cDNA Cloning and Molecular Modeling of Procerain B,a Novel Cysteine Endopeptidase Isolated from Calotropis procera

Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimensional structure of active procerain B was modeled by homology modeling using X-ray crystal structure of actinidin (PDB ID: 3P5U), a cysteine protease from the fruits of Actinidia arguta. The structural aspect of the enzyme is also discussed.     

Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii

A serine protease, named as "Milin" was purified to homogeneity from the latex of Euphorbia milii, a medicinal plant of Euphorbiaceae family. The molecular mass (SDS-PAGE), optimum pH and temperature of the enzyme were 51kDa, pH 8.0 and 60 degrees C, respectively. Milin retains full proteolytic activity over a wide range of pH (5.5-12) and temperature (up to 65 degrees C) with casein and azoalbumin as substrates. The activity of milin is inhibited by serine proteases inhibitors like PMSF, APMSF and DFP, but not by any other protease inhibitors such as E-64 and PCMB. Like the other serine proteases from the genus Euphorbia, the activity of milin was not inhibited by the proteinaceous inhibitor soyabean trypsin inhibitor (SBTI) even at very high concentrations that is naturally present in plants. The specific extinction coefficient (epsilon(280 nm)(1%)), molar extinction coefficient (a(m)) and isoelectric point of the enzyme were found to be 29, 152,500 M(-1) cm(-1) and pH 7.2, respectively. The enzyme is a glycoprotein with detectable carbohydrate moiety (7-8%) in its constitution, which is essential for the activity. The numbers of tryptophan, tyrosine and cysteine residues in the sequence of milin were estimated chemically and are 23, 14 and 14, respectively. Of the 14-cysteine residues, 12 constituted 6-disulfide linkages while two are free cysteines. The N-terminal sequence (first 12 amino acid residues) was determined and does not match with any sequence of known plant serine proteases. Perturbation studies by temperature, pH and chaotropes of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Thus, this serine protease may have potential applications in food industry.     

Purification and characterization of a novel protease from the latex of Pedilanthus tithymaloids

A novel protease was purified to homogeneity from the latex of Pedilanthus tithymaloids by a simple purification procedure involving ammonium sulfate precipitation and cation-exchange chromatography. The molecular weight of the protease was estimated to be approximately 63.1 kDa and the extinction coefficient (epsilon(1%)(280nm)) was 28.4. The enzyme hydrolyzes denatured natural substrates like casein, azoalbumin and azocasein with a high specific activity but little activity towards synthetic substrates. The pH and temperature optima were pH 8.0-9.5 and 65-70 degrees C, respectively. The proteolytic activity of the enzyme was inhibited by different protease-specific inhibitors (e.g., thiol, serine, metallo, etc.) up to a certain extent but not completely by any class of inhibitors. The enzyme was relatively stable towards pH change, temperature, denaturants and organic solvents. The enzyme consists of five disulfide bridges compared to three observed in most plant cysteine proteases. Overall, the striking features of this protease are its high molecular weight, high cysteine content and only partial inhibition of activity by different classes of protease inhibitors contrary to known proteases from other plant sources. The enzyme is named as pedilanthin as per the protease nomenclature.     

Purification of a novel cysteine protease,procerain B,from Calotropis procera with distinct characteristics compared to procerain

Proteases have applications in food, detergent and pharmaceutical industries. A novel protease has been purified from the latex of Calotropis procera and characterized. As another cysteine protease, procerain, is reported from the same source, the newly purified enzyme was named as procerain B. The enzyme shows distinct properties compared to procerain, in terms of cleavage recognition site, immunological properties and other physical properties like molecular weight, isoelectric point, etc. The newly purified enzyme shows a broad optimum pH (6.5–8.5) as well as broad optimum temperature (40–60 °C). Additionally, the enzyme retains its activity where most of other proteases are not active. Moreover, the enzyme appeared to be very efficient in hydrolysis of blood stain and may have potential application in detergent industries. Simple and economic purification of procerain B, together with easy availability of latex, makes the large-scale production of procerain B possible, thus enables to explore various industrial as well as biotechnological applications.     

Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult

Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively.     

