Cysteine proteinase activity in various developmental stages of Clonorchis sinensis: a comparative analysis

1. Cysteine proteinase activity in acidic extracts of various developmental stages of Clonorchis sinensis (metacercariae, 1-, 2-, and 3-month old worms) was examined. All the activities were maximum at acidic pH and showed inhibitor susceptibilities similar to the vertebrate cysteine proteinases. 2. Specific activity of cysteine proteinase(s) was highest in metacercariae with either CBZ-phe-arg-AFC or Azocoll as the substrate. The immature and mature worms had similar (but less than metacercariae) levels of activity. 3. A soluble cysteine proteinase with a native molecular weight of approximately 20,000 +/- 1414 was partially purified from 1-, 2-, and 3-month worms. The molecular weight of similar activity in metacercariae was approximately 32,000. 4. Results suggest developmental regulation of cysteine proteinase activity in the life cycle of C. sinensis.     

Partial purification and characterization of cysteine proteinases from various developmental stages of Paragonimus westermani

1. During development of Paragonimus westermani, larvae develop during migration within the host, and adult worms feed on pulmonary tissues, causing significant pathology in the mammalian host. In this report acidic extracts of various developmental stages (metacercariae and worms at one, two and three months of development) were examined for cysteine proteinase activity. 2. A soluble thiol-dependent proteinase activity with a native molecular weight of approximately 20,000 was isolated and partially purified. 3. The enzymes purified from the various developmental stages of the parasite had maximal activity at acidic pH and showed inhibitor susceptibilities similar to the vertebrate acidic cysteine proteinases. 4. Enzymatic activity was stable at pH 5.0 for at least two days when stored at 4 degrees C. 5. It is suggested that these enzymes may be involved in the nutrition of these parasites and/or during penetration and lysis of the tissues.     

Schistosoma mansoni: expression and role of cysteine proteinases in developing schistosomula

The adult stage of Schistosoma mansoni utilizes host hemoglobin as a nutrient source. A proteolytic enzyme (SMw32) that has "hemoglobinase" activity is secreted into the parasite gut where it appears to be rapidly activated by glutathione released from host red blood cells. In the present study the expression of this proteinase, in developing schistosomula, has been correlated with digestive tract development and a dramatic rise in enzyme activity as early as Days 8-10 of culture. No evidence of the SMw32 proteinase was found in eggs, cercariae, or in newly transformed larvae. Further, the proteinase expressed at Days 8-10 is indistinguishable from the adult worm enzyme. In the larvae, indirect immunofluorescence with an anti-SMw32 monoclonal antibody showed that the proteinase is found throughout the developing cecum. The importance of cysteine proteinases to parasite development was also studied using a specific enzyme inhibitor, Ep-459. In cultures containing Ep-459 most (75%) of the schistosomula failed to survive the 18-day study period. Moreover, those that did survive showed a decrease in their growth (body length). These data suggest that the SMw32 proteinase is a developmentally regulated enzyme and that cysteine proteinase activity is essential in providing nutrients for the growth and survival of this parasite in its mammalian host. Thus, this proteinase may be an important target for chemotherapeutic intervention.     

Isolation and properties of extracellular proteinases from Sporothrix schenckii.

Sporothrix schenckii, mainly in the yeast form of the organism, produced extracellular proteinases when cultivated in liquid media containing albumin or collagen as a nitrogen source, but did not do so in brain heart infusion medium. Isolation of two extracellular proteinases from albumin-containing medium was performed by chromatography on DEAE-Sepharose CL-6B and Sephacryl S-200. Proteinase I had a molecular weight of 36,500, an optimal pH at 6.0, and a pI at 4.8. Despite its activities in weakly acidic conditions, proteinase I demonstrated chymotrypsinlike characteristics, these being indicated by strong inhibitory activity by phenylmethylsulfonyl fluoride and chymostatin and good kinetic constants for a synthetic chymotrypsin substrate, Suc-Ala-Ala-Pro-Phe-MCA. Proteinase II had a molecular weight of 39,000, an optimal pH at 3.5, and a pI at 3.8. Proteinase II showed cathepsin D-like characteristics, these being indicated by strong inhibitory activity by pepstatin, an acidic optimal pH, and good kinetic constants for hemoglobin. These two enzymes hydrolyzed natural substrates such as stratum corneum, type I collagen, and elastin although not type IV collagen. Proteinase production and cell growth in collagen-containing medium and the enzymatic digestion of skin constituents by isolated proteinases suggested that these two proteinases cooperatively enable the organism to invade skin and to obtain peptides from insoluble proteins.     

