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:3.4.21.9 (enterokinase)
675 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Membrane-type serine protease 1 (MT-SP1), identical to matriptase, is a recently identified type II transmembrane serine protease. MT-SP1/matriptase is of considerable interest for the development, homeostasis, and cancer invasion and metastasis of epithelial tissues. The administration of inhibitors for MT-SP1/matriptase may be effective to suppress the development of tumors where the enzyme may be involved. In the present study, we produced a secreted form of recombinant MT-SP1/matriptase (ekMT-SP1s) that can be activated by enterokinase in vitro and investigated the inhibitory ability of various protease inhibitors toward the recombinant enzyme. The enterokinase-treated ekMT-SP1s (active ekMT-SP1s) cleaved various peptidyl-4-methylcoumaryl-7-amide (MCA) substrates with arginine (or lysine) residue at position P1, and the best substrate was t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA. The specificity for the synthetic and natural substrates of the active ekMT-SP1s was in good agreement with that of the natural enzyme. Endogenous protease inhibitors tested, except for antithrombin III, showed no or little inhibition on the cleavage of Boc-Gln-Ala-Arg-MCA by the active ekMT-SP1s. Aprotinin showed strong inhibitory activity toward the cleavage. Food-derived inhibitors, such as soybean trypsin inhibitor, Bowman-Birk inhibitor, and lima bean trypsin inhibitor inhibited it, while chicken ovomucoid did not. Synthetic inhibitors tested inhibited it, and among them, the inhibitory effect of FOY-305 was strongest. The present findings provide important information for the suppression of cancer invasion and metastasis for which MT-SP1/matriptase is responsible.
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PMID:Inhibition of membrane-type serine protease 1/matriptase by natural and synthetic protease inhibitors. 1288 93

Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human enteropeptidase (GenBank Accession No. U09860) was synthesized from 26 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. The fusion protein thioredoxin/human enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms. The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor. The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide with kinetic parameters K(m)=0.16 mM and k(cat)=115 s(-1) and small ester Z-Lys-SBzl with K(m)=140 microM, k(cat)=133 s(-1). L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with K(i)(*)=2.3 nM. L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions.
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PMID:Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli. 1296 50

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 EK-solCorin 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.
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PMID:Functional analysis of the transmembrane domain and activation cleavage of human corin: design and characterization of a soluble corin. 1455 95

To further elucidate the role of proteases capable of cleaving N-terminal proopiomelanocortin (N-POMC)-derived peptides, we have cloned two cDNAs encoding isoforms of the airway trypsin-like protease (AT) from mouse (MAT) and rat (RAT), respectively. The open reading frames comprise 417 amino acids (aa) and 279 aa. The mouse AT gene was located at chromosome 5E1 and contains 10 exons. The longer isoform, which we designated MAT1 and RAT1, has a simple type II transmembrane protein structure, consisting of a short cytoplasmic domain, a transmembrane domain, a SEA (63-kDa sea urchin sperm protein, enteropeptidase, agrin) module, and a serine protease domain. The human homolog of MAT1 and RAT1 is the human AT (HAT). The shorter isoform, designated MAT2 and RAT2, which contains an alternative N terminus, was formerly described in the rat as adrenal secretory serine protease (AsP) and has been shown to be involved in the processing of N-POMC-derived peptides. In contrast to the long isoform, neither MAT2 and RAT2 (AsP) contain a transmembrane domain nor a SEA domain but an N-terminal signal peptide to direct the enzyme to the secretory pathway. The C terminus, covering the catalytic triad, is identical in both isoforms. Immunohistochemically, MAT/RAT was predominantly expressed in tissues of the upper gastrointestinal and the respiratory tract-but also in the adrenal gland. Moreover, isoform-specific RT-PCR and quantitative PCR analysis revealed a complex expression pattern of the two isoforms with differences between mice and rats. These findings indicate a multifunctional role of these proteases beyond adrenal proliferation.
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PMID:The adrenal secretory serine protease AsP is a short secretory isoform of the transmembrane airway trypsin-like protease. 1469 Oct 9

