Bovine Cathepsin Research Articles

Revealing the inhibitory potential of Yersinia enterocolitica on cysteine proteases of the papain family

Cysteine proteases of the papain family, including mammalian cathepsins, play important physiological roles, however, their excessive activity may contribute to the development of various pathologies. Therefore, cysteine cathepsin inhibitors are being considered as promising drugs to treat cathepsin-driven diseases. Diverse saprophytic and parasitic microbes produce such inhibitors, which target the host's proteases playing pivotal roles in immune responses, thus leading to the survival of microbes within their host. Yersinia enterocolitica is a Gram-negative zoopathogenic coccobacillus, which has developed several mechanisms to evade the host's immune system. Nevertheless, the bacterium has not yet been shown to produce any cysteine protease inhibitors. Here we demonstrate that Y. enterocolitica strains of different bioserotypes and genotypes synthesize papain and human cathepsin L inhibitors, but not bovine cathepsin B inhibitors. By employing fluorimetry and zymography, the cell-surface inhibitors were shown to associate peripherally with the outer membrane, while the inhibitors present in cell-free extracts proved to: interact reversibly with their target enzymes, exhibit thermolability and stability in a range of pH values (5–9), and have high molecular weights. Batch affinity chromatography on papain–agarose resin was then undertaken to isolate putative inhibitors of cysteine proteases from the bacterial extract. The isolated 18 kDa protein was identified by LC–MS/MS as the periplasmic chaperone Skp. The Skp-containing eluate inhibited the activity of cysteine cathepsins produced by human dermal fibroblasts. The homologous Skp protein was also isolated from the extract of Escherichia coli. Our results point to a possible new biological role of the bacterial chaperone Skp.

Inhibition of cathepsin B activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent toxic isomer in the dioxin-like family. Due to its resistance to metabolic degradation, this ubiquitous environmental pollutant readily accumulates in multiple organs. Cathepsin B is a lysosomal cysteine protease playing an essential role in the intracellular protein turnover. Alterations in its expression, activity, and localization may facilitate the development of many pathologies, including cancer. TCDD, due to its extremely lipophilic nature, may diffuse through biological membranes and affect lysosomal enzymes, including cathepsins. Therefore, in this study we performed two enzymatic assays, spectrofluorimetry and gelatin zymography, in order to evaluate the effect of TCDD on purified bovine cathepsin B. We showed that the dioxin decreases the enzyme's activity in a dose-dependent manner. The reversibility of TCDD-induced inhibition of the protease was also examined, suggesting that TCDD does not bind covalently to the enzyme's active site, acting rather as a reversible inhibitor.

Identification of Semicarbazones, Thiosemicarbazones and Triazine Nitriles as Inhibitors of Leishmania mexicana Cysteine Protease CPB

Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.

Olive Flounder (Paralichthys olivaceus) Cystatin C: Cloning, mRNA Expression, and Enzymatic Characterization of Olive Flounder Cystatin C

Cystatins are endogenous inhibitors of mammalian lysosomal cysteine proteinases, such as cathepsins B, L, H, and S. Cystatin C belongs to the type 2 cystatin family. In this study, the 751-bp cystatin C cDNA (PoCystatin C) of olive flounder (Paralichthys olivaceus) was cloned by screening from the olive flounder cDNA library. The mRNA expression of the PoCystatin C gene was examined in various tissues from normal and lipopolysaccharide (LPS)-stimulated olive flounder by RT-PCR and was compared with inflammatory cytokines IL-1β, IL-6, and IL-8. PoCystatin C transcripts ubiquitously existed in all normal and LPS-stimulated tissues that were tested. The recombinant PoCystatin C protein was expressed in Escherichia coli BL21(DE3) in pCold™ TF DNA expression vector as a 70-kDa fusion protein. The protease inhibitory activities of recombinant PoCystatin C toward papain cysteine protease, piscine cathepsins (L, S, K, F, and X), and bovine cathepsin B were measured with the synthetic fluorogenic peptide substrates. PoCystatin C tightly inhibited papain cysteine protease, whereas cathepsins L, S, K, F, X, and B were inhibited with lower affinities. Our results indicate that the P. olivaceus cystatin C is a homolog of mammalian cystatin C due to its sequence, structure, tissue expression, and biochemical activity.