Schistosoma mansoni: differential expression of cathepsins L1 and L2 suggests discrete biological functions for each enzyme

Schistosoma mansoni cathepsins L1 (SmCL1) and L2 (SmCL2) were expressed as active recombinant proteinases in Saccharomyces cerevisiae. The recombinant enzymes exhibited substrate preferences characteristic of cathepsin-L-like cysteine proteinases. However, the enzymes differed in their substrate specificities; SmCL1 cleaved Boc-Val-Leu-Lys-NHMec with a higher efficiency than it cleaved Z-Phe-Arg-NHMec, whereas the opposite was true for SmCL2. The enzymes also differed in their pH profiles of activity; SmCL1 exhibited a broad pH profile with an optimum of pH 6. 5, while SmCL2 was active only in the acidic pH range with an optimum of 5.35. Immunoblot and RT-PCR analyses revealed that the native forms of both SmCL1 and SmCL2 are expressed in male and female worms, but at higher levels in adult female compared to male schistosomes. Additionally, both enzymes were observed in the excretory/secretory products of adult worms. The RT-PCR analysis indicated that neither enzyme is expressed in S. mansoni eggs or in miracidia, suggesting that the cathepsin-L-like activity that has been previously reported to be expressed in these stages may be the product of another gene(s). Cercariae do not express SmCL2, but appear to express SmCL1 in its inactive precursor form. Together with the findings of previous immunolocalization and phylogenetic analyses, the results reported here demonstrate that SmCL1 and SmCL2 are distinct cathepsin cysteine proteinases and strongly suggest that they play discrete biological roles.     

A monoclonal antibody from infected mice to a Schistosoma mansoni egg proteinase

A number of monoclonal antibodies were obtained by fusion of SP2/0 myeloma cells and spleen lymphocytes from mice infected with Schistosoma mansoni. These antibodies were tested for their ability to inhibit acidic, thiol-dependent proteinases previously isolated from Schistosoma mansoni eggs and adult worms. One of the monoclonal antibodies isolated inhibits egg proteinase activity measured in vitro with the use of a low m.w. synthetic substrate. This antibody, which is an IgG1 isotype, does not appreciably inhibit an acidic, thiol-dependent proteinase obtained from the adult stage of Schistosoma mansoni. Immunocytochemical methods with the monoclonal antibody have been used to localize the egg proteinase within a set of "penetration" glands in the unhatched miracidium.     

Strongyloides ransomi: proteolytic enzymes from larvae

The filariform larvae of Strongyloides ransomi can infect their hosts by penetration through skin. In this report, homogenates of these organisms were prepared and their proteolytic enzymes examined. Homogenates prepared in 0.2 M citrate, pH 4.0, contain two thiol-dependent proteinases with molecular weights of approximately 32,000 and 28,000. These proteinases have an acidic pH optimum and show substrate preferences and inhibitor susceptibilities similar to the vertebrate acidic cysteinyl proteinases. Homogenates prepared in 0.1 M Tris, pH 7.5, contain multiple proteolytic enzymes, active against both Azocoll and synthetic substrates. These enzymes do not require thiols for activity and they have an alkaline pH optimum. The enzymes are inhibited by both chelating agents and heavy metals, but not by serine-proteinase inhibitors. Extracts prepared in 0.1 M Tris-HCl, pH 7.5, contain endogenous proteinase inhibitors.     