Corin is a type II transmembrane serine protease and functions as the proatrial natriuretic peptide (pro-ANP) convertase in the heart. In the extracellular region of corin, there are two frizzled-like cysteine-rich domains, eight low density lipoprotein receptor (LDLR) repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain at the C terminus. To examine the functional importance of the domain structures in the propeptide of corin for pro-ANP processing, we constructed a soluble corin, EKshortCorin, that consists of only the protease domain and contains an enterokinase (EK) recognition sequence at the conserved activation cleavage site. After being activated by EK, EKshortCorin exhibited catalytic activity toward chromogenic substrates but failed to cleave pro-ANP, indicating that certain domain structures in the propeptide are required for pro-ANP processing. We then constructed a series of corin deletion mutants and studied their functions in pro-ANP processing. Compared with that of the full-length corin, a corin mutant lacking frizzled 1 domain exhibited approximately 40% activity, whereas corin mutants lacking single LDLR repeat 1, 2, 3, or 4 had approximately 49, approximately 12, approximately 53, and approximately 77% activity, respectively. We also made corin mutants with a single mutation at a conserved Asp residue that coordinates Ca(2+)-binding in LDLR repeats 1, 2, 3, or 4 (D300Y, D336Y, D373Y, and D410Y) and showed that these mutants had approximately 25, approximately 11, approximately 16, and approximately 82% pro-ANP processing activity, respectively. Our results indicate that frizzled 1 domain and LDLR repeats 1-4 are important structural elements for corin to recognize its physiological substrate, pro-ANP.
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PMID:Identification of domain structures in the propeptide of corin essential for the processing of proatrial natriuretic peptide. 1519 93

We report the identification and functional analysis of a type II transmembrane serine protease encoded by the mouse differentially expressed in squamous cell carcinoma (DESC) 1 gene, and the definition of a cluster of seven homologous DESC1-like genes within a 0.5-Mb region of mouse chromosome 5E1. This locus is syntenic to a region of human chromosome 4q13.3 containing the human orthologues of four of the mouse DESC1-like genes. Bioinformatic analysis indicated that all seven DESC1-like genes encode functional proteases. Direct cDNA cloning showed that mouse DESC1 encodes a multidomain serine protease with an N-terminal signal anchor, a SEA (sea urchin sperm protein, enterokinase, and agrin) domain, and a C-terminal serine protease domain. The mouse DESC1 mRNA was present in epidermal, oral, and male reproductive tissues and directed the translation of a membrane-associated 60-kDa N-glycosylated protein with type II topology. Mouse DESC1 was synthesized in insect cells as a zymogen that could be activated by exposure to trypsin. The purified activated DESC1 hydrolyzed synthetic peptide substrates, showing a preference for Arg in the P1 position. DESC1 proteolytic activity was abolished by generic inhibitors of serine proteases but not by other classes of protease inhibitors. Most interestingly, DESC1 formed stable inhibitory complexes with both plasminogen activator inhibitor-1 and protein C inhibitor that are expressed in the same tissues with DESC1, suggesting that type II transmembrane serine proteases may be novel targets for serpin inhibition. Together, these data show that mouse DESC1 encodes a functional cell surface serine protease that may have important functions in the epidermis, oral, and reproductive epithelium.
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PMID:Mouse DESC1 is located within a cluster of seven DESC1-like genes and encodes a type II transmembrane serine protease that forms serpin inhibitory complexes. 1532 53

Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoietic origin, notably mast cells. gamma-Tryptase, a recently described member of the family also known as transmembrane tryptase (TMT), is a membrane-bound serine protease found in the secretory granules or on the surface of degranulated mast cells. The 321 amino acid protein contains an 18 amino acid propeptide linked to the catalytic domain (cd), followed by a single-span transmembrane domain. gamma-Tryptase is distinguished from other human mast cell tryptases by the presence of two unique cysteine residues, Cys(26) and Cys(145), that are predicted to form an intra-molecular disulfide bond linking the propeptide to the catalytic domain to form the mature, membrane-anchored two-chain enzyme. We expressed gamma-tryptase as either a soluble, single-chain enzyme with a C-terminal His tag (cd gamma-tryptase) or as a soluble pseudozymogen activated by enterokinase cleavage to form a two-chain protein with an N-terminal His tag (tc gamma-tryptase). Both recombinant proteins were expressed at high levels in Pichia pastoris and purified by affinity chromatography. The two forms of gamma-tryptase exhibit comparable kinetic parameters, indicating the propeptide does not contribute significantly to the substrate affinity or activity of the protease. Substrate and inhibitor library screening indicate that gamma-tryptase possesses a substrate preference and inhibitor profile distinct from that of beta-tryptase. Although the role of gamma-tryptase in mast cell function is unknown, our results suggest that it is likely to be distinct from that of beta-tryptase.
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PMID:Expression and characterization of recombinant gamma-tryptase. 1681 34

