Proline-specific Endopeptidase Research Articles

Use of a proline-specific endopeptidase to reintroduce gluten in patients with non-coeliac gluten sensitivity: A randomized trial

A gluten-free diet (GFD) is the main therapy for non-coeliac gluten sensitivity (NCGS). However, the availability of novel enzymes with the ability to digest gluten could represent a therapeutic opportunity for NCGS patients to avoid a GFD. To evaluate the controlled reintroduction of gluten with or without the endopeptidase P1016 in NCGS patients. This is a randomized, double-blind, placebo-controlled monocentric study, Registered under ClinicalTrials.gov Identifier no. NCT01864993. Gluten was reintroduced incrementally over a 3-week period under nutritional control. NCGS patients were randomized into two groups and administered P1016 or placebo during gluten reintroduction. We evaluated symptoms (visual analogue scale, VAS), quality of life (SF-36) and mental health symptoms (SCL-90) on a weekly basis. We enrolled a total 23 patients who were allocated to a placebo group (n=11, age 38.4±2.9) or an intervention group (n=12, age 39.5±3.1). No effect of P1016 on symptoms was found. During gluten reintroduction, patients reported a significant increase in abdominal pain and a worsening of stool consistency. Furthermore, no differences were found between the groups regarding SCL-90 and SF-36 scores. Our results demonstrate a lack of effect of P1016 in the management of NCGS patients and the possible reintroduction of gluten.

Altered hippocampal gene expression, glial cell population, and neuronal excitability in aminopeptidase P1 deficiency

Inborn errors of metabolism are often associated with neurodevelopmental disorders and brain injury. A deficiency of aminopeptidase P1, a proline-specific endopeptidase encoded by the Xpnpep1 gene, causes neurological complications in both humans and mice. In addition, aminopeptidase P1-deficient mice exhibit hippocampal neurodegeneration and impaired hippocampus-dependent learning and memory. However, the molecular and cellular changes associated with hippocampal pathology in aminopeptidase P1 deficiency are unclear. We show here that a deficiency of aminopeptidase P1 modifies the glial population and neuronal excitability in the hippocampus. Microarray and real-time quantitative reverse transcription-polymerase chain reaction analyses identified 14 differentially expressed genes (Casp1, Ccnd1, Myoc, Opalin, Aldh1a2, Aspa, Spp1, Gstm6, Serpinb1a, Pdlim1, Dsp, Tnfaip6, Slc6a20a, Slc22a2) in the Xpnpep1−/− hippocampus. In the hippocampus, aminopeptidase P1-expression signals were mainly detected in neurons. However, deficiency of aminopeptidase P1 resulted in fewer hippocampal astrocytes and increased density of microglia in the hippocampal CA3 area. In addition, Xpnpep1−/− CA3b pyramidal neurons were more excitable than wild-type neurons. These results indicate that insufficient astrocytic neuroprotection and enhanced neuronal excitability may underlie neurodegeneration and hippocampal dysfunction in aminopeptidase P1 deficiency.

Successive digestion of tilapia collagen by serine proteinase and proline specific endopeptidase to produce novel angiotensin I-converting enzyme inhibitory peptides

Serine proteinase, purified from the hepatopancreas of Pacific white shrimp (Litopenaeus vannamei), was used to hydrolyze acid solubilized collagen (ASC) isolated from Nile tilapia (Oreochromis sp.) skin to produce angiotensin I-converting enzyme (ACE) inhibitory peptides (ACEIPs). A series of column chromatography assays were used to separate the ACEIPs. A peptide, NPARTCR, was isolated as it exhibited high ACE inhibition potential. Further digestion of this peptide by a proline specific endopeptidase (PSEP), produced a pentapeptide ARTCR with ACE inhibitory activity (IC50) of 77.0 μmol/L. Both NPARTCR and ARTCR inhibited ACE in a non-competitive manner. An in vivo study in rats demonstrated that ARTCR has ACE inhibitory activity via lowering systolic blood pressure in spontaneously hypertensive rats (SHRs). These results suggest that processing by-products from shrimp and tilapia are ideal raw materials for the production of serine proteinase and collagen, respectively. Serine proteinase and collagen are both ideal raw materials that can be used to derive ACE inhibitory active peptides against hypertension.

