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)

Previously we isolated a trypsin-like enzyme designated human airway trypsin-like protease from the sputum of patients with chronic airway diseases. This paper describes the cDNA cloning, characterization of the primary protein structure deduced from the cDNA, and gene expression of this enzyme in various human tissues. We obtained an entire 1517-base pair sequence of cDNA with an open reading frame encoding a polypeptide with 418-amino acid residues. The polypeptide consisted of a 232-residue catalytic region and a 186-residue noncatalytic region with a hydrophobic putative transmembrane domain near the NH2 terminus. The polypeptide was suggested to be a type II integral membrane protein in which the COOH-terminal catalytic region is extracellular. Therefore, this protein is thought to be synthesized as a membrane-bound precursor and to mature to a soluble and active protease by limited proteolysis. It showed 29-38% identity in the sequence of the catalytic region with human hepsin, enteropeptidase, acrosin, and mast cell tryptase. The noncatalytic region had little similarity to other known proteins. In Northern blot analysis a transcript of 1.9 kilobases was detectable most prominently in the trachea among 17 human tissues examined.
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PMID:Cloning and characterization of the cDNA for human airway trypsin-like protease. 956 16

Enteropeptidase inhibitor (DI) was isolated from bovine duodenum during purification of this enzyme. DI was purified by affinity chromatography on immobilised trypsin. DI preparations contain two main components: DI-9 (9 kD) and DI-20 (20 kD). The N-terminal amino acid sequence 1-19 of DI-9 is highly homologous to the Kunitz inhibitor (BPI). Molecular weights of DI-9 and BPI are the same (gel electrophoresis data). Fragment 1-19 of DI-9 differs from the corresponding region of BPI only at the position 17: DI-9 contains Ala-17 instead of Arg in BPI. The homology of N-terminal amino acid sequence 1-25 of DI-20 with the corresponding regions of some phospholipases A2 suggests that this protein is a new intestinal phospholipase A2. Inhibitor DI-9 and phospholipase DI-20 are probably isolated in a common lipoprotein complex. The only earlier known in vitro inhibitor of enteropeptidase, BPI, was localised in vivo in different tissues with this enzyme. In our opinion the Kunitz-type inhibitor DI-9 is, a physiological inhibitor of enteropeptidase.
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PMID:[An inhibitor of enteropeptidases and trypsin from the bovine duodenum]. 984 20

Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence [M. Matsushima et al. (1994) J. Biol. Chem. 269, 19976-19982]. In the present study, we purified the porcine enzyme approximately 2,200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.
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PMID:Purification and further characterization of enteropeptidase from porcine duodenum. 1022 May 88

Enteropeptidase is a membrane-bound serine protease that initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. The enzyme is remarkably specific and cleaves after lysine residues of peptidyl substrates that resemble trypsinogen activation peptides such as Val-(Asp)4-Lys. To characterize the determinants of substrate specificity, we solved the crystal structure of the bovine enteropeptidase catalytic domain to 2.3 A resolution in complex with the inhibitor Val-(Asp)4-Lys-chloromethane. The catalytic mechanism and contacts with lysine at substrate position P1 are conserved with other trypsin-like serine proteases. However, the aspartyl residues at positions P2-P4 of the inhibitor interact with the enzyme surface mainly through salt bridges with the Nzeta atom of Lys99. Mutation of Lys99 to Ala, or acetylation with acetic anhydride, specifically prevented the cleavage of trypsinogen or Gly-(Asp)4-Lys-beta-naphthylamide and reduced the rate of inhibition by Val-(Asp)4-Lys-chloromethane 22 to 90-fold. For these reactions, Lys99 was calculated to account for 1.8 to 2.5 kcal mol(-1) of the free energy of transition state binding. Thus, a unique basic exosite on the enteropeptidase surface has evolved to facilitate the cleavage of its physiological substrate, trypsinogen.
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PMID:Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide. 1049 81

Duodenase, a serine proteinase from bovine Brunner's (duodenal) glands that was predicted to be a natural activator of enteropeptidase zymogen, cleaves and activates recombinant single-chain bovine proenteropeptidase (kcat/Km = 2700 M(-1) s(-1)). The measured rate of proenteropeptidase cleavage by duodenase was about 70-fold lower compared with the rate of trypsin-mediated cleavage of the zymogen. The role of duodenase is supposed to be the primary activator of proenteropeptidase maintaining a certain level of active enteropeptidase in the duodenum. A new scheme of proteolytic activation cascade of digestive proteases is discussed.
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PMID:Activation of recombinant proenteropeptidase by duodenase. 1068 47

Many types of human tumor express trypsinogen-2, which may be a significant factor in the activation of pro-MMPs and the invasiveness of tumors. Prevention of trypsinogen-2 expression in cancer cells might be of benefit in cancer therapy. We describe here chemicals capable of down-regulating the expression of trypsinogen-2. Doxycycline (DOXY) and chemically modified tetracyclines (CMTs), previously known as inhibitors of the matrix metalloproteinase (MMP)-dependent proteinase cascade, down-regulated the mRNA and protein expression of trypsinogen-2 by COLO-205 human colon adenocarcinoma cells at therapeutically attainable concentrations (0. 1 to 1.0 microM). DOXY specifically inhibited the activation of pro-MMP-9 and cell migration induced by enteropeptidase, a specific activator of trypsinogen. Pro-MMP-9 activation and cell migration were also inhibited by tumor-associated trypsin inhibitor (TATI), which is a highly specific inhibitor of trypsin. CMT-3 as well as CMT-5 also inhibited cell migration, but an effect on the enteropeptidase-enhanced activation of pro-MMP-9 was not observed. Our results indicate that CMTs, DOXY and TATI inhibit cancer cell migration by down-regulating trypsinogen-2 expression or activity. Inhibition of trypsinogen-2 expression may represent a mechanism contributing to the ability of CMTs to suppress the pericellular proteolytic activity of some tumors.
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PMID:Down-regulation of trypsinogen-2 expression by chemically modified tetracyclines: association with reduced cancer cell migration. 1079 74

This review covers the unique catalytic and molecular properties of three proteolytic enzymes and a glycosidase from Aspergillus. An aspartic proteinase from A. saitoi, aspergillopepsin I (EC 3.4.23.18), favors hydrophobic amino acids at P1 and P'1 like gastric pepsin. However, aspergillopepsin I accommodates a Lys residue at P1, which leads to activation of trypsinogens like duodenum enteropeptidase. Substitution of Asp76 to Ser or Thr and deletion of Ser78, corresponding to the mammalian aspartic proteinases, cathepsin D and pepsin, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at 1. In addition, the double mutant T77D/G78(S)G79 of porcine pepsin was able to activate bovine trypsinogen to trypsin by the selective cleavage of the K6-I7 bond of trypsinogen. Deuterolysin (EC 3.4.24.39) from A. oryzae, which contains 1g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. It was concluded that His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn binding assay. Deuterolysin is a member of a family of metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand. Acid carboxypeptidase (EC 3.4.16.1) from A. saitoi is a glycoprotein that contains both N- and O-linked sugar chains. Site-directed mutagenesis of the cpdS, cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. A. saitoi carboxypeptidase indicated that Ser153, Asp357, and His436 residues were essential for the enzymic catalysis. The N-glycanase released high-mannose type oligosaccharides that were separated on HPLC. Two, which had unique structures of Man10 GlcNAc2 and Man11GlcNAc2, were characterized. An acidic 1,2-alpha-mannosidase (EC 3.2.1.113) was isolated from the culture of A. saitoi. A highly efficient overexpression system of 1,2-alpha-mannosidase fusion gene (f-msdS) in A. oryzae was made. A yeast mutant capable of producing Man5GlcNAc2 human-compatible sugar chains on glycoproteins was constructed. An expression vector for 1,2-alpha-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. The first report of production of human-compatible high mannose-type (Man5GlcNAc2) sugar chains in S. cerevisiae was described.
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PMID:Unique catalytic and molecular properties of hydrolases from Aspergillus used in Japanese bioindustries. 1083 Apr 77

