Loss Of Proteolytic Activity Research Articles

Periplasmic production of pernisine in Escherichia coli and determinants for its high thermostability.

Pernisine is a subtilisin-like serine proteinase secreted by the hyperthermophilic archaeon Aeropyrum pernix. The significant properties of this proteinase are remarkable stability and ability to degrade the infectious prion proteins. Here we show the production of pernisine in the periplasm of Escherichia coli. This strategy prevented the aggregation of pernisine in the cytoplasm and increased the purity of the isolated pernisine. The thermostability of this recombinant pernisine was significantly increased compared with previous studies. In addition, several truncated pernisine variants were constructed and expressed in E. coli to identify the minimally active domain. The catalytic domain of pernisine consists of the αẞα structurally similar core flanked by the N-terminal and C-terminal outer regions. The deletion of the C-terminal α helix did not affect the pernisine activity at 90°C. However, the complete deletion of the C-terminal outer region resulted in loss of proteolytic activity. The pernisine variant, in which the N-terminal outer region was deleted, had a reduced activity at 90°C. These results underline the importance of the Ca2+ binding sites predicted in these outer regions for stability and activity of pernisine. KEY POINTS: • Aggregation of produced pernisine was prevented by translocation into periplasm. • Thermostability of mature pernisine was increased. • The outer regions of the catalytic core are required for pernisine thermostability.

Development of mutant human immunoreactive trypsinogen 1 (IRT1) and mutant human immunoreactive trypsinogen 2 (IRT2) for use in immunoassays

Immunoreactive Trypsinogen (IRT) is a protein-based pancreatic proenzyme that has an important role in protein digestion in humans. In human body, once IRT present in the small intestine, the proteolytic cleavage activates trypsinogen into trypsin. When IRT is in the active form, it is capable to cleave antibodies, other proteins and even itself while it is desired to use in immunoassays. According to the literature, there are three important IRT isoforms called Immunoreactive Trypsinogen 1 (IRT1), Immunoreactive Trypsinogen 2 (IRT2), and Immunoreactive Trypsinogen 3 (IRT3). However, trypsinogen 1 (cationic trypsinogen, IRT1) and trypsinogen 2 (anionic trypsinogen, IRT2) are the major isoforms in human pancreatic juice and used in the diagnosis of cystic fibrosis (CF). In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutated and expressed in P. pastoris X-33 strain. The loss of proteolytic activity has been proven with the BAEE test. Antigenic properties of K23D IRTs and the effect of proteolytic inactivation on their performance in immunoassays were assessed with ELISA and Western Blot. In ELISA results K23D mutated IRTs showed higher signals than Wild-Type forms.

The structure of tripeptidyl peptidase I (TPP1) provides insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (LINCL) is an autosomal recessive disease caused by mutations in the TPP1 gene resulting in functional defect of the gene product. Clinical symptoms usually begin after two years of age and comprise developmental stagnation, ataxia and epilepsy, finally leading to a complete loss of motor function, vision and speech by the age of ten. More than 50% of the LINCL associated genetic alterations are missense mutations that result in conformational distrubances. To elucidate the structural basis for these TPP1 mutants, we crystallized fully glycosylated TPP1. X-ray crystallographic analysis at 2.35Å resolution reveals a globular structure with a subtilisin-like fold, a S475-E272-D360 catalytic triad and an octahedrally coordinated Ca2+ binding site that are characteristic features of the S53 sedolisin family of serine proteases. On the basis of the resolved TPP1 structure we characterized the molecular effects of 28 missense mutations. These mutations either disrupt protein folding leading to instability or compromise the active center resulting in loss of proteolytic activity. Our results extend the understanding of the molecular mechanisms underlying LINCL and offer novel therapeutic options.