Digestive proteinase activity of the Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae)

The khapra beetle, Trogoderma granarium, is one of the most important stored product pests worldwide. A study of digestive proteinases in T. granarium was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. The pH of guts was determined by addition of pH indicator solutions to broken open gut regions. The last instar larvae were dissected in cold distilled water and the whole guts were cleaned from adhering unwanted tissues. The pooled gut homogenates were centrifuged and the supernatants were used in the subsequent enzyme assay. Total proteinases activity of the gut homogenates was determined using the protein substrate azocasein. Optimal azocasein hydrolysis by luminal proteinases of the larvae of T. granarium was highly alkaline in pH 10-10.5, although the pH of luminal contents was slightly acidic (pH 6.5). The extract showed the highest activity at 55 degrees C (pH 6.5), 45 degrees C (pH 8) and 30 degrees C (pH 10). The proteolytic activity was strongly inhibited in the presence of phenylmethylsulphonyl fluoride (82.33+/-4.37% inhibition). This inhibition was decreased with increasing of the pH of assay incubating medium. N-p-tosyl-L-lysine chloromethyl ketone (51.6+/-3.3% inhibition) and N-tosyl-L-phenylalanine chloromethyl ketone (27.23+/-4.37 % inhibition) showed inhibitory effect on proteolysis. Addition of thiol activators dithiothreitol and L-cysteine had not enhanced azocaseinolytic activity. The data suggest that protein digestion in the larvae of T. granarium is primarily dependent on serine proteinases; trypsin- and chymotrypsin-like proteinases.     

Purification and substrate specificity of two cysteine proteinases of Giardia lamblia.

The proteinase activity present in homogenates of trophozoites of Giardia lamblia, active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures. These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of Mr = 95,000 and 35,000 +/- 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (Mr = 95,000) and proteinase II (Mr = 35,000) were active against the beta-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1-6, 8-18, and 20-30 of the insulin beta-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.     

Novel aspartyl proteinase associated to fat body histolysis during Ceratitis capitata early metamorphosis

During larva to adult transition, the larval fat body of the Medfly (Ceratitis capitata) progressively disintegrates to be replaced by the adult one, after imago ecdysis. Here we show that a temporal correlation exists among the microscopy images of fat body progressive disintegration, the activation of fat body lysosomes (as judged by acid phosphatase activity), and the activity of a novel fat body aspartyl proteinase. The enzyme was purified and partially characterized. This proteinase exhibited a wide range of acid isoforms with isoelectric points from 5.6 to 7.3, an optimum pH of 3.0 for hemoglobin digestion, and was completely inhibited by pepstatin A. The apparent molecular weight was estimated (42 +/- 1 kDa) and the protein was characterized as N-glycosylated, judging from affinity to Concanavalin A. From the biochemical characteristics, the enzyme that we called "Early Metamorphosis Aspartyl Proteinase" (EMAP) appears to be similar to mammalian Cathepsin D. However, the N-terminal sequence of EMAP showed no similarity with any known animal Cathepsins and exhibited an important instability to neutral and alkaline pH. This feature seems to be a peculiar characteristic of insect aspartyl proteinases. The temporal activity profile of EMAP during metamorphosis correlated well with the microscopy images of fat body cell autolytic death. Our data support the notion that EMAP is a metamorphosis-specific lysosomal proteinase, mostly expressed during larval fat body histolysis.     

