Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Serine proteinase inhibitors play a major role in the turnover of connective tissues. In this study, we isolated and determined partial amino-terminal amino acid sequence of trypsin/elastase/plasmin inhibitors (M(r) 33,000 and 31,000) from the extracellular matrix of SV40-transformed human skin fibroblasts. The antitrypsin activity of the inhibitors was monitored by substrate reverse zymography. Polyclonal antisera to alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 2-antiplasmin, inter-alpha-trypsin inhibitor, plasminogen activator inhibitors-1 and -2, and a monoclonal antibody to protease nexin-1 did not label the 33-, 31-, and 27-kDa inhibitors. A computer search for amino acid sequence homology indicated that the 31-kDa inhibitor is novel. In contrast, the sequence of the 33-kDa inhibitor shared 70 to 90% homology with the amino-terminal sequence of a recently characterized 32-kDa trypsin/tissue factor inhibitor called tissue factor pathway inhibitor-2. The 33- and 31-kDa inhibitors bind to heparin-Sepharose and were recovered from the affinity beads as well as from the t12 FB extracellular matrix with 1 M NaCl. Based on these results, we propose that the extracellular matrix of human mesenchymal cells sequester a family of novel serine proteinase inhibitors.
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PMID:Novel extracellular matrix-associated serine proteinase inhibitors from human skin fibroblasts. 787 99

The urokinase-urokinase receptor system plays a dominant role in the degradation and invasion of extracellular matrix (ECM) by tumor cells. In this system, urokinase bound to its cell receptor converts plasminogen to plasmin, a broad-spectrum serine protease that participates in the degradation and invasion of connective tissues by tumor cells. In this study, we evaluated whether these activities of plasmin are inhibited by a newly characterized human 32 kDa recombinant serine protease inhibitor called trypsin/tissue factor pathway inhibitor-2 (rTFPI-2). We found that rTFPI-2 dose-dependently inhibited fluid-phase plasmin as well as plasmin generated on the ECM and/or the cell surface of HT-1080 fibrosarcoma cells. The degradation of radiolabeled matrix as well as Matrigel invasion by these tumor cells is also inhibited by rTFPI-2 in a dose-dependent fashion. We have reported that rTFPI-2 is identical to 33 kDa extracellular matrix-associated serine protease inhibitor (33 kDa MSPI), whereas the 31 and 27 kDa MSPIs are under-glycosylated forms of the 33 kDa MSPI. We therefore evaluated the ability of MSPIs from the ECM of dermal fibroblasts to inhibit plasmin and found that the plasmin activity was effectively blocked by the MSPIs. We have also evaluated whether the HT-1080 cells synthesize and secrete the MSPIs and found that the synthesis and secretion of the MSPIs was undetectable in these cells. Collectively, our results suggest that rTFPI-2/33 kDa MSPI inhibits plasmin on the tumor cell and ECM surfaces as well as the degradation and invasion of matrix by HT-1080 fibrosarcoma cells.
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PMID:HT-1080 fibrosarcoma cell matrix degradation and invasion are inhibited by the matrix-associated serine protease inhibitor TFPI-2/33 kDa MSPI. 961 Jul 35

Human tissue factor pathway inhibitor-2 (TFPI-2)/matrix-associated serine protease inhibitor (MSPI), a Kunitz-type serine protease inhibitor, inhibits plasmin, trypsin, chymotrypsin, plasma kallikrein, cathepsin G, and factor VIIa-tissue factor complex. The mature protein has a molecular mass of 32-33 kDa, but exists in vivo as two smaller, underglycosylated species of 31 and 27 kDa. TFPI-2/MSPI triplet is synthesized and secreted by a variety of cell types that include epithelial, endothelial, and mesenchymal cells. Because the majority (75-90%) of TFPI-2/MSPI is associated with the extracellular matrix (ECM), we examined which components of the ECM bind TFPI-2/MSPI. We found that TFPI-2/MSPI bound specifically to heparin and dermatan sulfate. Interaction of these two glycosaminoglycans (GAGs) with TFPI-2/MSPI involved one or more common protein domains, as evidenced by cross-competition experiments. However, binding affinity for TFPI-2/MSPI with heparin was 250-300 times greater than that for TFPI-2/MSPI with dermatan sulfate. Binding of TFPI-2/MSPI to GAGs was inhibited by NaCl or arginine but not by glucose, mannose, galactose, 6-aminohexanoic acid, or urea, suggesting that arginine-mediated ionic interactions participate in the GAG binding of TFPI-2/MSPI. This supposition was supported by the observation that only NaCl or arginine could elute the TFPI-2/MSPI protein triplet from an ECM derived from human dermal fibroblasts. Reduced TFPI-2/MSPI did not bind to heparin, suggesting that proper disulfide pairings and conformation are essential for matrix binding. To determine whether heparin modulates the activity of TFPI-2/MSPI, we determined the rate of inhibition of plasmin by the inhibitor with and without heparin and found that TFPI-2/MSPI is more active in the presence of heparin. Collectively, our results demonstrate that conformation-dependent arginine-mediated ionic interactions are responsible for the TFPI-2/MSPI triplet binding to fibroblast ECM, heparin, and dermatan sulfate and that heparin augmented the rate of inhibition of plasmin by TFPI-2/MSPI.
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PMID:Matrix localization of tissue factor pathway inhibitor-2/matrix-associated serine protease inhibitor (TFPI-2/MSPI) involves arginine-mediated ionic interactions with heparin and dermatan sulfate: heparin accelerates the activity of TFPI-2/MSPI toward plasmin. 1049 84