Cubic liquid crystalline glyceryl monooleate matrices for oral delivery of enzyme

In situ cubic phase transforming system of glyceryl monooleate (GMO) has been prepared which offers protection to the metaloenzyme, seratiopeptidase (STP), in gastric environment and provides delayed and controlled release with no initial burst after oral administration. Effect of magnesium trisilicate (MTS) on floating, proteolytic activity and drug release was studied. Gelucire ® 43/01 was incorporated in the system to provide prolonged lag time. The drug-loaded matrices required 100 mg of MTS to overcome floatability of GMO matrix. Plain GMO matrices showed 85.3% loss of proteolytic activity in acidic medium, whereas matrices containing MTS showed retention of activity (111.6%). The hydrophobic nature of MTS induced formation of cubic phase at faster rate and the existence of cubic phase was confirmed by polarizing light microscopy. Furthermore, MTS provided alkaline microenvironment, which prevented acid-catalyzed hydrolysis and protein unfolding. The magnesium ions restored the activity of STP. The release of STP was decreased with increasing amount of MTS in the matrix. Gelucire did not affect proteolytic activity. The water uptake of matrices with gelucire was decelerated due to formation of hexagonal phase. However, the rate of STP release from these matrices was very slow due to incorporation of gelucire into lipid bilayers, which provided resistance to movement of STP. Thus, microenvironment-controlled in situ cubic phase transforming GMO matrices provided protection to STP and controlled release.

Intramolecular Dimerization Is Required for the Chlamydia-Secreted Protease CPAF To Degrade Host Transcriptional Factors

We previously identified a chlamydial protein designated CPAF (chlamydia protease/proteasome-like activity factor) that is secreted into host cell cytosol for degrading host transcription factors required for major histocompatibility complex antigen expression. Here we report that CPAF, synthesized as a 70-kDa proprotein, is processed into two fragments (designated CPAFn and CPAFc) to form intramolecular dimers that are much more stable than the naïve CPAF. Precipitation with antibodies that recognized CPAF dimers removed the proteolytic activity responsible for degrading host transcription factor RFX5 from chlamydia-infected host cell cytosol, while precipitation with antibodies that recognized free CPAF fragments alone did not remove this activity. Separation of CPAFn from CPAFc resulted in a loss of proteolytic activity. Furthermore, neither expressed full-length CPAF that was not processed nor coexpressed CPAFn and CPAFc fragments that failed to form dimers degraded RFX5. These observations demonstrate that intramolecular dimerization is required for CPAF to degrade host transcription factors, a strategy that is utilized by an obligate intracellular bacterial species to evade host defenses.

Hypochlorous Acid Generated by Myeloperoxidase Modifies Adjacent Tryptophan and Glycine Residues in the Catalytic Domain of Matrix Metalloproteinase-7 (Matrilysin): AN OXIDATIVE MECHANISM FOR RESTRAINING PROTEOLYTIC ACTIVITY DURING INFLAMMATION

Dysregulation of matrix metalloproteinase (MMP) activity is implicated in tissue destruction under inflammatory conditions. An important mechanism controlling enzymatic activity might involve reactive oxygen species generated by phagocytes. Myeloperoxidase, a heme protein secreted by neutrophils, monocytes, and macrophages, uses hydrogen peroxide to generate hypochlorous acid (HOCl). We demonstrate that HOCl inhibits the activity of human matrilysin (MMP-7) in vitro, suggesting that it might limit proteolytic activity during inflammation. When MMP-7 was exposed to HOCl generated by myeloperoxidase, the proteinase lost activity. High performance liquid chromatographic analysis of the tryptic digest of the HOCl-treated proteinase demonstrated the absence of two peptides that were present in the untreated enzyme. Tandem mass spectrometric analysis revealed that both of the lost peptides contained methionine and tryptophan-glycine residues. The methionine residue of one of the peptides had been oxidized to methionine sulfoxide. In contrast, the major product from the other peptide was 4 atomic mass units smaller than its precursor (WG-4). This novel oxidation product was derived though modification of adjacent tryptophan and glycine residues in the catalytic domain of the enzyme. Loss of proteolytic activity was associated with conversion of the precursor peptide to WG-4 but not with methionine oxidation. In contrast, hydrogen peroxide failed to oxidize MMP-7 or to inactivate the enzyme. Thus, HOCl inactivates MMP-7, perhaps by site-specific conversion of tryptophan-glycine to WG-4. This inactivation mechanism is distinct from the well studied mechanisms involving tissue inhibitors of metalloproteinases. Our findings suggest that local pericellular production of HOCl by phagocytes is a physiological mechanism for governing MMP activity during inflammation.