Treatment of both native and deamidated gluten peptides with an endo-peptidase from Aspergillus niger prevents stimulation of gut-derived gluten-reactive T cells from either children or adults with celiac disease.

Celiac disease (CD) is characterized by an inappropriate immunological reaction against gluten driven by gluten-specific CD4+ T cells. We screened 25 proteases and tested 10 for their potential to degrade gluten in vitro. Five proteases were further tested for their ability to prevent the proliferative response by a gluten-specific CD4+ T cell clone and seven gluten-reactive T cell lines to protease-digested gluten peptides. A proline-specific endo-peptidase from Aspergillus niger (AnP2) was particularly efficient at diminishing proliferation after stimulation with cleaved antigen, and could completely block the response against both native and deamidated gluten peptides. We found that AnP2 was efficient down to a 1:64 protease:substrate ratio (w:w). When AnP2 was tested in assays using seven gluten-reactive T cell lines from individual CD patients (three adults and four children), the response to gluten was diminished in all cases. Our study indicates a therapeutic benefit of AnP2 to CD patients.

Gene cloning and enzymatic characterization of an endoprotease Endo-Pro-Aspergillus niger

A novel endoprotease Endo-Pro-Aspergillus niger (endoprotease EPR) was first successfully expressed at high level in the methylotrophic yeast Pichia pastoris and the purification procedure was established. The endoprotease EPR is 95 % identity with proline specific endopeptidase from A. niger CBS513.88 (EMBL; AX458699), while sharing low identity with those from other microorganisms. The purified endoprotease EPR was a monomer of 60 kDa. Furthermore, the peptide mass fingerprinting (PMF) analysis confirmed that the purified protein was an endoprotease Endo-Pro-Aspergillus niger. A three-dimensional model revealed that the active site of the enzyme was located in Ser(179)-Asp(458)-His(491), based on template 3n2zB with sequence identity of 17.6 %. The optimum pH and temperature of the endoprotease EPR were pH 4-5 and 35 °C, and the stabilities were pH 3-7 and 15-60 °C, respectively. Furthermore, the endoprotease EPR had the ability to digest peptides with the C-terminal of proline as well as alanine, and was also capable of hydrolyzing larger peptides. The properties of the endoprotease EPR made it a highly promising candidate for future application in the field of brewing and food process.

Degree of hydrolysis of some vegetable proteins used as fining agents and its influence on polyphenol removal from red wine

The increasing demand for economic and efficient wine fining leads to the search for alternatives to traditionally used proteins, preferably new products with the side effect of non-allergenicity. A comparative fining trial on laboratory scale with equal quantities of vegetable proteins, partly used for the first time as fining agents for wine in Germany, was accomplished. Spectrum of wheat, rice, maize and potato proteins was expanded by enzymatic hydrolysis, additionally to evaluate influence of protein structure on fining effects. Proteins were modified by two different enzymes, proline-specific endopeptidase (PeP) and alcalase. Molecular weight distributions and degree of hydrolysis (DH) were compared to color intensity and HPLC data of phenolic marker substances primarily responsible for bitterness and astringency. With a total of 20 different varieties of fining agents, statistical evaluation was performed for a rapid exploratory analysis of taste improvement without the need for sensorial tests. Overall, influence on phenol composition and color intensity decreased with the degree of proteolysis. The effect of maize proteins and corresponding hydrolyzates was rather low, whereas maize proteins were nearly not influenced by the use of 2 different peptidases. Wheat proteins with highest proline content (about 9–10%) and molecular weight fractions of 14 and 30–45 kDa had a pronounced effect on color intensity. Rice and potato proteins with only half of proline content and molecular weight fractions mainly ranging between 10 and 32 kDa showed less effect on wine color, but preferably on phenolic marker compounds mainly responsible for bitter and astringent taste sensations.