Olive flounder (Paralichthys olivaceus) cystatin B: Cloning, tissue distribution, expression and inhibitory profile of piscine cystatin B

Among the cystatin superfamily, cystatin B, also known as stefin B, is an intracellular inhibitor that regulates the activities of cysteine proteases, such as papain and cathepsins. In this study, the 536 bp cystatin B cDNA (referred to hereafter as PoCystatin B) was cloned from olive flounder (Paralichthys olivaceus) using a combination of the rapid amplification of cDNA ends (RACE) approach and olive flounder cDNA library screening. To determine the tissue distribution of PoCystatin B mRNA, the expression of PoCystatin B in normal and lipopolysaccharide (LPS)-stimulated flounder tissues were compared with that of the inflammatory cytokines interleukin (IL)-1β, IL-6, and IL-8 by reverse transcription (RT)-polymerase chain reaction (PCR). The results of the RT-PCR analysis revealed ubiquitous PoCystatin B expression in normal and LPS-stimulated tissues. To characterize the enzymatic activity of PoCystatin B protein, recombinant PoCystatin B protein was overexpressed in Escherichia coli BL21(DE3) cells in the pCold™ TF DNA expression vector as a soluble fusion protein of 67-kDa. PoCystatin B inhibited papain cysteine protease, bovine cathepsin B, and fish cathepsins F and X to a greater extent, whereas fish cathepsins L, S, and K were inhibited to a lesser extent. These results indicate that the enzymatic characteristics of the olive flounder cystatin B are similar to those of mammalian cystatin B proteins, and provide a better understanding of the mechanisms of regulation of cathepsins and cystatins in marine organisms.

Cold-Adapted Digestive Aspartic Protease of the Clawed Lobsters Homarus americanus and Homarus gammarus: Biochemical Characterization

Aspartic proteinases in the gastric fluid of clawed lobsters Homarus americanus and Homarus gammarus were isolated to homogeneity by single-step pepstatin-A affinity chromatography; such enzymes have been previously identified as cathepsin D-like enzymes based on their deduced amino acid sequence. Here, we describe their biochemical characteristics; the properties of the lobster enzymes were compared with those of its homolog, bovine cathepsin D, and found to be unique in a number of ways. The lobster enzymes demonstrated hydrolytic activity against synthetic and natural substrates at a wider range of pH; they were more temperature-sensitive, showed no changes in the K(M) value at 4°C, 10°C, and 25°C, and had 20-fold higher k(cat)/K(M) values than bovine enzyme. The bovine enzyme was temperature-dependent. We propose that both properties arose from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low habitat temperatures. This is supported by the fast denaturation rates induced by temperature.

Structural Basis for Specificity of Propeptide-Enzyme Interaction in Barley C1A Cysteine Peptidases

C1A cysteine peptidases are synthesized as inactive proenzymes. Activation takes place by proteolysis cleaving off the inhibitory propeptide. The inhibitory capacity of propeptides from barley cathepsin L and B-like peptidases towards commercial and barley cathepsins has been characterized. Differences in selectivity have been found for propeptides from L-cathepsins against their cognate and non cognate enzymes. Besides, the propeptide from barley cathepsin B was not able to inhibit bovine cathepsin B. Modelling of their three-dimensional structures suggests that most propeptide inhibitory properties can be explained from the interaction between the propeptide and the mature cathepsin structures. Their potential use as biotechnological tools is discussed.

Bovine cathepsin D activity under high pressure

The stability and catalytic activity of bovine cathepsin D in Bis-Tris buffer (pH 6.0) in different pressure–temperature domains (0.1–650 MPa, 20–75 °C) were investigated and described with mathematical models. Cathepsin D inactivation followed first-order kinetics at all pressure–temperature conditions tested. The protease was largely pressure stable at room temperature and heat stable at ambient pressure up to 300 MPa and 55 °C, respectively, causing less than 10% inactivation after 10 min treatment. Pressure and temperature act synergistically on the enzyme inactivation under most conditions. However, at 100 MPa a significant stabilisation of the enzyme against temperature-induced inactivation was observed. Pressure drastically inhibited the cleavage of a synthetic substrate by cathepsin D in Bis-Tris buffer (pH 6.0) causing a reduction of the catalytic rate of more than 50% at 100–400 MPa. Maximal substrate cleavage by cathepsin D was identified at 60 °C and ambient pressure conditions after 20 min treatment.

Proteomics Evaluation of Chemically Cleavable Activity-based Probes

Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system.