Processing, stability, and kinetic parameters of C5a peptidase from Streptococcus pyogenes.

A recombinant streptococcal C5a peptidase was expressed in Escherichia coli and its catalytic properties and thermal stability were subjected to examination. It was shown that the NH2-terminal region of C5a peptidase (Asn32-Asp79/Lys90) forms the pro-sequence segment. Upon maturation the propeptide is hydrolyzed either via an autocatalytic intramolecular cleavage or by exogenous protease streptopain. At pH 7.4 the enzyme exhibited maximum activity in the narrow range of temperatures between 40 and 43 degrees C. The process of heat denaturation of C5a peptidase investigated by fluorescence and circular dichroism spectroscopy revealed that the protein undergoes biphasic unfolding transition with Tm of 50 and 70 degrees C suggesting melting of different parts of the molecule with different stability. Unfolding of the less stable structures was accompanied by the loss of proteolytic activity. Using synthetic peptides corresponding to the COOH-terminus of human complement C5a we demonstrated that in vitro peptidase catalyzes hydrolysis of two His67-Lys68 and Ala58-Ser59 peptide bonds. The high catalytic efficiency obtained for the SQLRANISHKDMQLGR extended peptide compared to the poor hydrolysis of its derivative Ac-SQLRANISH-pNA that lacks residues at P2'-P7' positions, suggest the importance of C5a peptidase interactions with the P' side of the substrate.

Porcine kallikrein-4 activation, glycosylation, activity, and expression in prokaryotic and eukaryotic hosts.

Kallikrein-4 (KLK4) is a serine proteinase believed to be important in the normal development of dental enamel. We isolated native KLK4 from developing pig enamel and expressed four recombinant forms. Pig KLK4 was expressed in bacteria with and without the propeptide, and in two eukaryotic systems. Recombinant pig KLK4 was secreted as a zymogen by '293' cells and purified. The proKLK4 was activated in vitro by thermolysin and recombinant pig enamelysin, but not by native KLK4. These results were confirmed using a fluorescent peptide analog of the KLK4 propeptide-enzyme junction. Native KLK4 appears as a doublet at 37 kDa and 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Removal of N-linked oligosaccharides by digestion with deglycosidase-F reduced the doublet to a single band at approximately 28 kDa, demonstrating that the active enzyme is glycosylated, and that the 37 kDa and 34 kDa forms differ only in their number of glycosylations. Deglycosylation was also associated with a loss of proteolytic activity. We digested recombinant pig amelogenin with native KLK4 and characterized the cleavage products by N-terminal sequencing and mass spectrometry. Eleven cleavage sites in the amelogenin protein were identified, demonstrating that KLK4 degrades amelogenin and is likely to participate in the degradation of enamel proteins in vivo.

Autolysis of the proteinase from Pseudomonas fluorescens.

The gene encoding the proteinase from Pseudomonas fluorescens was cloned and sequenced in an effort to identify the cleavage sites involved in its autolysis at 50 degrees C. A single open reading frame consisting of 1449 nucleotides, encoding a protein of 482 amino acids, was found. Analysis of the N-terminal amino acid sequence of the purified proteinase indicated the presence of a prosequence consisting of 13 amino acid residues. The molecular weight of the mature protein was calculated as 48,900 based on the deduced amino acid sequence, which was consistent with that of the purified proteinase as determined by sodium dodecylsulfate-PAGE. Greater than 90% loss of proteolytic activity was observed upon heating at 50 degrees C for 2 min compared with the unheated enzyme. Incubation of the proteinase at 50 degrees C led to disappearance of the intact enzyme, as shown by sodium dodecyl sulfate-PAGE, whereas it was stable in the presence of the protease inhibitor o-phenanthroline. Autolytic fragments were fractionated by reverse-phase HPLC and subjected to N-terminal amino acid sequence analysis in an effort to determine the cleavage sites. The cleavage profile was not definitive; however, amino acid residues with small side chain groups, such as glycine or alanine, were frequently found adjacent to the cleavage sites.