Polyserase-1 (polyserine protease-1)/TMPRSS9 (transmembrane serine protease 9) is a type II transmembrane serine protease (TTSP) that possesses unique three tandem serine protease domains. However, the physiological function of each protease domain remains poorly understood. We discovered a new splice variant of polyserase-1, termed Serase-1B, which contains 34 extra amino acids consisting a SEA module (a domain found in sea urchin sperm protein, enterokinase and agrin) adjacent to the transmembrane domain and the first protease domain with a mucin-like box at the C-terminus. The tissue distribution of this enzyme by RT (reverse transcription)-PCR analysis revealed high expression in the liver, small intestine, pancreas, testis and peripheral blood CD14+ and CD8+ cells. To investigate the role of Serase-1B, a full-length form recombinant protein was produced. Interestingly, recombinant Serase-1B was partly secreted as a soluble inactive precursor and it was also activated by trypsin. This activated enzyme selectively cleaved synthetic peptides for trypsin and activated protein C, and it was inhibited by several natural serine protease inhibitors, such as aprotinin, alpha2-antiplasmin and plasminogen activator inhibitor 1. In addition, Serase-1B efficiently converted pro-uPA (urokinase-type plasminogen activator) into active uPA and this activation was strongly inhibited by these natural inhibitors. Furthermore, this activation was also negatively regulated by glycosaminoglycans. Our results indicate that Serase-1B is a novel member of TTSPs that might be involved in uPA/plasmin-mediated proteolysis and possibly implicated in biological events such as fibrinolysis and tumour progression.
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PMID:Serase-1B, a new splice variant of polyserase-1/TMPRSS9, activates urokinase-type plasminogen activator and the proteolytic activation is negatively regulated by glycosaminoglycans. 1687 79

We cloned two distinct cDNAs for enteropeptidase (EP) from the intestine of the medaka, Oryzias latipes, which is a small freshwater teleost. The mRNAs code for EP-1 (1,036 residues) and EP-2 (1,043 residues), both of which have a unique, conserved domain structure of the N-terminal heavy chain and C-terminal catalytic serine protease light chain. When compared with mammalian EP serine proteases, the medaka enzyme exhibited extremely low amidolytic activity for small synthetic peptide substrates. Twelve mutated forms of the medaka EP protease were produced by site-directed mutagenesis. Among them, one mutant protease, E173A, was found to have considerably reduced nonspecific hydrolytic activities both for synthetic and protein substrates without serious reduction of its Asp-Asp-Asp-Asp-Lys (D(4)K)-cleavage activity. For the cleavage of fusion proteins containing a D(4)K-cleavage site, the medaka EP proteases were shown to have advantages over their mammalian counterparts. Based on our present data, we propose that the E173A mutant is the most appropriate protease to specifically cleave proteins containing the D(4)K cleavage sequence.
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PMID:Specificity of the medaka enteropeptidase serine protease and its usefulness as a biotechnological tool for fusion-protein cleavage. 1743 97

TTSPs [type II TMPRSSs (transmembrane serine proteases)] are a growing family of trypsin-like enzymes with, in some cases, restricted tissue distribution. To investigate the expression of TTSPs in the nervous system, we performed a PCR-based screening approach with P10 (postnatal day 10) mouse spinal cord mRNA. We detected the expression of five known TTSPs and identified a novel TTSP, which we designated neurobin. Neurobin consists of 431 amino acids. In the extracellular part, neurobin contains a single SEA (sea-urchin sperm protein, enterokinase and agrin) domain and a C-terminal serine protease domain. RT-PCR (reverse transcription-PCR) analysis indicated the expression of neurobin in spinal cord and cerebellum. Histochemical analysis of brain sections revealed distinct staining of Purkinje neurons of the cerebellum. Transiently overexpressed neurobin was autocatalytically processed and inserted into the plasma membrane. Autocatalytic activation could be suppressed by mutating Ser(381) in the catalytic pocket to an alanine residue. The protease domain of neurobin, produced in Escherichia coli and refolded from inclusion bodies, cleaved chromogenic peptides with an arginine residue in position P(1). Serine protease inhibitors effectively suppressed the proteolytic activity of recombinant neurobin. Ca2+ or Na+ ions did not significantly modulate the catalytic activity of the protease. Recombinant neurobin processed 17-kDa FGF-2 (fibroblast growth factor-2) at several P(1) lysine and arginine positions to distinct fragments, in a heparin-inhibitable manner, but did not cleave FGF-7, laminin or fibronectin. These results indicate that neurobin is an authentic TTSP with trypsin-like activity and is able to process FGF-2 in vitro.
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PMID:Neurobin/TMPRSS11c, a novel type II transmembrane serine protease that cleaves fibroblast growth factor-2 in vitro. 1821 25


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