Purification and characterization of a cysteine proteinase from silkworm eggs

Eggs of the silkworm, Bombyx mori, contain a high level of a proteinase which is most active in acidic pH region. The proteinase was purified from an extract of eggs by a six-step procedure which included conventional chromatographic fractionations. The molecular mass of the proteinase was estimated to be 350 kDa by gel filtration and 47 kDa by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels, suggesting an octameric structure. The amino acid composition was found to resemble that of mammalian lysosomal cysteine proteinases, in particular cathepsin L. The NH2-terminal 10-residue sequence is Val-Gln-Phe-Phe-Asp-Leu-Val-Lys-Glu-Glu-. The enzyme appears to be a member of the class of cysteine proteinases since it was strongly inhibited by sulfhydryl-reactive compounds and N-[N-(1,3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). The enzyme hydrolyzed various protein substrates, such as hemoglobin, vitellogenin, vitellin, and lipophorin, with maximal activity around pH 3-3.5. The specificity of the cleavage sites in the oxidized B chain of insulin was rather well defined and there was high affinity for hydrophobic residues at the P2 and P3 positions. The cysteine proteinase is thought to be involved in protein degradation during embryonic development of silkworm eggs.     

Schistosoma mansoni: purification and characterization of the major acidic proteinase from adult worms

We report purification of the major digestive proteinase from adult worms of Schistosoma mansoni. This enzyme is a thiol proteinase with a pH optimum of 5 and is activated by thiol reagents. It was purified 300-fold using a combination of gel chromatography and chromatofocusing. It readily hydrolyzed hemoglobin with an apparent Km of 0.29 microM and a specific activity of 27 micrograms degraded/min/mg enzyme at 37 C. Peptides with positively charged amino acids were preferentially cleaved. The enzyme degraded Boc-Arg-Arg-7-amino-4-methyl coumarin with a kcat/Km of 9083 M-1 sec-1. Lengthening the peptide chain to 3 amino acids or substituting glycine for the amino terminal arginine resulted in decreased activity. The enzyme was inhibited by chloromethylketone-derivatized peptides of similar sequence and by leupeptin. The purified proteinase exhibits microheterogeneity in different preparations with forms ranging in molecular weight from 30,000 to 35,000, and pI 5.7-6.0.     

Cysteine proteinases from Schistosoma haematobium adult worms.

To identify and characterize cysteine proteinases from Schistosoma haematobium, lyophilized adult worms were homogenized, and enzymes were isolated and purified. From extracts prepared in acidic buffer, 3 putative cysteine proteinases were identified either directly or indirectly. The first proteinase (ShCP1) was identified by labeling with a radioiodinated inhibitor, Z-Tyr-Ala-CHN2, as a 35-kDa protein. However, it could not be detected by silver staining, amino acid sequencing, or by a monoclonal antibody specific for a similar molecule from Schistosoma mansoni. A second cysteine proteinase, ShCP2, was purified by gel filtration and dialysis. This 32-kDa molecule was thiol-dependent and was labeled with Z-Tyr-Ala-CHN2. The amino terminal amino acid sequence of ShCP2 showed remarkable similarity (up to 77%) to that of S. mansoni cathepsin B (SmCP2) as well as to mammalian cysteine proteinases. Both ShCP1 and ShCP2 reacted with polyclonal antibodies against S. mansoni, suggesting the existence of shared antigenic epitopes. A third activity, ShCP3, was identified as possibly a distinct proteinase based on its similarities to a 28-kDa cysteine proteinase from S. mansoni. This preliminary investigation demonstrates that the overall profile of cysteine proteinases in S. haematobium is very similar to that of S. mansoni.     

Aspartyl proteinase from cucumber (Cucumis sativus) seeds. Preparation and characteristics

Aspartyl proteinase (EC 3.4.23) from cucumber seeds was purified by ammonium sulphate fractionation, chromatography on immobilized pepstatin and gel filtration on Sephacryl S-200. The preparation obtained, homogeneous on polyacrylamide-gel electrophoresis in acidic and alkaline media, has a molecular mass of 42,000, pI of 5.2, and shows the highest activity with denatured haemoglobin at pH 3.2. The proteinase is stable in slightly alkaline medium, whereas it is inactivated in acidic medium, especially in the presence of NaCl. The enzyme activity is affected neither by the inhibitors of serine proteinases, sulfhydryl-proteinases and metalloproteinases, nor by divalent metal ions, whereas the enzyme is inactivated by the inhibitors of aspartyl proteinases: 1,2,3-epoxy(p-nitrophenoxy)propane, diazoacetyl-DL-norleucine and pepstatin.     