Trypsinogen is a serine proteinase produced mainly by the pancreas, but it has recently been found to be expressed also in several cancers such as ovarian and colon cancer and in vascular endothelial cells. In this study, we found that trypsinogen-1 and -2 are present at high concentrations (median levels, 0.4 and 0.5 mg/L, respectively) in human seminal fluid and purified them to homogeneity by immunoaffinity and anion exchange chromatography. Purified trypsinogen isoenzymes displayed a M(r) of 25 to 28 kd in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Most of the trypsinogen-1 purified from seminal fluid was enzymatically active whereas trypsinogen-2 occurred as the proform, which could be activated by enteropeptidase in vitro. Immunohistochemically, trypsinogen protein was detected in the human prostate, urethra, utriculus, ejaculatory duct, seminal vesicles, deferent duct, epididymal glands, and testis. Expression of trypsinogen mRNA in the same organs was demonstrated by in situ hybridization. Trypsinogen mRNA was also detected in the prostate and seminal vesicles by reverse transcriptase-polymerase chain reaction and Northern blotting. Isolated trypsin was shown to activate the proenzyme form of prostate-specific antigen. These results suggest that trypsinogen isoenzymes found in seminal fluid are produced locally in the male genital tract and that they may play a physiological role in the semen.
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PMID:Expression and characterization of trypsinogen produced in the human male genital tract. 1110 74

Enteropeptidase (enterokinase) is a serine protease highly specific for recognition and cleavage of the target sequence of Asp-Asp-Asp-Asp-Lys (D4K). The three-dimensional structure of the enteropeptidase shows that the N-terminal amino acid is buried inside the protein providing molecular interactions necessary to maintain the conformation of the active site. To determine the influence of the N-terminal amino acid of enteropeptidase light chain (EK(L)) on the enzymatic activity, we constructed various mutants including 17 different single amino acid substitutions and three different extensions at the N-terminal end. The mutants of recombinant enteropeptidase (rEK(L)) were expressed in Saccharomyces cerevisiae and secreted into culture medium. Among 20 different mutants tested, the only mutant with the Ile --> Val substitution exhibited significant activity. The kinetic properties of the mutant protein were very similar to those of the wild-type rEK(L). Based on the three-dimensional structure where the N-terminal Ile is oriented into hydrophobic pocket, the results suggest that Val could substitute Ile without affecting the active conformation of the enzyme. The results also explain why all trypsin-like serine proteases carry either Ile or Val at the N-termini and none other amino acid residues are found. Moreover, this finding provides a mental framework for expressing the N-terminally engineered enteropeptidase in Escherichia coli, utilizing the known property of the methionine aminopeptidase that exhibits poor activity toward the N-terminal Met-Ile bond, but offers efficient cleavage of the Met-Val bond.
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PMID:Engineered recombinant enteropeptidase catalytic subunit: effect of N-terminal modification. 1191 64

Enteropeptidase (enterokinase) (EC 3.4.21.9), a highly specific processing protease, initiating a cascade of reactions activating the digestion enzymes. Catalyzing trypsinogen activation enteropeptidase exhibits unique properties for high efficiency hydrolysis of the polypeptide chain after lysine-15 residue in the -DDDDK15- sequence. In 1998 we found an unusual calcium-dependent autolysis of the enteropeptidase heavy chain leading to the drastic loss of its activity towards trypsinogen: after lysine-360 (-NNYEK360-INCN-), -), arginine-384 (-NEWER384-TQGS-), arginine-422 (-GRRER422-VGLL-) and lysine-465 (-QNMEK465-TIFQ-) residues. We used hepta-nona-peptides as the model substrates for autolysys: human angiotensin II--DRVYIHPF and cattle hemoglobin b-chain fragments: LTAEEKA and MLTAEEKAA. Kinetic parameters of enteropeptidase hydrolysis for these substrates were determined. Recent study demonstrates the ability of enteropeptidase to hydrolyze peptide bonds formed by carboxyl groups of Lys or Arg residues if less than four but at least one negative charged amino acid residue is in any of substrate P2-P5 positions. Ca(2+)-dependent autolysis of enteropeptidase heavy chain and of trypsin were compared; the second one serves as the natural defense mechanism against the undesirable premature proenzymes activation in pancreas leading to pancreatitis. The corresponding enteropeptidase inactivation in low Ca2+ environment ought to be the component of the same protective mechanism.
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PMID:[Hydrolysis by enteropeptidase of nonspecific (model) peptide sequences and possible physiological role of this phenomenon]. 1269 55


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