Human tissue factor pathway inhibitor-2 (TFPI-2) is a 32-kDa serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G, and plasma kallikrein but not urokinase and tissue-type plasminogen activators or thrombin. After discovering that TFPI-2 expression is down-regulated or lost during tumor progression, we investigated the role of TFPI-2 in the invasiveness of the prostate cancer cell line (LNCaP). We stably transfected LNCaP cells with a 0.7-kb vector expressing TFPI-2 in the sense orientation and measured the expression of TFPI-2 protein and mRNA by these cells by western and northern blotting. Neither TFPI-2 protein nor mRNA was expressed by parental LNCaP cells or vector-transfected controls, but levels of both protein and mRNA were significantly increased in the sense-TFPI-2 clones. The sense clones were less invasive than the control cells in Matrigel invasion and spheroid migration assays. This is the first demonstration that upregulation of TFPI-2 plays a significant role in the invasive behavior of human prostate cancer cells.
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PMID:Overexpression of tissue factor pathway inhibitor-2 (TFPI-2), decreases the invasiveness of prostate cancer cells in vitro. 1111 49

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G and plasma kallikrein but not urokinase (uPA) or tissue-type plasminogen activator and thrombin. Earlier studies from our and other laboratories have shown that the production of TFPI-2 is downregulated during the progression of various cancers. To investigate the role of TFPI-2 in the invasion and metastasis of lung tumors, the human lung cancer cell line A549, which produces high levels of TFPI-2, was stably transfected with a vector capable of expressing an antisense transcript complementary to the full-length TFPI-2 mRNA. Northern blot analysis was used to quantify the TFPI-2 mRNA transcript, and western blot analysis was used to measure TFPI-2 protein levels in parental cells and stably transfected (vector and antisense) clones. The levels of TFPI-2 mRNA and protein were significantly less in antisense clones than in the parental and vector controls. The invasive potential of the parental cells and stably transfected vector clones in vitro, as measured by the Matrigel invasion assay, was also markedly less than that of antisense clones. Further characterization of these clones showed that more cells migrated from antisense clones than from parental and vector clones. These data suggest that TFPI-2 is critical for the invasion and metastasis of lung cancer and that the downregulation of TFPI-2 production may be a feasible approach to increase invasiveness and metastasis.
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PMID:In vitro modulation of human lung cancer cell line invasiveness by antisense cDNA of tissue factor pathway inhibitor-2. 1131 97

Human tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein (PP5) and matrix-associated serine protease inhibitor (MSPI), is a 32-kDa extracellular matrix (ECM) protein consisting of three tandomly arranged Kunitz-type domains that inhibits plasmin, trypsin, chymotrypsin, cathepsin G and plasma kallikrein but not urokinase and tissue-type plasminogen activators or thrombin. Earlier studies in our laboratory revealed that the production of TFPI-2 is reduced or absent during the tumor progression of human gliomas. In the present study, we investigated the role of TFPI-2 in the invasiveness of the amelanotic melanoma cell line C-32. We stably transfected C-32 cells with a vector capable of expressing TFPI-2 in a sense orientation (0.7 kb). TFPI-2 protein production was then determined by western blotting and the mRNA level by northern blotting in parental and stably transfected (vector and sense) clones. The levels of TFPI-2 protein and mRNA were significantly higher in the sense clones, but neither was detected in parental and vector control clones. In addition, in vitro Matrigel invasion/migration assays revealed that the invasive behavior of sense clones was inhibited compared with the behavior of parental and vector clones. This is the first study to show that the upregulation of TFPI-2 plays a significant role in reducing the invasive behavior of human amelanotic melanomas.
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PMID:Role of tissue factor pathway inhibitor-2 (TFPI-2) in amelanotic melanoma (C-32) invasion. 1144 60

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G, and plasma kallikrein but not urokinase-type plasminogen activator, tissue plasminogen activator, or thrombin. Preliminary findings in our laboratory suggested that the expression of TFPI-2 is downregulated or lost during tumor progression in human gliomas. To investigate the role of TFPI-2 in the invasiveness of brain tumors, we stably transfected the human high-grade glioma cell line SNB19 and the human low-grade glioma cell line Hs683 with a vector capable of expressing a transcript complementary to the full-length TFPI-2 mRNA in either sense (0.7 kb) or antisense (1 kb) orientations. Parental cells and stably transfected cell lines were analysed for TFPI-2 protein by Western blotting and for TFPI-2 mRNA by Northern blotting. The levels of TFPI-2 protein and mRNA were higher in the sense clones (SNB19) and decreased in the antisense (Hs683) clones than in the corresponding parental and vector controls. In spheroid and matrigel invasion assays, the SNB19 parental cells were highly invasive, but the sense-transfected SNB-19 clones were much less invasive; the antisense-transfected Hs683 clones were more invasive than their parental and vector controls. After intracerebral injection in mice, the sense-transfected SNB19 clones were less able to form tumors than were their parental and vector controls, and the antisense-Hs683 clones but not the parental or vector controls formed small tumors. This is the first study to demonstrate that down- or upregulation of TFPI-2 plays a significant role in the invasive behavior of human gliomas.
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PMID:A novel function of tissue factor pathway inhibitor-2 (TFPI-2) in human glioma invasion. 1168 73

Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz-type serine proteinase inhibitor that is secreted by all cells of the vasculature, and presumably plays a role in the regulation of plasmin-mediated matrix remodeling. In this report, we describe the cloning and expression of a full-length cDNA for bovine TFPI-2 that exhibits 72% sequence identity with that of human TFPI-2. Following a 22 residue signal peptide, the mature protein contains 212 amino acids with 18 cysteines, three putative N-glycosylation sites, and one putative O-glycosylation site. The deduced sequence of mature bovine TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxy-terminal tail highly enriched in basic amino acids. Recombinant bovine TFPI-2 was expressed in HEK 293 cells and resolved into two isoforms, designated as alpha-TFPI-2 (M(r) 33 kDa) and beta-TFPI-2 (M(r) 31 kDa), which presumably represent differentially glycosylated forms of the inhibitor. Similar to human TFPI-2, both bovine TFPI-2 isoforms exhibited strong inhibitory activity towards trypsin and plasmin, and weak inhibitory activity towards the factor VIIa-tissue factor complex.
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PMID:Molecular cloning, expression, and characterization of bovine tissue factor pathway inhibitor-2. 1292 85

Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits the plasmin- and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion, and metastasis. To directly assess its role in tumor growth and metastasis in vivo, we stably transfected HT-1080 fibrosarcoma cells expressing either fully active wild-type human TFPI-2 (WT) or inactive R24Q TFPI-2 (QT) and examined their ability to form tumors and metastasize in athymic mice in comparison to mock-transfected cells (MT). MT and QT fibrosarcoma tumors grew 2 to 3 times larger than WT tumors. Tumor metastasis was confined to the lung and was observed in 75% of mice treated with either MT or QT cells, whereas only 42% of mice treated with WT cells developed lung metastases. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of each tumor group revealed 3- to 6-fold lower levels of murine vascular endothelial growth factor gene expression in WT tumors in relation to either MT or QT tumors. Comparative tumor gene expression analysis revealed that several human genes implicated in oncogenesis, invasion, metastasis, apoptosis, and angiogenesis had significantly altered levels of expression in WT tumors. Our collective data demonstrate that secretion of inhibitory TFPI-2 by a highly metastatic tumor cell markedly inhibits its growth and metastasis in vivo by regulating pericellular extracellular matrix (ECM) remodeling and angiogenesis.
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PMID:The effect of human tissue factor pathway inhibitor-2 on the growth and metastasis of fibrosarcoma tumors in athymic mice. 1452 59

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type proteinase inhibitor that regulates a variety of serine proteinases involved in coagulation and fibrinolysis through their non-productive interaction with a P(1) residue (Arg-24) in its first Kunitz-type domain (KD1). Previous kinetic studies revealed that TFPI-2 was a more effective inhibitor of plasmin than several other serine proteinases, but the molecular basis for this specificity was unclear. In this study, we employed molecular modeling and mutagenesis strategies to produce several variants of human TFPI-2 KD1 in an effort to identify interactive site residues other than the P(1) Arg that contribute significantly to its inhibitory activity and specificity. Molecular modeling of KD1 based on the crystal structure of bovine pancreatic trypsin inhibitor revealed that KD1 formed a more energetically favorable complex with plasmin versus trypsin and/or the factor VIIa-tissue factor complex primarily due to strong ionic interactions between Asp-19 (P(6)) and Arg residues in plasmin (Arg-644, Arg-719, and Arg-767), Arg-24 (P(1)) with Asp-735 in plasmin, and Arg-29 (P(5)') with Glu-606 in plasmin. In addition, Leu-26 through Leu-28 (P(2)'-P(4)') in KD1 formed strong van der Waals contact with a hydrophobic cluster in plasmin (Phe-583, Met-585, and Phe-587). Mutagenesis of Asp-19, Tyr-20, Arg-24, Arg-29, and Leu-26 in KD1 resulted in substantial reductions in plasmin inhibitory activity relative to wild-type KD1, but the Asp-19 and Tyr-20 mutations revealed the importance of these residues in the specific inhibition of plasmin. In addition to the reactive site residues in the P(6)-P(5)' region of KD1, mutation of a highly conserved Phe at the P(18)' position revealed the importance of this residue in the inhibition of serine proteinases by KD1. Thus, together with the P(1) residue, the nature of other residues flanking the P(1) residue, particularly at P(6) and P(5)', strongly influences the inhibitory activity and specificity of human TFPI-2.
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PMID:Structure-function analysis of the reactive site in the first Kunitz-type domain of human tissue factor pathway inhibitor-2. 1497 Feb 25


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