Heterogeneity of Proteinase Inhibitors in the Water-Soluble Organic Matrix from the Oyster Nacre

We extracted proteinase inhibitors from the nacre of the oyster Pinctada margaritifera with water. Mixing the nacre powder with water for 20 h led to a water-soluble fraction [0.24% (wt/wt) of nacre]. After dialysis of the water-soluble matrix through 6- to 8-kDa and 0.5-kDa membranes, the proteinase inhibitors were divided into low and high molecular weight fractions that contained inhibitors of papain, bovine cathepsin B, and human cathepsin L. We studied the heterogeneity of the inhibitors after separating the low molecular weight fraction according to charge and hydrophobicity. After multistep purification, mass spectrometry analysis revealed that a potent inhibitory fraction contained several molecules. This observation demonstrates the difficulties encountered in attempting to isolate individual metabolites from the complex mixture of molecules present in nacre matrix. Interestingly, the low molecular weight fraction contained specific inhibitors that could discern between cathepsin B and cathepsin L. The nacre organic inhibitors were active against several cysteine proteinases, yet they were more specific in relation to serine proteinases, because only proteinase K was inhibited. These results demonstrate, for the first time, the presence of active proteinase inhibitors in the mollusc shell, and it is possible that these inhibitors may play a role in either protection of proteins involved in shell formation or in defense against parasites, or both.

Cathepsin D from Atlantic cod ( Gadus morhua L.) liver. Isolation and comparative studies

The isolated cathepsin D-like enzyme from Atlantic cod ( Gadus morhua L.) liver was shown to be a monomer with a molecular mass of approximately 40 kDa. It was inhibited by Pepstatin A and had an optimum for degradation of haemoglobin at pH 3.0. The purified enzyme had lower temperature stability than bovine cathepsin D. Antibodies raised against the purified enzyme and against two C-terminal peptides of cod cathepsin D recognized a 40 kDa protein in immunoblotting of the samples from the purification process. Both antisera showed cross reactivity with a similar sized protein in liver from cod, saithe ( Pollachius virens L.), Atlantic herring ( Clupea harengus L.) and Atlantic salmon ( Salmo salar L.). A protein of same size was detected in wolffish ( Anarhichas lupus L.) liver with the antibody directed against the purified enzyme. This antibody also recognized the native enzyme and detected the presence of cathepsin D in muscle of cod, saithe, herring and salmon. These antibodies may be useful in understanding the mechanisms of post mortem muscle degradation in fish by comparing immunohistochemical localization and enzyme activity, in particular in cod with different rate of muscle degradation. They may also be used for comparing muscle degradation in different fish species.

Peptides containing Acylated C‐terminal Gem diamines: Novel Irreversible Inactivators of the Cysteine and Serine Proteinases†

This study reports on the synthesis of peptides containing C-terminal acylated gem-diamines and their utilization for the preparation of irreversible inactivators of the serine and cysteine proteinases. We have succeeded in obtaining an inhibitor Acetyl-Val-Pro-g-Val-CO-O-C(6)H(4)-NO(2) of neutrophil and pancreatic elastases that functions in a time-dependent manner, indicative of the action of an irreversible inactivator, functioning, most probably, through the formation of a long-lived acyl enzyme intermediate. In addition, we have demonstrated the irreversible inhibition of the cysteine proteinase bovine cathepsin B, by chloroacetyl and bromoacetyl derivatives of a dipeptide gem-diamine, Cbz-Phe-g-Ala-CO-CH(2)Hal (Hal = Br, Cl).

Tick vitellin is dephosphorylated by a protein tyrosine phosphatase during egg development: Effect of dephosphorylation on VT proteolysis

Vitellin (VT) is a phospholipoglycoprotein that is the main component of arthropod egg yolk. Phosphorylation is a recurrent feature of every VT molecule described so far. However, the role played by such post-translational modification during egg development is not yet clear. In the eggs of the hard tick Boophilus microplus, VT is a phosphotyrosine-containing protein. VT–phosphotyrosine residues are gradually removed during tick embryogenesis due to the action of a 45 kDa egg tyrosine phosphatase. This enzyme is strongly inhibited by ammonium molybdate, sodium vanadate and cupric ion. The role of phosphotyrosine residues in VT proteolytic degradation was evaluated. Western blots probed with a monoclonal anti-phosphotyrosine antibody demonstrated that the high molecular mass VT subunits (VT 1 and VT 2) are the main targets of dephosphorylation during egg development. Both dephosphorylation and proteolysis of VT 1 and VT 2 are blocked by ammonium molybdate in total egg homogenates. When purified VT was dephosphorylated in vitro with lambda phosphatase and then incubated in the presence of bovine cathepsin D, VT proteolysis increased dramatically. Altogether, these data are the first to show that phosphotyrosine residues are present in a yolk protein, and that such residues might be involved in the regulation of VT breakdown during egg development.