α;2‐Macroglobulin Inhibition of Endogenous Proteases in Fish Muscle

ABSTRACTKinetic parameters for α;2‐macroglobulin inhibition of selected proteases (collagenase, cathepsin D, trypsin and chymotrypsin) were determined along with the extent of inhibition of the corresponding fish muscle proteases. Protease inhibition by α;2‐macroglobulin occurred in the presence of large macromolecular substrates but not in the presence of synthetic substrates. The inhibitor remained active at refrigerated temperatures (4–7°C). The α;2‐macroglobulin did not have any inhibitory effects on proteases in intact fish muscle tissue possibly due to lack of penetration of the tissues, but showed various levels of inhibition of proteases in tissue homogenates. Inhibitor concentration of 0.1% per weight of tissue homogenate resulted in about 17% loss of proteolytic activity while 0.4% inhibitor concentration caused complete loss of protease activity.

Protease stabilization by highly concentrated anionic surfactant mixtures

AbstractWe have found that anionic surfactants such as linear alkylbenzene sulfonate (LAS) can solubilize proteases in a substantially nonaqueous environment without loss of proteolytic activity. Moreover, in mixtures of anionic and nonionic surfactants with a moderate amount of water (water less than 30 wt%), controlled levels of LAS and water solubilize proteases; yet, in these concentrated surfactant mixtures, enzymes maintain their activity for extended periods. Experimental design techniques have been used to delineate the relationship between protease stability and the water, pH and anionic surfactant levels in these surfactant concentrates. As the sum of water and LAS levels is increased, maximum enzyme stability is observed, after which stability falls off. At low water and LAS levels (sum of both <20%), protease solubility is low, while at high levels of water and LAS (sum of LAS and water >45%), denaturation predominates. Additionally, we have developed a new and simple method to predict protease stability by which a synthetic peptide is used to measure protease activity directly in the surfactant concentrate. From the application of this new technique to our system and to commercial liquid detergent formulations, it is apparent that water facilitates the loss of activity of proteases in surfactant concentrates by increasing the rate of autolysis.

Regulation of proteolytic activity of Vibrio vulnificus by iron-containing compounds

Previous studies have shown that the Vibrio vulnificus is able to use hemoglobin and the hemoglobin-haptoglobin complex to reverse iron limitation. In addition, we report here that free hemin, as well as hemin bound to albumin, will reverse iron-limited growth. While protease negative mutants were able to use hemin and hemoglobin, they were unable to compete as successfully for albumin-bound hemin. Using Luria broth with the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDA), no changes in proteolytic activity were seen. In contrast, growth in a low iron defined medium resulted in a loss of proteolytic activity. This loss was reversed by the addition of hemin or hemoglobin but not by inorganic iron or globin alone. Since one portal of entry of this organism is pre-existing wounds, hemoglobin released during cell damage may be important in activating extracellular proteases leading to the massive cell damage seen with this vibrio.

Identification of the primary antimicrobial domains in human neutrophil cathepsin G.

Lysosomal cathepsin G from human neutrophils is a chymotrypsin-like protease which also possesses antimicrobial activity. The antimicrobial activity, however, is independent of protease activity, because treatment of this enzyme with the irreversible serine protease inhibitor diisopropylfluorophosphate has no effect on its antimicrobial action. In this study, we found that digestion of cathepsin G with clostripain caused a loss of proteolytic activity in this neutrophil proteinase. However, bactericidal activity in in vitro assays against Staphylococcus aureus and Neisseria gonorrhoeae was retained. Fractionation of the clostripain-digested cathepsin G mixture yielded two distinct antimicrobial peptides. The sequences of these peptides were IIGGR and HPQYNQR (residues 1-5 and 77-83 in cathepsin G, respectively). Synthetic peptides corresponding to these sequences were also prepared and found to exert broad-spectrum antimicrobial activity in vitro, displaying conditions of temperature- and pH-dependent optima for antimicrobial action resembling that of the full-length enzyme. Depending on the target bacterial strain, these peptides exhibited antimicrobial activity between 5.0 x 10(-5) and 4.0 x 10(-4) M. Significantly, replacement of certain residues within these peptides with either alanine or valine significantly reduced their antibacterial capacities. Our studies suggest that cathepsin G has two antimicrobial sequences, either or both of which may contribute to its bactericidal activity.

