) were identical to those found to be important for the interaction of substrate factor X with the tissue factor.factor VIIa complex. The residues form a continuous surface-exposed patch with an area of about 500 A(2), which appears to be located outside the tissue factor-factor VII contact zone. In agreement, the two monoclonal antibodies 5G6 and D3H44-F(ab')(2), whose epitopes overlap with this identified region, inhibited the rates of factor VII activation by 86% and 95%, respectively. These antibodies also strongly inhibited the conversion of (125)I-labeled factor VII when cell membrane-expressed, full-length tissue factor (1-263) was employed. Together the results suggest the usage of a common surface region of tissue factor in its dual role-as a cofactor for factor Xa-mediated factor VII activation and as a cofactor for factor VIIa-mediated factor X activation. The finding that factor Xa and factor X may engage in similar, if not identical, molecular interactions with tissue factor further indicates that factor Xa and factor X are similarly oriented toward their respective interaction partners in the ternary catalytic complexes.
Tissue factor (TF) binds the zymogen (VII) and activated (VIIa) forms of coagulation factor VII with high affinity. The structure determined for the sTF-VIIa complex [Banner, D. W., et al. (1996) Nature 380, 41-46] shows that all four domains of VIIa (Gla, EGF-1, EGF-2, and protease) are in contact with TF. Although a structure is not available for the TF-VII complex, the structure determined for free VII [Eigenbrot, C., et al. (2001) Structure 9, 675-682] suggests a significant conformational change for the zymogen to enzyme transition. In particular, the region of the protease domain that must contact TF has a conformation that is altered from that of VIIa, suggesting that the VII protease domain interacts with TF in a manner different from that of VIIa. To test this hypothesis, a panel of 12 single-site sTF mutants, having substitutions of residues observed to contact the proteolytic domain of VIIa, have been evaluated for binding to both zymogen VII and VIIa. Affinities were determined by surface plasmon resonance measurements using a noninterfering anti-TF monoclonal antibody to capture TF on the sensor chip surface. Dissociation constants (K(D)) measured for binding to wild-type sTF are 7.5 +/- 2.4 nM for VII and 5.1 +/- 2.3 nM for VIIa. All of the sTF mutants except S39A and E95A exhibited a significant decrease (>2-fold) in affinity for VIIa. The changes in affinity measured for VII or VIIa binding with substitution in sTF were comparable in magnitude. We conclude that the proteolytic domain of both VII and VIIa interacts with this region of sTF in a nearly identical fashion. Therefore, zymogen VII can readily adopt a VIIa-like conformation required for binding to TF.
Highly potent bifunctional inhibitors of Factor VIIa (FVIIa) were generated by linking two distinct peptides, recently shown to bind to two discrete exosites on the FVIIa protease domain [Dennis, Eigenbrot, Skelton, Ultsch, Santell, Dwyer, O'Connell and Lazarus (2000) Nature (London) 404, 465-470; Dennis, Roberge, Quan and Lazarus (2001) Biochemistry 40, 9513-9521; Roberge, Santell, Dennis, Eigenbrot, Dwyer and Lazarus (2001) Biochemistry 40, 9522-9531]. Fusion peptides consisting of an N-terminal A-series peptide followed by flexible linkers, an E-series peptide, and the Z-domain of protein A were expressed in Escherichia coli and purified using IgG-Sepharose affinity chromatography. The fusion peptides were potent anticoagulants and had steep concentration dependence curves in tissue factor-dependent prothrombin time (PT) assays in comparison to the individual peptides or their noncovalent combination. This phenomenon was dependent on the length of the linker joining the A- and E-peptides. The fusion of the peptides increased the extent of inhibition of Factor X (FX) activation to 100% at saturating peptide concentrations, but did not improve the binding affinity for Factor VIIa (FVIIa) at the A- and E- binding sites or the IC(50) for the inhibition of FX activation. Differences between the peptides in the PT fold prolongation in normal and FVII-deficient plasma, in conjunction with the inhibition of (125)I-FVII activation, suggest that the enhanced effects of the fusion peptides involve the inhibition of FVII autoactivation.
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