Proteolytic activation of internalized cholera toxin within hepatic endosomes by cathepsin D

We have defined the in vivo and in vitro metabolic fate of internalized cholera toxin (CT) in the endosomal apparatus of rat liver. In vivo, CT was internalized and accumulated in endosomes where it underwent degradation in a pH-dependent manner. In vitro proteolysis of CT using an endosomal lysate required an acidic pH and was sensitive to pepstatin A, an inhibitor of aspartic acid proteases. By nondenaturating immunoprecipitation, the acidic CT-degrading activity was attributed to the luminal form of endosomal cathepsin D. The rate of toxin hydrolysis using an endosomal lysate or pure cathepsin D was found to be high for native CT and free CT-B subunit, and low for free CT-A subunit. On the basis of IC(50) values, competition studies revealed that CT-A and CT-B subunits share a common binding site on the cathepsin D enzyme, with native CT and free CT-B subunit displaying the highest affinity for the protease. By immunofluorescence, partial colocalization of internalized CT with cathepsin D was confirmed at early times of endocytosis in both hepatoma HepG2 and intestinal Caco-2 cells. Hydrolysates of CT generated at low pH by bovine cathepsin D displayed ADP-ribosyltransferase activity towards exogenous Gsalpha protein suggesting that CT cytotoxicity, at least in part, may be related to proteolytic events within endocytic vesicles. Together, these data identify the endocytic apparatus as a critical subcellular site for the accumulation and proteolytic degradation of endocytosed CT, and define endosomal cathepsin D an enzyme potentially responsible for CT cytotoxic activation.

The inactivation of bovine cathepsin B by novel N-chloro-acetyl-dipeptides: Application of the Houghten ‘tea bag’ methodology to inhibitor synthesis

In this study, a series of N-chloro-acetylated dipeptides were synthesised by the application of Houghten’s methodology of multiple analog peptide syntheses (MAPS). The peptides, all of which contain a C-terminal free acid, were tested as inactivators of bovine cathepsin B, in an attempt at exploiting the known and, amongst the cysteine proteinases, unique carboxy dipeptidyl peptidase activity of the protease. We have succeeded in obtaining a number of effective inactivators, the most potent of which—chloroacetyl-Leu-Leu-OH, inactivates the enzyme with an apparent second-order rate constant of 3.8 × 10 4 M −1 min −1. In contrast, the esterified analog, chloroacetyl-Leu-Leu-OMe, inactivates the enzyme some three orders of magnitude less efficiently, lending credence to our thesis that a free carboxylic acid moiety is an important determinant for inhibitor effectiveness. This preliminary study has highlighted a number of interesting features about the specificity requirements of the bovine proteinase and we believe that our approach has great potential for the rapid delineation of the subsite specificities of cathepsin B-like proteases from various species.

A rapid purification procedure for cathepsin L from bovine species and production of specific antibodies

AbstractTwo approaches have been carried out for the production of polyclonal antibodies specific of bovine cathepsin L (EC 3.4.22.15). The first one consisted in the purification of cathepsin L from bovine liver, through the development of an original protocol that can be achieved in 4 days, and further injection in rabbits. The purification scheme included the preparation of an enriched lysosomal extract, selective precipitation with ammonium sulfate and different chromatographic steps including size‐exclusion chromatography, cation‐ and anion‐exchange chromatography and the use of an affinity column for the removal of trace amounts of bovine serum albumin. The final enzyme preparation was purified 8400‐fold and consisted predominantly in the double‐chain form of cathepsin L. This was proved by the presence of a main protein band with Mr of 25 kDa, corresponding to the cathepsin L heavy chain, in SDS‐PAGE under reducing and denaturant conditions, and was further confirmed by immunoblotting. The second approach consisted in the generation of polyclonal antibodies against the peptide sequence 99PECSAANDTGFVDIPQR115, specific of the bovine cathepsin L heavy chain. Both types of antibodies were proved to specifically recognize cathepsin L. However, while antibodies raised against the whole protein recognized native cathepsin L, the antipeptide was able to recognize the enzyme in its denatured form. Both of them are adequate for the future development of immunoassays, allowing a rapid and accurate quantification of cathepsin L levels directly in crude extracts of meat and meat‐derived products. Copyright © 2004 Society of Chemical Industry

Hydrolysis of bovine caseins by cathepsin B, a cysteine proteinase indigenous to milk

