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)

Enteropeptidase (EP) is a serine proteinase and activates trypsinogen to trypsin, thus playing an important role in food digestion. Nevertheless, the localization of EP is still controversial, likely due to a lack of studies using specific antibodies against EP. The aim of this study was to define cellular localization of EP in human duodenum and expression in tumor cells at the duodenal region. Immunohistochemical staining for resected tissues was performed with two antibodies against recombinant EP light and heavy chains, respectively. In situ hybridization was done with two RNA probes that include either the light or the heavy chain sequences of proEP, respectively. The two antibodies reacted with enterocytes, accentuated on the brush border, and goblet cells, with increasing intensity from the bottom of crypts to the top of villi. Paneth cells, neuroendocrine cells, Brunner's glands, lymphocytes, smooth muscle, or connective tissue did not react with the antibodies. The two RNA probes detected EP mRNA expression only in enterocytes and goblet cells. EP is produced in enterocytes and goblet cells, and the localization on the brush border of the cells is reasonable for the physiological activation of digestive enzymes. Interestingly, the antibodies reacted with tumor cells in duodenal polyps and adenocarcinoma at the duodenum but not in Brunner's gland adenoma. EP seems to be a marker of differentiated enterocytes and goblet cells, which suggests the existence of a common progenitor of these cells. Furthermore, EP may be a useful marker of tumor cells originating from these cells.
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PMID:Expression of enteropeptidase in differentiated enterocytes, goblet cells, and the tumor cells in human duodenum. 1290 31

Certain serine proteases signal to cells by cleaving protease-activated receptors (PARs) and thereby regulate hemostasis, inflammation, pain and healing. However, in many tissues the proteases that activate PARs are unknown. Although pancreatic trypsin may be a physiological agonist of PAR(2) and PAR(4) in the small intestine and pancreas, these receptors are expressed by cells not normally exposed pancreatic trypsin. We investigated whether extrapancreatic forms of trypsin are PAR agonists. Epithelial cells lines from prostate, colon, and airway and human colonic mucosa expressed mRNA encoding PAR(2), trypsinogen IV, and enteropeptidase, which activates the zymogen. Immunoreactive trypsinogen IV was detected in vesicles in these cells. Trypsinogen IV was cloned from PC-3 cells and expressed in CHO cells, where it was also localized to cytoplasmic vesicles. We expressed trypsinogen IV with an N-terminal Igkappa signal peptide to direct constitutive secretion and allow enzymatic characterization. Treatment of conditioned medium with enteropeptidase reduced the apparent molecular mass of trypsinogen IV from 36 to 30 kDa and generated enzymatic activity, consistent with formation of trypsin IV. In contrast to pancreatic trypsin, trypsin IV was completely resistant to inhibition by polypeptide inhibitors. Exposure of cell lines expressing PAR(2) and PAR(4) to trypsin IV increased [Ca(2+)](i) and strongly desensitized cells to PAR agonists, whereas there were no responses in cells lacking these receptors. Thus, trypsin IV is a potential agonist of PAR(2) and PAR(4) in epithelial tissues where its resistance to endogenous trypsin inhibitors may permit prolonged signaling.
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PMID:Trypsin IV, a novel agonist of protease-activated receptors 2 and 4. 1472 24

A comparative study of secondary specificities of enteropeptidase and trypsin was performed using peptide substrates with general formula A-(Asp/Glu)n-Lys(Arg)-(downward arrow)-B, where n = 1-4. This was the first study to demonstrate that, similar to other serine proteases, enteropeptidase has an extended secondary binding site interacting with 6-7 amino acid residues surrounding the peptide bond to be hydrolyzed. However, in the case of typical enteropeptidase substrates containing four negatively charged Asp/Glu residues at positions P2-P5, electrostatic interaction between these residues and the secondary site Lys99 of the enteropeptidase light chain is the main factor that determines hydrolysis efficiency. The secondary specificity of enteropeptidase differs from the secondary specificity of trypsin. The chromophoric synthetic enteropeptidase substrate G5DK-F(NO2)G (kcat/Km = 2380 mM(-1) x min(-1)) is more efficient than the fusion protein PrAD4K-P26 (kcat/Km = 1260 mM(-1) x min(-1)).
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PMID:Study of secondary specificity of enteropeptidase in comparison with trypsin. 1537 72