Substitution mutations of the highly conserved arginine 87 of HIV-1 protease result in loss of proteolytic activity

The 297bp gene coding for the HIV-1 protease was chemically synthesized and expressed in E. coli (1). Single amino acid substitutions (Arg 87 -> Lys; Arg 87 -> Glu) were introduced in the C-terminally located conserved region GlyArgAsn of the protease gene in the wild-type clone. The products of the mutant and the wild-type clones were expressed at approximately similar levels at 30 minutes of induction but the mutant protease proteins accumulated as a function of time of induction unlike the wild-type protease which declined after 60 minutes. The mutants were completely devoid of proteolytic activity as determined in assays employing as substrates a synthetic nonapeptide and a gag related recombinant polyprotein.

Protein oxidation and loss of protease activity may lead to cataract formation in the aged lens

Over 95% of the dry mass of the eye lens consists of specialized proteins called crystallins. Aged lenses are subject to cataract formation, in which damage, cross-linking, and precipitation of crystallins contribute to a loss of lens clarity. Cataract is one of the major causes of blindness, and it is estimated that over 50,000,000 people suffer from this disability. Damage to lens crystallins appears to be largely attributable to the effects of UV radiation and/or various active oxygen species (oxygen radicals, 1O 2, H 2O 2, etc.). Photooxidative damage to lens crystallins is normally retarded by a series of antioxidant enzymes and compounds. Crystallins which experience mild oxidative damage are rapidly degraded by a system of lenticular proteases. However, extensive oxidation and cross-linking severely decrease proteolytic susceptibility of lens crystallins. Thus, in the young lens the combination of antioxidants and proteases serves to prevent crystallin damage and precipitation in cataract formation. The aged lens, however, exhibits diminished antioxidant capacity and decreased proteolytic capabilities. The loss of proteolytic activity may actually be partially attributable to oxidative damage which proteases (like any other protein)_can sustain. We propose that the rate of crystallin damage increases as antioxidant capacity declines with age. The lower protease activity of aged lens cells may be insufficient to cope with such rates of crystallin damage, and denatured crystallins may begin to accumulate. As the concentration of oxidatively denatured crystallins rises, cross-linking reactions may produce insoluble aggregates which are refractive to protease digestion. Such a scheme could explain many events which are known to contribute to cataract formation, as well as several which have appeared to be unrelated. This hypothesis is also open to experimental verification and intervention.

Structural and functional determinants of Mucor miehei protease. II. Circular dichroic studies on the native and periodate-oxidized enzyme

Structural investigations of the glycoprotein, Mucor miehei protease, by CD, suggested that the native enzyme contains a significant amount of β structure and little, if any, α-helix within the pH range 2.5 to 7.0. Comparison of the CD spectra of the protease with those of other proteins suggested somewhat distorted β arrangements. Exposure of the enzyme to periodate oxidation resulted in a rapid loss of about 40% of the carbohydrate but no detectable change in structure or enzymatic activity. The remainder of the carbohydrate was resistant to further oxidation; any additional decrease being associated with changes in structure and loss of proteolytic activity.