The presence of the lysosomal cysteine proteinase cathepsin B in milk has been demonstrated recently. The potential significance of this enzyme to proteolysis in milk and dairy products was evaluated by the study of its proteolytic specificity towards the caseins. Bovine cathepsin B (1.4 units mL−1) was added separately to β-casein or αs1-casein (5 mg mL–1, in 100 mm Na acetate buffer, pH 6.0, containing 1.5 mm dithiothreitol). Samples were taken over a 24 h period and analysed by urea polyacrylamide gel electrophoresis and RP-HPLC; peptides were identified by N-terminal sequencing and mass spectrometry. Cathepsin B cleaved β-casein at 32 sites and αs1-casein at 35 sites, extensively hydrolysing both proteins. Thus, cathepsin B has a very broad specificity against the caseins, with an apparent preference for bonds incorporating the amino acids Leu, Val, Gln, Pro and Ser. Cathepsin B cleaved some bonds in common with chymosin (including the Phe23–Phe24 bond of αs1-casein), plasmin and the cell envelope-associated proteinases of Lactococcus, suggesting that it could be involved in proteolysis in dairy products, particularly if manufactured from high somatic cell count milk.

Purification of bovine cathepsin B: proteomic characterization of the different forms and production of specific antibodies.

Cathepsin B (EC 3.4.22.1) has been highly purified (14,225 fold) from bovine kidney by a rapid procedure that included the preparation of an enriched lysosomal extract, a selective fractionation with ammonium sulphate, size-exclusion chromatography, two cation-exchange chromatographies, and anion-exchange chromatography on diethylaminoethyl-Sephacel. After the last purification step, two hydrolytic peaks against Z-Phe-Arg-AMC were separated from each other, a minor peak corresponding to the cathepsin B single-chain form and a major one representing the double-chain form of cathepsin B. The single-chain form showed a molecular mass of 31 kDa on sodium dodecyl sulphate - polyacrylamide gel electrphoresis (PAGE) under reducing conditions, whereas the heavy chain of the double-chain form appeared as a doublet with molecular masses of 23.4 and 25 kDa, respectively. The identity of the different cathepsin B isoforms and the quality of the final enzyme preparation were confirmed by using two types of antibodies, one against a synthetic peptide sequence and one against purified cathepsin B. The proteomic study confirmed the identity of the different SDS-PAGE protein bands as cathepsin B isoforms and allowed the comparison and study of some structural differences between them at the level of their primary structures.

Immunological similarity between Schistosoma and bovine cathepsin D

IgG antibodies from sera of rabbits immunized with a mixture of three synthetic peptides of highly conserved surface-exposed sequences between Schistosoma japonicum and S. mansoni cathepsin D, and a rabbit anti-bovine cathepsin D serum strongly recognized a 45 kDa molecule on immunoblots of adult S. mansoni worm saline extracts (AWSE). This recognition was abolished by immunoadsorption with two of the three selected peptides. The anti-peptide antibodies fixed onto Protein A-Sepharose specifically immunoprecipitated a S. mansoni AWSE component that was able to degrade bovine hemoglobin at pH 3.8. This reaction was inhibited by 7 μM pepstatin A, a classical aspartyl protease inhibitor, suggesting that the parasite cathepsin D was immunoprecipitated. The anti-peptide antibodies also recognized on a dot-blot assay a purified, commercially obtained bovine cathepsin D preparation but not the purified human counterpart. On the other hand, the anti-bovine cathepsin D serum recognized the two above-mentioned schistosome peptides. In addition, S. mansoni-infected patient sera recognized on immunoblots the bovine but not the human cathepsin D. These results, together with a comparative analysis of the selected peptide sequence regions between the schistosome and the two mammal enzymes, allowed us to pinpoint to one amino acid the cross-reactivity between parasite and bovine cathepsin D and the lack of it with human cathepsin D. This difference might be of relevance for immunodiagnosis.

Effect of high pressure on the reaction between bovine myofibrils and cathepsin D

This study deals with the effect of high pressure [50-500 MPa] and time of treatment [20-900 s] on the reaction between myofibrils and cathepsin D from bovine post mortem meat, using Surface Response Methodology. We shown that every high pressure treatment enhanced activity of cathepsin D as evaluated on haemoglobin as a substrate or on control meat myofibrils. We also put in evidence that cathepsin D could carry out the hydrolysis of high pressure treated myofibrils. At last we studied the action of pressurised cathepsin D on pressurised myofibrils, and proved that the hydrolysis was increasing up to 170 MPa and then decreased; above 300 MPa the activity was lower than with control cathepsin D and control myofibrils. Thus above 300 MPa recognition of natural substrate is affected by high pressure induced modifications. These results may help to explain why high pressure treatment of post rigor meat is not able to increase tenderness.