Strongyloides spp.: demonstration and partial characterization of acidic collagenolytic activity from infective larvae

Infective larvae of Strongyloides spp. have been shown to contain azocollytic enzymes which may aid in host skin penetration. Attempts to demonstrate classical, neutral pH-active collagenase activity in Strongyloides ratti were unsuccessful. In the current study, we investigated the presence of acidic collagenolytic activity in the infective larvae of Strongyloides ransomi, S. ratti, and S. stercoralis. All three species demonstrated collagenolytic activity in acidic homogenates as well as in neutral freeze-thaw fractions. Biochemical characterization of this collagenolytic activity from S. ratti and S. ransomi indicated that it was active over an acidic pH range, although it was stable at a neutral pH. This, along with molecular weight estimates and inhibitor susceptibilities, suggested that the collagenolytic activity was similar to vertebrate acidic cysteinyl proteinases. These studies also indicated that this activity is similar to the acidic cysteinyl proteinases in extracts of S. ransomi.     

Purification and properties of three endopeptidases from baker's yeast

Three endopeptidases, proteinases A, B, and Y, were purified from baker's yeast, Saccharomyces cerevisiae. Two molecular forms of proteinase A (PRA), Mr 45,000 and 54,000, (estimated on SDS-PAGE) were obtained. Both forms were inhibited by pepstatin and other acid proteinase inhibitors. The enzyme digested hemoglobin most rapidly at pH 2.7-3.2 and casein at pH 2.4-2.8 and 5.5-6.0. The optimum pH for hydrolysis of protein substrates could be shifted to about 5 with 4-6 M urea. Urea also stimulated the enzyme activity by 30-50%. As other acid proteinases, the enzyme preferentially cleaved peptide bonds of X-Tyr and X-Phe type. A proteinase B (PRB) preparation of approximately Mr 33,000 possessed milk clotting activity and showed an inhibition pattern typical for seryl-sulfhydryl proteases. The purified enzyme could be stabilized with 40% glycerol and stored at -20 degrees C without significant loss of activity for several months. The third endopeptidase, designated PRY, of Mr 72,000 when estimated by Sephadex G-100 gel filtration, had properties resembling PRA and PRB. Similar to PRB, it could be inhibited by up to 90% with phenylmethylsulfonyl fluoride and para-chloromercuribenzoate and preferentially hydrolyzed the Leu15-Tyr16 peptide bond of the oxidized beta-chain of insulin. On the other hand, contrary to PRB, it had neither milk clotting activity nor esterolytic activity toward N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine ethyl ester and was stable during storage at -20 degrees C without glycerol. The enzyme also showed a lower pH optimum for hydrolysis of casein yellow than PRB.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure and function of acid proteases. VIII. Purification and characterization of cathepsins D from Japanese monkey lung.

Two kinds of cathepsin D were found in Japanese monkey lung and were named cathepsins D-I and D-II. Cathepsin D-I was partially purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. It had properties common to other ordinary cathepsins D in terms of the elution position from a DEAE-cellulose column at pH 8.0, the pH-dependence of activity toward acid-denatured hemoglobin, and the molecular weight of 35,000 as determined by Sephadex G-100 gel filtration. On the other hand, cathepsin D-II was purified about 1,000-fold by a combination of ammonium sulfate fractionation and column chromatographies on DEAE-cellulose and Sephadex G-100. It was a very acidic protein as judged from its elution position from a DEAE-cellulose column at pH 8.0, and the high mobility toward the anode on disc gel electrophoresis at pH 8.6. Its molecular weight was determined to be 35,000 by Sephadex G-100 gel filtration and 39,000 by SDS-polyacrylamide gel electrophoresis. It was optimally active at pH 2.8 against acid-denatured hemoglobin as a substrate, showing 80% of the optimal activity at pH 1.0, and almost no activity above pH 4.0. This pH-profile of activity was similar to that of monkey pepsin C (gastricsin). It did not hydrolyze N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine, a synthetic substrate for pepsin, but was inhibited by a series of pepsin inhibitors such as pepstatin, 1,2-epoxy-3-(p-nitrophenoxy)propane, p-bromophenacyl bromide, and diazoacetyl-DL-norleucine methyl ester, although the diazo reagent was a rather weak inhibitor of the enzyme. The amino acid composition of cathepsin D-II was found to be fairly different from those of other cathepsins D. However, it showed a striking resemblance to that of Japanese monkey pepsinogen C, suggesting some evolutionary relationship between them.     