Intracellular Protease from Streptococcus durans

An intracellular protease from Streptococcus durans was isolated and partially purified and characterized. A 67-fold purification was obtained with retention of 53% of the original activity. Purification was complicated by the presence of nucleic acids which could not be removed without loss of proteolytic activity. Ribonuclease inactivated the protease, but attempts to reactivate the protease by adding ribonucleic acid extracted from S. durans were unsuccessful. Disc electrophoresis of the enzyme in polyacrylamide gel showed one protein zone with an adjoining nucleic acid zone.The protease exhibited normal reaction kinetics with respect to substrate concentrations, enzyme concentration, and reaction rate. The proteolytic activity was affected by the ionic strength of the assay reaction mixture, and an ionic strength of 0.1 was optimal. Proteolytic activity was optimal at pH 7.5, with secondary activity occurring at pH 5.5 to 6.Divalent Ca, Co, Cu, Mg, Mn, Ni, Zn, and di- and trivalent Fe had no effect on the activity of the protease purified 67-fold, but Hg inhibited the protease about 15%. Treatment of the assay reaction mixture could be partially restored by the addition of the aforementioned ions, except Fe, Cu, and Hg.The protease withstood heating at 97 to 99C for 60 minutes at pH 6 and 7.5, but lost 21% of activity at pH 8.5. Casein and β-lactoglobulin were acted upon by the enzyme, but there was no proteolytic activity detected on bovine serum albumin and hemoglobin, or in the milk-clot test.p-Chloromercuribenzoate was the most effective inhibitor of sulfhydryl groups, followed by N-ethylmaleimide and iodoacetamide. Reducing agents and diisopropyl fluorophosphate had no effect on the proteolytic activity.

Some properties of the extracellular proteolytic enzymes of the milk-spoiling organismPseudomonas aeruginosaATCC 10145

SummaryThe proteolytic enzymes present in the culture supernatant ofPseudomonas aeruginosaATCC 10145 were active in the pH range 5·5–9·0 with a maximum activity at pH 7·3. Heating for 15 sec at 72°C resulted in a 36% loss of proteolytic activity whereas heating for 30 min at 63°C resulted, in a 6% loss. Boiling for 2 min completely inactivated the proteolytic enzymes. At 2°C the proteolytic enzymes were stable for at least a month and casein was readily hydrolysed at this temperature.

Secretion of Pepsinogen by the Pyloric Glands of the Dog, With Some Observations on the Histology of the Gastric Mucosa

Summary Histologic study of the gastric mucosa of the dog, according to the Marks-Drysdale staining technique, showed an abrupt disappearance of peptic cells marking the boundary between fundic and pyloric glands. Below this boundary the parietal cells became progressively more sparse and showed changes in form and staining reactions, but were still present within a few millimeters of the pyloroduodenal junction. The principal cell type of the pyloric glands, the mucous glandular cells, gave a positive periodic acid-Schiff staining reaction and, in the necks of the glands, showed a transition to mucous neck cells indistinguishable from their counterpart in the fundic glands. Pouches of the pyloric gland area were prepared in 8 dogs. The line of transection of the stomach was 5 cm. or less from the pylorus. Biopsies from this line of transection showed the characteristics described above for the pyloric glands. No peptic cells were seen in sections prepared by the Marks-Drysdale technique or the CambelSgouris modification of the Bowie technique. The juice secreted by these pouches was scant, viscous, and alkaline. It invariably contained a protease active on radioiodina ted serum albumin substrate at pH 2.0. The pH activity curve of pyloric juice was essentially identical with that of crystalline swine pepsin. Exposure of pyloric juice to alkali caused no loss of proteolytic activity unless the juice had first been acidified; the protease of pyloric juice thus behaves, in this regard, like pepsinogen. Crystalline pepsin added to pyloric juice could be fully recovered in terms of proteolytic activity, indicating that a pepsin inhibitor is not present in the juice. Feeding caused a decrease in concentration of proteolytic activity of the juice. Injection of bethanechol produced no significant change in concentration of proteolytic activity.

Crystalline papain: VI. Extensive stepwise hydrolysis of mercuripapain by leucine aminopeptidase without loss of proteolytic activity

Crystalline papain: VI. Extensive stepwise hydrolysis of mercuripapain by leucine aminopeptidase without loss of proteolytic activity