Differences in the unfolding of procerain induced by pH, guanidine hydrochloride, urea, and temperature

The structural and functional aspects along with equilibrium unfolding of procerain, a cysteine protease from Calotropis procera, were studied in solution. The energetic parameters and conformational stability of procerain in different states were also estimated and interpreted. Procerain belongs to the alpha + beta class of proteins. At pH 2.0, procerain exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is predominantly a beta-sheet conformation and exhibits strong ANS binding. GuHCl and temperature denaturation of procerain in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts in the molecular structure of procerain, possibly domains, with different stability that unfolds in steps. Moreover, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. At lower pH, procerain unfolds to the acid-unfolded state, and a further decrease in the pH drives the protein to the A state. The presence of 0.5 M salt in the solvent composition directs the transition to the A state while bypassing the acid-unfolded state. GuHCl-induced unfolding of procerain at pH 3.0 seen by various methods is cooperative, but the transitions are noncoincidental. Besides, a strong ANS binding to the protein is observed at low concentrations of GuHCl, indicating the presence of an intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8 M), procerain retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to unfolding by urea at lower pH, and the transitions are cooperative and coincidental. Further, the properties of the molten globule-like state and the intermediate state are different, but both states have the same conformational stability. This indicates that these intermediates may be located on parallel folding routes of procerain.     

Indicain, a dimeric serine protease from Morus indica cv. K2

A high molecular mass serine protease has been purified to homogeneity from the latex of Morus indica cv. K2 by the combination of techniques of ammonium sulfate precipitation, hydrophobic interaction chromatography, and size-exclusion chromatography. The protein is a dimer with a molecular mass of 134.5 kDa and with two monomeric subunits of 67.2 kDa and 67.3 (MALDI-TOF), held by weak bonds susceptible to disruption on exposure to heat and very low pH. Isoelectric point of the enzyme is pH 4.8. The pH and temperature optima for caseinolytic activity were 8.5 and 80 degrees C, respectively. The extinction coefficient (epsilon280(1%)) of the enzyme was estimated as 41.24 and the molecular structure consists of 52 tryptophan, 198 tyrosine and 42 cysteine residues. The enzyme activity was inhibited by phenylmethylsulfonylflouride, chymostatin and mercuric chloride indicating the enzyme to be a serine protease. The enzyme is fairly stable and similar to subtilases in its stability toward pH, strong denaturants, temperature, and organic solvents. Polyclonal antibodies specific to enzyme and immunodiffusion studies reveal that the enzyme has unique antigenic determinants. The enzyme has activity towards broad range of substrates comparable to those of subtilisin like proteases. The N-terminal residues of indicain (T-T-N-S-W-D-F-I-G-F-P) exhibited considerable similarity to those of other known plant subtilases, especially with cucumisin, a well-characterized plant subtilase. This is the first report of purification and characterization of a subtilisin like dimeric serine protease from the latex of M. indica cv. K2. Owing to these unique properties the reported enzyme would find applications in food and pharma industry.     

Ginger rhizome as a potential source of milk coagulating cysteine protease

A milk coagulating protease was purified ∼10.2-fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, together with ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was estimated to be ∼36 kDa by SDS-PAGE, and exhibited a pI of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of ∼60 °C. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg²⁺ and Cu²⁺. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher for α_s-casein followed by β- and κ-casein. The purified enzyme differed in molecular mass, pI, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry.     

Purification and biochemical characterization of a highly active cysteine protease ervatamin A from the latex of Ervatamia coronaria

Ervatamia coronaria, a flowering plant (family Apocynaceae) indigenous to India, has medicinally important applications. A search for biochemical constituents of the latex of the plant yielded at least three thiol proteases with distinctly different properties. One of them, a highly active protease (ervatamin A), was purified to homogeneity by ion exchange and gel filtration chromatography. The enzyme exhibited high proteolytic activity toward natural substrates and amidolytic activity toward synthetic substrates. The pH and temperature optima for proteolytic activity were 8–8.5 and 50–55°C, respectively. Proteolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. The estimated molecular mass of the enzyme was 27.6 kDa. The extinction coefficient (1% 280) of the enzyme was estimated as 21.9, and the protein molecule consists of 8 tryptophan, 11 tyrosine and 7 cysteine residues. Isoelectric point of the purified enzyme was 8.37. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and a typical color in ELISA. The N-terminal sequence of the enzyme showed conserved amino acid residues to other plant cysteine proteases. Ervatamin A shows high activity in relation to the other thiol proteases isolated from the same source.     

Granulosain I,a Cysteine Protease Isolated from Ripe Fruits of <Emphasis Type="Italic">Solanum granuloso</Emphasis><Emphasis Type="Italic">-leprosum</Emphasis> (Solanaceae)

A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.     