We previously reported that gill group IB secretory phospholipase A(2) (sPLA(2)) exists as an inactive pro-sPLA(2) with the dipeptide Ala-Arg, at the N-terminus of mature sPLA(2) in mucous cells. Pro-sPLA(2) should be activated after being secreted to the surface of gill epithelia by trypsin-like protease. To clarify the above hypothesis, we investigated the existence of pro-sPLA(2) activating protease (PAP) in the gills of the red sea bream, using gill pro-sPLA(2) as a substrate. PAP was solubilized from the membrane fraction of the gills with 2% sodium cholate and partially purified by benzamidine-Sepharose chromatography and reversed-phase HPLC. Partially purified proteases, PAP1 and PAP2 showed a high molecular mass of about 200 kDa by gelatin zymography. PAP1 and PAP2 had optimal pH from 7 to 9 and were inhibited by trypsin inhibitors. These properties of PAP1 and PAP2 suggest that both enzymes belong to the membrane-associated trypsin-like serine protease family, such as enteropeptidase and corin. This is the first report verifying the existence of the activating protease of group IB pro-sPLA(2) isoforms in a non-digestive tissue.
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PMID:Partial purification and characterization of pro-phospholipase A2 activating proteases from gill membranes of the red sea bream, Chrysophrys major. 1582 Jan 42

The activation peptide of vertebrate trypsinogens contains a highly conserved tetra-aspartate sequence (Asp(19-22) in humans) preceding the Lys-Ile scissile bond. A large body of research has defined the primary role of this acidic motif as a specific recognition site for enteropeptidase, the physiological activator of trypsinogen. In addition, the acidic stretch was shown to contribute to the suppression of autoactivation. In the present study, we determined the relative importance of these two activation peptide functions in human cationic trypsinogen. Individual Ala replacements of Asp(19-22) had minimal or no effect on trypsinogen activation catalyzed by human enteropeptidase. Strikingly, a tetra-Ala(19-22) trypsinogen mutant devoid of acidic residues in the activation peptide was still a highly specific substrate for human, but not for bovine, enteropeptidase. In contrast, an intact Asp(19-22) motif was critical for autoactivation control. Thus, single Ala mutations of Asp(19), Asp(20) and Asp(21) resulted in 2-3-fold increased autoactivation, whereas the Asp(22) --> Ala mutant autoactivated at a 66-fold increased rate. These effects were multiplicative in the tri-Ala(19-21) and tetra-Ala(19-22) mutants. Structural modeling revealed that the conserved hydrophobic S2 subsite of trypsin and the unique Asp(218), which forms part of the S3-S4 subsite, participate in distinct inhibitory interactions with the activation peptide. Finally, mutagenesis studies confirmed the significance of the negative charge of Asp(218) in autoactivation control. The results demonstrate that in human cationic trypsinogen the Asp(19-22) motif per se is not required for enteropeptidase recognition, whereas it is essential for maximal suppression of autoactivation. The evolutionary selection of Asp(218), which is absent in the large majority of vertebrate trypsins, provides an additional mechanism of autoactivation control in the human pancreas.
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PMID:The tetra-aspartate motif in the activation peptide of human cationic trypsinogen is essential for autoactivation control but not for enteropeptidase recognition. 1597 May 97

The effects of calcium ions on hydrolysis of low molecular weight substrates catalyzed by different forms of enteropeptidase were studied. A method for determining activity of truncated enteropeptidase preparations lacking a secondary trypsinogen binding site and displaying low activity towards trypsinogen was developed using N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (Z-Lys-S-Bzl). The kinetic constants for hydrolysis of this substrate at pH 8.0 and 25 degrees C were determined for natural enteropeptidase (K(m) 59.6 microM, k(cat) 6660 min(-1), k(cat)/K(m) 111 microM(-1) x min(-1)), as well as for enteropeptidase preparation with deleted 118-783 fragment of the heavy chain (K(m) 176.9 microM, k(cat) 6694 min(-1), k(cat)/K(m) 37.84 microM(-1) x min(-1)) and trypsin (K(m) 56.0 microM, k(cat) 8280 min(-1), k(cat)/K(m) 147.86 microM(-1) x min(-1)). It was shown that the enzymes with trypsin-like primary active site display similar hydrolysis efficiency towards Z-Lys-S-Bzl. Calcium ions cause 3-fold activation of hydrolysis of the substrates of general type GD(4)K-X by the natural full-length enteropeptidase. In contrast, the hydrolysis of substrates with one or two Asp/Glu residues at P2-P3 positions is slightly inhibited by Ca2+. In the case of enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 fragment), the activating effect of calcium ions was not detected for all the studied substrates. The results of hydrolysis experiments with synthetic enteropeptidase substrates GD(4)K-F(NO(2))G, G(5)DK-F(NO(2))G (where F(NO(2)) is p-nitrophenyl-L-phenylalanine residue), and GD(4)K-Nfa (where Nfa is beta-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions.
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PMID:Effect of calcium ions on enteropeptidase catalysis. 1627 Oct 29