Trypanosoma cruzi: partial characterization of minor cruzipain isoforms non-adsorbed to Concanavalin A-Sepharose

The present paper reports the partial characterization of a subset of atypical cruzipain molecules which do not bind to Concanavalin A-Sepharose column. They are present in different strains of epimastigote forms of Trypanosoma cruzi and represent a 2-4% of total cruzipain. They were purified by affinity chromatography on Cystatin-Sepharose, recognized by the polyclonal anti-cruzipain serum, and their activity in gelatin-containing gels was completely abolished by E-64, TLCK, leupeptin, and aprotinin but not by PMSF, pepstatin A, EDTA or 1,10-phenantroline. These cysteine proteinases, as well as cruzipain showed to be endoproteinases able to hydrolize azocasein, hemoglobin, and bovine serum albumin at acidic pHs. However, evidences are presented indicating that this subset of cruzipain isoforms were also able to use the same blocked chromogenic peptidyl substrates than cruzipain at similar optimal alkaline pH values although with a different order of preference. Moreover, they showed a different oligosaccharide pattern after enzymatic treatment by high pH anion exchange chromatography, suggesting that this structural difference may account for the atypical behaviour in the lectin column.     

Purification and characterization of a subtilisin-like proteinases secreted in the stationary growth phase of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> H2

Proteinases secreted during the early and late stationary phases have been isolated from the culture liquid of Bacillus amyloliquefaciens H2 using CM-cellulose ion-exchange chromatography with subsequent FPLC on a Mono S column. Considering the character of hydrolysis of specific chromogenic substrates and the type of inhibition, these enzymes were identified as subtilisin-like proteinases. The molecular weight of both proteinases is 29 kD. The proteolytic activity of the proteinases secreted during the early and late stationary phases towards the synthetic substrate Z-Ala-Ala-Leu-pNA was maximal at pH 8.5 and 9.0, respectively. The maximal activity of both proteinases was observed at 37 degrees C, and the proteins were stable within the pH range of 7.2-9.5. The subtilisin-like proteinases from B. amyloliquefaciens were shown to catalyze synthesis of peptide bonds.     

Purification and characterization of aspartic proteinase from sunflower seeds

Aspartic proteinases were purified from sunflower seed extracts by affinity chromatography on a pepstatin A-EAH Sepharose column and by Mono Q column chromatography. The final preparation contained three purified fractions. SDS-PAGE showed that one of the fractions consisted of disulfide-bonded subunits (29 and 9 kDa), and the other two fractions contained noncovalently bound subunits (29 and 9 kDa). These purified enzymes showed optimum pH for hemoglobinolytic activity at pH 3.0 and were completely inhibited by pepstatin A like other typical aspartic proteinases. Sunflower enzymes showed more restricted specificity on oxidized insulin B chain and glucagon than other aspartic proteinases. The cDNA coding for an aspartic proteinase was cloned and sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 440 amino acid residues with a molecular mass of 47,559 Da. The difference between the molecular size of purified enzymes and of the mature enzyme was due to the fact that the purified enzymes were heterodimers formed by the proteolytic processing of the mature enzyme. The derived amino acid sequence of the enzyme showed 30-78% sequence identity with that of other aspartic proteinases.