Sulfated chitooligosaccharides as prolyl endopeptidase inhibitor

Prolyl endopeptidase (PEP, EC 3.4.21.26) is a proline-specific endopeptidase with a serine-type mechanism, which digests small peptide-like hormones, neuroactive peptides, and various cellular factors. PEP has been involved in neurodegenerative disorders, therefore, the discovery of PEP inhibitors can revert memory loss caused by amnesic compounds. In this study, we prepared hetero-chitooligosaccharides (COSs) with different molecular sizes using ultrafiltration (UF) membrane reactor system from hetero-chitosan with different degrees of deacetylation (DD; 90%, 75% and 50% deacetylation), and synthesized sulfated COSs (SCOSs). PEP inhibitory activities of SCOSs were evaluated and the results showed that 50% deacetylated SCOSs (50-SCOSs) exhibited higher inhibitory activities than those of 90% and 75% deacetylated SCOSs (90-SCOSs and 75-SCOSs). Among the 50-SCOSs (50-SCOS I, 5000–10,000 Da; 50-SCOS II, 1000–5000 Da; 50-SCOS III, below 1000 Da), 50-SCOS II possessed the highest inhibitory activity and IC 50 value was 0.38 mg/ml. Kinetics studies with 50-SCOS II indicated a competitive enzyme inhibition with a K i value of 0.78 mg/ml. It was concluded that the 50-SCOS II may be useful for PEP inhibitor and for developing a new type PEP inhibitor from carbohydrate based materials.

Chemical Synthesis of Human β-Defensin (hBD)-1, -2, -3 and -4: Optimization of the Oxidative Folding Reaction

Reduced human β-defensin (hBD)-1, -2, -3 and -4 synthesized by Boc chemistry were subjected to oxidative folding reaction under optimal conditions. Among the factors affecting the oxidative folding in the presence of reduced and oxidized glutathione (GSH/GSSG), the buffer concentration and reaction temperature were essential for the predominant formation of the native disulfide structure. The homogeneity of the four synthetic hBDs was confirmed by analytical procedures using RP-HPLC, IEX-HPLC, capillary zone electrophoresis (CZE) and MALDI-TOF MS as well as sequencing, although high temperature (70 °C) was used for the RP-HPLC analysis of hBD-3 and hBD-4 to exclude the influence of equilibrium with the respective conformers having native disulfide pairing. All synthetic hBDs were shown to possess the native disulfide structure by sequential analyses and mass measurements with cystine segments obtained by enzymatic digestion. Upon digestion of hBD-1 and hBD-4 with proline specific endopeptidase, the Cys-X bond was found to be reproducibly cleaved together with the Pro-X bond although the cleavage of Cys-X afforded the appropriate cystine segments for determining the disulfide structure of hBD-1 and hBD-4. With respect to antimicrobial activity against E. coli, the four synthetic hBDs of high homogeneity possessed the same potencies as those reported previously.

Potent antagonistic action of synthetic analogues of APGWGNamide, an antagonist of molluscan neuropeptide APGWamide

Fifty-five kinds of analogues of APGWGNamide (Ala–Pro–Gly–Trp–Gly–Asn–NH 2), which is an antagonist of molluscan neuropeptide APGWamide, were synthesized and their antagonistic activities were examined on two molluscan smooth muscles. Among all the analogues tested, on spontaneous contraction of the crop of the land snail, Euhadra congenita, APGWG( l-biphenylalanine, Bip)amide showed the most potent antagonistic activity and its potency was 50–100 times higher than that of APGWGNamide. Likewise, on phasic contraction of the anterior byssus retractor muscle (ABRM) of the sea mussel, Mytilus edulis, the effect of APGWG( d-homophenylalanine, dHfe) was the most potent and showed 5–10 times stronger activity than that of APGWGNamide . In the tolerance test to known exo- and endopeptidases or the crop tissue homogenate, APGWGNamide was not only easily degraded by a proline-specific endopeptidase but also by the homogenate. Two kinds of potent antagonists were thus developed: APGWG(Bip)amide and APGWG(dHfe)amide, which will be useful tools for investigation of the function of APGWamide in the snail and the mussel, respectively.

Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties

The major N-linked carbohydrate structures were determined for recombinant human plasma lecithin:cholesterol acyltransferase (LCAT). The analysis of the structure of oligosaccharides by fast atom bombardment mass spectrometry (FAB-MS) and linkage analysis was preceded by reduction and carboxymethylation of the intact glycoproteins and digestion with trypsin and proline specific endopeptidase. The N-glycans were subsequently released from the glycopeptides by PNGase F digestion and the oligosaccharides were separated using a C18 Sep-pak® cartridge. The data from the combination of FAB spectrometry and linkage analysis show that the N-linked glycans present on recombinant LCAT (rLCAT) were composed primarily of triantennary and tetraantennary structures with and without core fucosylation. A minor population of glycans (less than 5%) contained up to three repeats of N-acetyllactosamine in one or more antennae. The LCAT activities of both recombinant and circulating forms of plasma LCAT were determined using low molecular weight and lipoprotein substrates. The catalytic behavior of these two enzyme forms were found to be very similar if not identical. These findings validate the concept that the recombinant enzyme can serve as an appropriate model for structure/function studies of LCAT and provide the foundation for subsequent structural studies.—Lacko, A. G., A. J. Reason, C. Nuckolls, B. J. Kudchodkar, M. P. Nair, G. Sundarrajan, P. H. Pritchard, H. R. Morris, and A. Dell. Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties. J. Lipid Res. 1998. 39: 807–820.

Engineering cyclophilin into a proline-specific endopeptidase.

Designing an enzyme requires, among a number of parameters, the appropriate positioning of catalytic machinery within a substrate-binding cleft. Using the structures of cyclophilin-peptide complexes, we have engineered a new catalytic activity into an Escherichia coli cyclophilin by mutating three amino acids, close to the peptide binding cleft, to form a catalytic triad similar to that found in serine proteases. In conjunction with cyclophilin's specificity for proline-bearing peptides, this creates a unique endopeptidase, cyproase 1, which cleaves peptides on the amino-side of proline residues. When acting on an Ala-Pro dipeptide, cyproase 1 has an efficiency (kcat/Km) of 0.7 x 10(4) M(-1) s(-1) and enhances the rate of reaction (kcat/kuncat) 8 x 10(8)-fold. This activity depends upon a deprotonated histidine and is inhibited by nucleophile-specific reagents, as occurs in natural serine proteases. Cyproase 1 can hydrolyse a protein substrate with a proline-specific endoprotease activity.

Structure-activity relationships of boronic acid inhibitors of dipeptidyl peptidase IV. 1. Variation of the P2 position of Xaa-boroPro dipeptides.

A series of prolineboronic acid (boroPro) containing dipeptides were synthesized and assayed for their ability to inhibit the serine protease dipeptidyl peptidase IV (DPPIV). Inhibitory activity, which requires the (R)-stereoisomer of boroPro in the P1 position, appears to tolerate a variety of L-amino acids in the P2 position. Substitution at the P2 position which is not tolerated include the D-amino acids, alpha,alpha-disubstituted amino acids, and glycine. Specificity against DPPII and proline specific endopeptidase is reported. A correlation between the ability to inhibit DPPIV in cell culture and in the human mixed lymphocyte reaction is demonstrated. A synthesis of prolineboronic acid is reported as well as conditions for generating the fully unprotected boronic acid dipeptides in either their cyclic or acyclic forms.

Enzymatic peptide synthesis by the recombinant proline-specific endopeptidase from Flavobacterium meningosepticum and its mutationally altered Cys-556 variant.

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) was investigated for its potential as a catalyst in peptide synthesis. Using an activated peptide ester or a peptide amide as the acyl component, the enzyme catalyzed kinetically controlled aminolysis and transpeptidation respectively, with various amino acid amides as acyl acceptors. To a certain extent the nucleophile preference reflected the amino acid preference in the S1'-position of the enzyme in peptide hydrolysis: the highest fractions of aminolysis were obtained using amino acid amides with hydrophobic side-chains (e.g. Leu-NH2, Phe-NH2). PSE also catalyzed the thermodynamically controlled condensation of short peptides with a free carboxyterminus and various amino acid amides. This enabled us to examine the acceptance of different acyl components in the substrate-binding site of the enzyme with regard to their amino acid composition: In the S1 position proline was clearly favored, but alanine was also accepted, whereas the S2 subsite accepted various amino acids rather unspecifically. Since PSE was shown to be extremely sensitive against water-miscible organic solvents, an alternative approach was used to increase yields in enzymatic peptide synthesis: a derivative of PSE in which the catalytic Ser-556 is converted to a Cys was constructed by protein engineering. This mutant (PSEcys) exhibited a dramatically increased peptide ligase activity in aqueous solution.

Rapid purification of proline-specific endopeptidase fromFlavobacterium meningosepticum heterologously expressed inEscherichia coli

Two fast and efficient purification methods for the preparation of large amounts of proline-specific endopeptidase (PSE) [EC 3.4.21.26] fromFlavobacterium meningosepticum heterologously expressed inEscherichia coli are described. Overproduction and accumulation of PSE in the periplasmic space ofE. coli means that a single gel chromatography step or ion exchange chromatography step was sufficient to obtain homogenously pure PSE preparations. With these procedures, up to 490 μg of purified enzyme per gE. coli cells were obtained. The different purification methods are discussed.

Enzymatic peptide synthesis by the recombinant proline-specific endopeptidase from Flavobacterium meningosepticum and its mutationally altered Cys-556 variant

Enzymatic peptide synthesis by the recombinant proline-specific endopeptidase from Flavobacterium meningosepticum and its mutationally altered Cys-556 variant

Determination of three disulfide bonds in a major house dust mite allergen, Der f II.

Der f II is a major mite allergen consisting of 129 amino acid residues. Der f II contains six cysteine residues, suggesting the existence of three disulfide bonds which would stabilize this small protein. As the first step in revealing the relationship between the structure and the allergenic property of Der f II, the formation of disulfide bonds was examined. Der f II purified from Dermatophagoides farinae was treated with lysyl-endopeptidase or proline-specific endopeptidase, and the peptide fragments thus generated were separated by reverse phase high performance liquid chromatography. Determination of the amino acid sequence of each peptide collected in this way proved the existence of three disulfide bonds between Cys8 and Cys119, Cys21 and Cys27, and Cys73 and Cys78.

Thyrotropin‐Releasing Hormone Mediates Growth Hormone Release Induced by Milk and Nursing in Neonatal Rats

Previous evidence from this laboratory suggested that growth hormone (GH) release induced by milk in vitro and by nursing in vivo from neonatal rat pituitary glands is mediated by an alternative GH-releasing factor(s) (GRF) distinct from GH-releasing hormone (GHRH(1-43) ). In the present experiments we tested whether thyrotropin-releasing hormone (TRH) could fulfil the criteria of this alternative GRF in neonatal rats. The water-soluble fraction of rat milk (infranatant, prepared by ultracentrifugation) and its methanol/acetic acid extract (milk-borne peptides) stimulated GH release from perifused pituitary glands obtained from 2-day-old rats. Dialysis of the infranatant (mol wt cut-off: 2,000) against 500 volumes of culture medium at 4°C eliminated its GH-releasing activity in the perifusion system, while the infranatant retained its full GRF-like activity when incubated at 4°C without dialysis. The milk-borne GRF eluted as a single peak and coeluted with TRH in a combined gel permeation chromatography (Sephadex G-10) and perifusion set-up. Prolactin secretion was also stimulated simultaneously with the release of GH induced either by milk or by TRH. In a stepwise C(18) reversed-phase chromatography, milk-borne GRF was highly hydrophilic and coeluted with synthetic TRH. The in vitro GH-releasing bioactivities of synthetic TRH and a milk extract purified in C(18) reversed-phase chromatography were abolished by proline-specific endopeptidase. Thus, TRH and milk-borne GRF displayed similar molecular weights, hydropathic characteristics and proteolytic enzyme resistance. In vivo, nursing (which has been reported as a potent stimulus of GH secretion even in the absence of milk-intake) increased serum GH levels in 2-day-old pups. A supramaximal dose of TRH (10 ng/g intraperitoneally) stimulated GH release in 2-day-old pups separated from their mothers for 6 h to a similar extent as nursing. Nursing-induced levels of serum GH were not further elevated by TRH. This failure of TRH to further increase serum GH levels was not due to a maximal GH output by the neonatal pituitary gland, since the GH release induced by the serotonin precursor 5-hydroxy-L-tryptophan was augmented either by TRH or by nursing. These data provide evidence that the milk-borne GRF-like activity in vitro is indistinguishable from TRH, and suggest that TRH (probably of hypothalamic origin) might be the mediator of the nursing-induced release of GH in vivo as a physiological GRF in neonatal rats.

Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme

A three-disulfide form of hen egg white lysozyme with Cys6 and Cys127 blocked by carboxymethyl groups was prepared, purified, and characterized for eventual use in protein folding experiments. Trypsin digestion followed by proline-specific endopeptidase digestion facilitated the unambiguous assignment of the disulfide bond pairings and the modified residues in this derivative. 3SS-lysozyme demonstrated nearly full enzymatic activity at its pH optimum, pH 5.5. The 3SS-lysozyme derivative and unmodified lysozyme were shown to be identical by CD spectroscopy at pH 3.6. Immunochemical binding assays demonstrated that the conformation of lysozyme was perturbed predominantly only locally by breaking and blocking the disulfide bond between Cys6 and Cys127. Both 3SS-lysozyme and unmodified lysozyme exhibited reversible thermally induced transitions at pH 2.0, but the Tm of 3SS-lysozyme, 18.9 degrees C, was found to be 34 degrees lower than that of native lysozyme under the same conditions. The conformational chemical potential of the denatured form of unmodified lysozyme was determined from the transition curves to be approximately 6.7 kcal/mol higher than that of the denatured form of 3SS-lysozyme, at pH 2.0 and 35 degrees C, if the conformational chemical potential for the folded forms of both 3SS-lysozyme and unmodified lysozyme is arbitrarily assumed to be 0.0 kcal/mol. A calculation of the increase in the theoretical loop entropy of denatured 3SS-lysozyme resulting from the cleavage of the Cys6-Cys127 disulfide bond, however, yielded a value of only 5.4 kcal/mol for the difference in conformational chemical potential. This suggests that, in addition to the entropic component, there is also an enthalpic contribution to the difference in the conformational chemical potential corresponding to approximately 1.3 kcal/mol. Thus, it is concluded that the reduction and blocking of the disulfide bond between Cys6 and Cys127 destabilizes 3SS-lysozyme relative to unmodified lysozyme predominantly by stabilizing the denatured conformation by increasing its chain entropy.

Differences in defining residues relevant to antibody binding by ELISA and proteolysis protection at the level of peptide antigenic determinants

The interaction of monoclonal antibody (mAb) 1C3 with bovine β-casein was analyzed by two methods. The competitive enzyme-linked immunosorbent assay in which mAb 1C3 bound competitively to a peptide in solution or β-casein adsorbed to wells in microtitration plates showed that β-casein (f194–200) (peptide of residues 194 to 200 of β-casein) was the shortest peptide among the tested peptides that had binding ability for mAb 1C3. Protection against proteinase digestion was analyzed by incubating proline-specific endopeptidase, carboxypeptidase Y or aminopeptidase T with β-casein (f193–202) in the presence of mAb 1C3 or nonspecific mAb 31A4. Proteolysis of peptide bonds between residues 200 and 201, and 201 and 202 was depressed in the presence of mAb 1C3. However, peptide bonds between 193 and 194, and 194 and 195 were cleaved in the presence of mAb 1C3 as easily as in the presence of mAb 31A4, suggesting that the region of residues 200 to 202 was obscured by, or within the antibody binding site, but that the region of residues 193 to 195 was not. The apparent antibody binding site shown from the protection against proteolysis by mAb was clearly not identical to the shortest antigen peptide, β-casein (f194–200), indicated from the competitive binding assay.

An improved noninfections murine skin model of organized granulomatous inflammation

An improved model of granulomatous inflammation in skin was developed by second passage skin grafting of isolated, lyophilized skin granulomas, originally elicited in naive mice by inoculations of lyophilized hepatic schistosome egg granulomas. The tissue reaction is caused by a single exposure to a noninfectious, acellular granulomagenic stimulus and occurs in healthy mice free of systemic disease. The model should prove useful for isolation of granuloma initiation factor(s). Furthermore, because there is a time lag before new granuloma formation begins, a window exists for analytical dissection of the initiation process. In this study we described the responses of host cells by autoradiography, and light and electron microscopy. The activity of angiotensin-converting enzyme and proline-specific endopeptidase showed a modulation during granuloma formation. In addition we found that severe immunosuppression with high dose cyclosporine therapy did not alter granuloma formation, supporting the idea that initiation of organized granulomas is T-cell independent.