The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis

The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.     

A novel extracellular protease from Pseudomonas aeruginosa MCM B-327: enzyme production and its partial characterization

The focus of this study was on production, purification and characterization of dehairing protease from Pseudomonas aeruginosa MCM B-327, isolated from vermicompost pit soil. Optimum protease activity, 395 U mL(-1), was observed in the medium containing soybean meal and tryptone, at pH 7 and 30 °C. The crude enzyme exhibited dehairing activity. As compared to chemical method, enzymatic method of dehairing showed reduction in COD, TDS and TSS by 34.28%, 37.32% and 51.58%, respectively. Zymogram of crude enzyme on native-PAGE presented two bands with protease activity of molecular weights of 56 and 67 kDa. Both proteases showed dehairing activity. Out of these, 56kDa protease (PA02) was purified 3.05-folds with 2.71% recovery. The enzyme was active in pH range 7-9 and temperature 20-50 °C with optimum pH of 8 and temperature 35°C. Moreover, the enzyme activity of PA02 protease was not strongly inhibited by specific inhibitor showing the novel nature of enzyme compared to serine, cysteine, aspartyl and metalloproteases. Kinetic studies indicated that substrate specificity of PA02 protease was towards various natural and synthetic proteolytic substrates but inactive against collagen and keratin. These findings suggest protease secreted by P. aeruginosa MCM B-327 may have application in dehairing for environment-friendly leather processing.     

Purification and characterization of a serine protease from Cucumis trigonus Roxburghi

Kachri fruit, Cucumis trigonus Roxburghi, contains high protease activity and has been used as meat tenderizer in the Indian subcontinent. A 67 kDa serine protease from Kachri fruit was purified by DEAE-Sepharose and CM-Sepharose chromatography, whose optimum activity was at pH 11 and 70 degrees C. Its activity was strongly inhibited by PMSF, but not by EDTA, pepstatin, or cysteine protease inhibitors. The substrate specificity of the purified protease towards synthetic peptides was comparable to cucumisin, the first characterized subtilisin class plant protease from the sarcocarp of melon fruit (Cucumis melo). These characteristics, along with the N-terminal amino acid sequence, indicated that the isolated protease from Cucumis trigonus Roxburghi is a cucumisin homologue, which belongs to the serine protease family.     

Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding

Streptococcus pyogenes, an important pathogen in humans, secretes an IgG specific endopeptidase named IdeS. To elucidate the mechanism that is responsible for this specificity, we have here characterized the activity of IdeS in detail. Both gamma chains of human IgG or its Fc fragment were cleaved in the hinge region after Gly236 by IdeS, but other proteins or synthetic peptides containing sequences such as the P(4)-P(1) segment in the IgG cleavage site, or long peptides resembling the IgG hinge, were not hydrolyzed at all. This is likely due to a second binding site interacting with the Fc part of IgG. The lack of IdeS activity on peptide substrates necessitated the development of an assay with IgG as the substrate for kinetic studies. IdeS showed a sigmoidal velocity curve at physiological IgG concentrations, and a declining enzyme rate at higher IgG concentrations. This atypical velocity curve suggests product inhibition and/or allosteric control, which again indicates the presence of an exosite involved in substrate binding. The pseudoequilibrium constant for IdeS hydrolysis of IgG was 90 microM. The enzyme exhibited activity in the pH range of 5.1-7.6, with an optimum at pH 6.6. IdeS was stable above pH 10 but not at acidic pH. It exhibited an activity maximum around 37 degrees C and a decreased thermal stability at 42 degrees C. Iodoacetate and iodoacetamide inhibited IdeS, as expected for a cysteine protease, and biochemical evidence verified this classification. E-64 and chicken cystatin, specific inhibitors of family C1 and C13 cysteine proteases, were without effect on enzyme activity, as were class specific serine, aspartic, and metallo protease inhibitors. No significant similarities were found in protein sequence comparisons with known enzyme families, suggesting that IdeS represents a novel family of cysteine proteases.     