The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.
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PMID:Biochemical characterization of human enteropeptidase light chain. 1648 14

Compatible solutes are small organic osmoprotectants that have the capability to stabilize proteins. In coupled assays, the effect of the solutes ectoine, hydroxyectoine and betaine on the activation of the zymogens trypsinogen and chymotrypsinogen, catalyzed by enteropeptidase and trypsin, respectively, was studied. To different extents, all solutes protected the zymogens against activation. Ectoine (800 mM) was the most potent solute in reducing the formation of trypsin to 4% of the control value and of chymotrypsin to 23%. In separate experiments, the ability of the solutes to preserve proteolytic activity during incubation was investigated. After 4 h, trypsin and chymotrypsin completely lost their activity, but in the presence of ectoine, approximately 50% residual activity was maintained. It is proposed that a conformational shift of the protein towards folded, native-like states induced by preferential exclusion of the solute is responsible for the stabilizing and chaperone-like effects.
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PMID:Compatible solutes as protectants for zymogens against proteolysis. 1679 60

Comparative substrate analysis of full-length bovine enteropeptidase and trypsin, bovine and human enteropeptidase light chains was performed using model N-terminal dodecapeptides corresponding to wild-type human trypsinogen and pancreatitis-associated mutant trypsinogens K23R and D22G. The substitution of Lys residue by Arg at P1 leads to 2-fold increase in the efficiency of enteropeptidase hydrolysis; the absence of the negatively charged residue at P2 reduces the efficiency of such hydrolysis by two orders of magnitude. The difference in efficiency of peptide chain hydrolysis after Lys/Arg residues by enteropeptidase compared to trypsin is equal to the difference in hydrolysis by serine proteases of different primary specificity of their specific substrates.
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PMID:The ways of realization of high specificity and efficiency of enteropeptidase. 1734 25

DESC1 was identified using gene-expression analysis between squamous cell carcinoma of the head and neck and normal tissue. It belongs to the type II transmembrane multidomain serine proteinases (TTSPs), an expanding family of serine proteinases, whose members are differentially expressed in several tissues. The biological role of these proteins is currently under investigation, although in some cases their participation in specific functions has been reported. This is the case for enteropeptidase, hepsin, matriptase and corin. Some members, including DESC1, are associated with cell differentiation and have been described as tumor markers. TTSPs belong to the type II transmembrane proteins that display, in addition to a C-terminal trypsin-like serine proteinase domain, a differing set of stem domains, a transmembrane segment and a short N-terminal cytoplasmic region. Based on sequence analysis, the TTSP family is subdivided into four subfamilies: hepsin/transmembrane proteinase, serine (TMPRSS); matriptase; corin; and the human airway trypsin (HAT)/HAT-like/DESC subfamily. Members of the hepsin and matriptase subfamilies are known structurally and here we present the crystal structure of DESC1 as a first member of the HAT/HAT-like/DESC subfamily in complex with benzamidine. The proteinase domain of DESC1 exhibits a trypsin-like serine proteinase fold with a thrombin-like S1 pocket, a urokinase-type plasminogen activator-type S2 pocket, to accept small residues, and an open hydrophobic S3/S4 cavity to accept large hydrophobic residues. The deduced substrate specificity for DESC1 differs markedly from that of other structurally known TTSPs. Based on surface analysis, we propose a rigid domain association for the N-terminal SEA domain with the back site of the proteinase domain.
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PMID:Crystal structure of the catalytic domain of DESC1, a new member of the type II transmembrane serine proteinase family. 1738 11


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