Characterization of the proteolytic system present in Vasconcellea quercifolia latex

Vasconcellea quercifolia (Caricaceae) latex contains several cysteine endopeptidases with high proteolytic activity. Cysteine endopeptidases are the main active compounds used by the plant as a defense mechanism. A proteolytic preparation from V. quercifolia (“oak leaved papaya”) latex was purified by cation exchange chromatography. From SDS-PAGE and blotting of the selected fractions, the N-terminal amino acid sequences of polypeptides were determined by Edman’s degradation. The analysis by peptide mass fingerprinting (PMF) of the enzymes allowed their characterization and confirmed the presence of seven different cysteine proteinases in the latex of V. quercifolia. Moreover, the comparison between the tryptic maps with those deposited in databases using the MASCOT tool showed that none of the isolated proteases matched with another plant protease. Notably, a propeptidase was detected in the plant latex, which is being the first report in this sense. Furthermore, the cDNA of one of the cysteine proteases that is expressed in the latex of V. quercifolia was cloned and sequenced. The consensus sequence was aligned using the ClustalX web server, which allowed detecting a high degree of identity with cysteine proteases of the Caricaceae family and establishing the evolutionary relationship between them. We also observed a high conservation degree for those amino acid residues which are essential for the catalytic activity and tridimensional structure of the plant proteases belonging to the subfamily C1A. The PMF analysis strongly suggests that the sequence obtained corresponds to the VQ-III peptidase.     

Characterization of a cysteine protease from wheat Triticum aestivum (cv. Giza 164)

Enzymes, especially proteases, have become an important and indispensable part of the processes used by the modern food and feed industry to produce a large and diversified range of products for human and animal consumption. A cysteine protease, used extensively in the food industry, was purified from germinated wheat Triticum aestivum (cv. Giza 164) grains through a simple reproducible method consisting of extraction, ion exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 61000+/-1200-62000+/-1500 by SDS-PAGE and gel filtration. The cysteine protease had an isoelectric point and pH optimum at 4.4 and 4.0, respectively. The enzyme exhibited more activity toward azocasein than the other examined substrates with K(m) 2.8+/-0.15 mg azocasein/ml. In addition, it had a temperature optimum of 50 degrees C and based on a heat stability study 55% of its initial activity remained after preincubation of the enzyme at 50 degrees C for 30 min prior to substrate addition. All the examined metal cations inhibited the enzyme except Co(2+), Mg(2+), Mn(2+) and Li(+). The proteolytic activity of the enzyme was inhibited by thiol-specific inhibitors, whereas iodoacetate and p-hydroxymercuribenzoate caused a competitive inhibition with Ki values 6+/-0.3 mM and 21+/-1.2 microM, respectively. Soybean trypsin inhibitor had no effect on the enzyme. The enzyme activity remained almost constant for 150 days of storage at -20 degrees C. The properties of this enzyme, temperature and pH optima, substrate specificity, stability and sensitivity to inhibitors or activators, meet the prerequisites needed for food industries.     

Protease activity of 80 kDa protein secreted from the apicomplexan parasite Toxoplasma gondii

This study describes the characterization of 80 kDa protease showing gelationlytic property among three proteases in the excretory/secretory proteins (ESP) from Toxoplasma gondii. The protease activity was detected in the ESP but not in the somatic extract of RH tachyzoites. This protease was active only in the presence of calcium ion but not other divalent cationic ions such as Cu(2+), Zn(2+), Mg(2+), and Mn(2+), implying that Ca(2+) is critical factor for the activation of the protease. The 80 kDa protease was optimally active at pH 7.5. Its gelatinolytic activity was maximal at 37 degrees C, and significant level of enzyme activity of the protease remained after heat treatment at 56 degrees C for 30 min or 100 degrees C for 10 min. This thermostable enzyme was strongly inhibited by metal chelators, i.e., EDTA, EGTA, and 1,10- phenanthroline. Thus, the 80 kDa protease in the ESP secreted by T. gondii was classified as a calcium dependent neutral metalloprotease.     

A novel protease from Entamoeba histolytica homologous to members of the family S28 of serine proteases

Serine proteases are one of the biologically most important and widely distributed enzyme families. A protease capable of degrading the substrate Suc-AAF-AMC was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified by ion-exchange chromatography and electroelution, and appeared on 2D-PAGE as a spot of 60 kDa and pI of 4.65. Data obtained from zymogram suggest the active protease is present either as homodimer (130 kDa) or homotetramer (250 kDa). The optimal temperature of the enzyme was 37 degrees C, and it exhibited activity over a broad pH range. The protease was strongly inhibited by TPCK and chelating agents. The enzymatic activity was restored upon addition of calcium. BLAST analysis with the sequence of internal peptides of the protein revealed two open reading frames within the genome of E. histolytica, homologous to members of the family S28, clan SC of serine proteases.     

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S₂ and for positively charged residues in S₁. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S₂ pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms.