Isolation and characterization of recombinant Drosophila Copia aspartic proteinase

The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe-Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 degrees C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15-Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.     

Degradation of a peptide in pitcher fluid of the carnivorous plant Nepenthes alata Blanco

Carnivorous plants acquire substantial amounts of nitrogen from insects. The tropical carnivorous plant Nepenthes produces trapping organs called pitchers at the tips of tendrils elongated from leaf ends. Acidic fluid is secreted at the bottoms of the pitchers. The pitcher fluid includes several hydrolytic enzymes, and some, such as aspartic proteinase, are thought to be involved in nitrogen acquisition from insect proteins. To understand the nitrogen-acquisition process, it is essential to identify the protein-degradation products in the pitcher fluid. To gain insight into protein degradation in pitcher fluid, we used the oxidized B-chain of bovine insulin as a model substrate, and its degradation by the pitcher fluid of N. alata was investigated using liquid chromatography-mass spectrometry (LC-MS). LC-MS analysis of the degradation products revealed that the oxidized B-chain of bovine insulin was initially cleaved at aromatic amino acids such as phenylalanine and tyrosine. These cleavage sites are similar to those of aspartic proteinases from other plants and animals. The presence of a series of peptide fragments as degradation products suggests that exopeptidase(s) is also present in the pitcher fluid. Amino acid analysis and peptide fragment analysis of the degradation products demonstrated that three amino acids plus small peptides were released from the oxidized B-chain of bovine insulin, suggesting that insect proteins are readily degraded to small peptides and amino acids in the pitcher fluid of N. alata.     

Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600.

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.     

Purification and Characterization of Serine Proteinase 2 from Bacillus intermedius 3-19

A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and kcat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.     

Isolation and Characterization of Glutamyl Endopeptidase 2 from Bacillus intermedius 3-19

The culture filtrate of Bacillus intermedius 3-19 was used for isolation by chromatography on CM-cellulose and Mono S columns of a proteinase that is secreted during the late stages of growth. The enzyme is irreversibly inhibited by the inhibitor of serine proteinases diisopropyl fluorophosphate, has two pH optima (7.2 and 9.5) for casein hydrolysis and one at pH 8.5 for Z-Glu-pNA hydrolysis. The molecular weight of the enzyme is 26.5 kD. The K(m) for Z-Glu-pNA hydrolysis is 0.5 mM. The temperature and pH dependences of the stability of the proteinase were studied. The enzyme was identified as glutamyl endopeptidase 2. The N-terminal sequence (10 residues) and amino acid composition of the enzyme were determined. The enzyme hydrolyzes Glu4-Gln5, Glu17-Asp18, and Cys11-Ser12 bonds in the oxidized A-chain of insulin and Glu13-Ala14, Glu21-Arg22, Cys7-Gly8, and Cys19-Gly20 bonds in the oxidized B-chain of insulin.     

Localization and characterization of hemoglobin-degrading aspartic proteinases from the malarial parasite Plasmodium falciparum.

Three hemoglobin-degrading proteinases were partially purified from food vacuoles isolated from trophozoite-stage forms of the malarial parasite Plasmodium falciparum. Two of the proteinases (M1 and M2) were solubilized by repeated sonication. The remaining proteinase (M3) was solubilized by treatment of the particulate fraction with taurocholic acid, suggesting that proteinase M3 is a membrane-bound proteinase whereas proteinases M1 and M2 are weakly associated with parasite membrane. The location of these proteinases suggests that they may participate in the digestion of host cytosolic protein. After partial purification, but not before, proteinases M1, M2 and M3 are highly sensitive to pepstatin, supporting their designation as aspartic proteinases. These aspartic proteinases show broad specificity for protein substrates. Native hemoglobin, acid denatured hemoglobin and oxidatively damaged hemoglobin are comparable substrates. Hemoglobin within the food vacuole was shown to be primarily native hemoglobin. Chemical modification studies indicate that these three aspartic proteinases have similar properties. The peptide maps from degradation of hemoglobin, however, suggest that aspartic proteinases M1, M2 and M3 are distinct proteinases.     

Protein chemical characterization of Mucor pusillus aspartic proteinase. Amino acid sequence homology with the other aspartic proteinases, disulfide bond arrangement and site of carbohydrate attachment

The amino acid sequence of Mucor pusillus aspartic proteinase was determined by analysis of fragments obtained from cleavage of the enzyme by CNBr and limited tryptic digestion. The proteinase is a single polypeptide chain protein containing 361 amino acid residues, cross-linked by two disulfide bonds. A sugar moiety composed of two GlcNAc residues and four neutral sugar residues is asparagine-linked to the chain. The sequence of M. pusillus proteinase is highly homologous with the M. miehei proteinase (83% identity). The homology with other aspartic proteinases is low (22-24%) and indicates that the Mucor proteinases diverged at an early evolutionary phase. The most conservative regions of the molecule are those involved in catalysis and forming the binding cleft and the core region of the molecule.     

Structure and function of plant aspartic proteinases.

Aspartic proteinases of the A1 family are widely distributed among plant species and have been purified from a variety of tissues. They are most active at acidic pH, are specifically inhibited by pepstatin A and contain two aspartic residues indispensible for catalytic activity. The three-dimensional structure of two plant aspartic proteinases has been determined, sharing significant structural similarity with other known structures of mammalian aspartic proteinases. With a few exceptions, the majority of plant aspartic proteinases identified so far are synthesized with a prepro-domain and subsequently converted to mature two-chain enzymes. A characteristic feature of the majority of plant aspartic proteinase precursors is the presence of an extra protein domain of about 100 amino acids known as the plant-specific insert, which is highly similar both in sequence and structure to saposin-like proteins. This insert is usually removed during processing and is absent from the mature form of the enzyme. Its functions are still unclear but a role in the vacuolar targeting of the precursors has been proposed. The biological role of plant aspartic proteinases is also not completely established. Nevertheless, their involvement in protein processing or degradation under different conditions and in different stages of plant development suggests some functional specialization. Based on the recent findings on the diversity of A1 family members in Arabidopsis thaliana, new questions concerning novel structure-function relationships among plant aspartic proteinases are now starting to be addressed.     

Shewasin A, an active pepsin homolog from the bacterium Shewanella amazonensis

The view has been widely held that pepsin-like aspartic proteinases are found only in eukaryotes, and not in bacteria. However, a recent bioinformatics search [Rawlings ND & Bateman A (2009) BMC Genomics10, 437] revealed that, in seven of ～ 1000 completely sequenced bacterial genomes, genes were present encoding polypeptides that displayed the requisite hallmark sequence motifs of pepsin-like aspartic proteinases. The implications of this theoretical observation prompted us to generate biochemical data to validate this finding experimentally. The aspartic proteinase gene from one of the seven identified bacterial species, Shewanella amazonensis, was expressed in Escherichia coli. The recombinant protein, termed shewasin A, was produced in soluble form, purified to homogeneity, and shown to display properties remarkably similar to those of pepsin-like aspartic proteinases. Shewasin A was maximally active at acidic pH values, cleaving a substrate that has been widely used for assessment of the proteolytic activity of other aspartic proteinases, and displayed a clear preference for cleaving peptide bonds between hydrophobic residues in the P1*P1' positions of the substrate. It was completely inhibited by the general inhibitor of aspartic proteinases, pepstatin, and mutation of one of the catalytic Asp residues (in the Asp-Thr-Gly motif of the N-terminal domain) resulted in complete loss of enzymatic activity. It can thus be concluded unequivocally that this Shewanella gene encodes an active pepsin-like aspartic proteinase. It is now beyond doubt that pepsin-like aspartic proteinases are not confined to eukaryotes, but are encoded within some species of bacteria. The distinctions between the bacterial and eukaryotic polypeptides are discussed and their evolutionary relationships are outlined.     

Proteinases of small intestine enterocytes of swine. Purification and properties of aspartyl proteinase similar to cathepsin D

A new aspartic proteinase was isolated from porcine intestine mucosa by affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100. The enzyme was purified 1600-fold and appeared homogeneous upon polyacrylamide gel electrophoresis. The proteinase has a Mr 60 000 +/- 4000 Da. During sodium dodecyl sulfate polyacrylamide gel electrophoresis the enzyme produced a single protein band (Mr 30 000 +/- 3000 Da). Isoelectric focusing revealed that the enzyme has several multiple forms (pI 6.9, 7.5, 8,0). The enzyme is a glycoprotein containing 5.9% of carbohydrates; the mannose to galactose ratio is 1:3. The amino acid composition of the enzyme was studied. The proteinase splits an oxidized insulin B-chain and synthetic substrates. The pH optimum is 3.2. The enzyme is immunologically identical to porcine spleen cathepsin D.     

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.     

Proteolytic enzymes of Neurospora crassa. Purification and some properties of five intracellular proteinases.

Five intracellular proteolytic enzymes from Neurospora crassa were isolated and partially characterized: an acidic and an alkaline endopeptidase, one carboxypeptidase and two aminopeptidases. All these proteinases were purified from the same crude extract to homogenity by heat treatment, precipitation with ammonium sulfate, chromatography on DEAE-cellulose, CM-cellulose, DEAE-Sephadex, hydroxyapatite and by gel filtration. The acid proteinase hydrolysed acid-denatured haemoglobin at pH 3.0. The alkaline proteinase and the carboxypeptidase are serine proteinases that require a sulfhydryl group for activity. The aminopeptidases are both metallo-proteinases; one posseses broad specifity to the B-chain of oxidized insulin, the other posseses only narrow specifity and can only split the N-terminal basic amino acids of peptides.     

Digestive proteinases in Lasioderma serricorne (Coleoptera: Anobiidae)

The cigarette beetle, Lasioderma serricorne (Fabricius), is a common pest of stored foods. A study of digestive proteinases in L. serricorne was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. Optimal casein hydrolysis by luminal proteinases of L. serricorne was in pH 8.5-9.0 buffers, although the pH of luminal contents was slightly acidic. Results from substrate and inhibitor analyses indicated that the primary digestive proteinases were serine proteinases. The most effective inhibitors of caseinolytic hydrolysis were from soybean (both Bowman Birk and Kunitz), with some inhibition by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and leupeptin. Casein zymogram analysis identified at least eight proteolytic activities. Activity blot analyses indicated one major proteinase activity that hydrolysed the trypsin substrate N-alpha-benzoyl-L-arginine rho-nitroanilide, and three major proteinase activities that hydrolysed the chymotrypsin substrate N-succinyl ala-ala-pro-phe rho-nitroanilide. The absence of cysteine, aspartic, and metallo proteinases in L. serricorne digestion was evidenced by the lack of activation by thiol reagents, alkaline pH optima, and the results from class-specific proteinase inhibitors. The data suggest that protein digestion in L. serricorne is primarily dependent on trypsin- and chymotrypsin-like proteinases.     

Action and Specificity of a Streptomyces Alkalophilic Proteinase

Action of the crystalline alkalophilic proteinase of a Streptomyces sp. which was active at pH values around 13, was investigated. The enzyme attacked various keratinous proteins such as wool, hair, feather etc., dissolving them with hydrolysis degrees from 2 to 6%, and the degradation of several keratins was observed under a microscope and scanning electron-microscope. Specificity of the enzyme was also studied using oxidized insulin B-chain. The enzyme cleaved most easily the peptide linkages between -Ser · His-, -Leu · Val-, -Phe · Tyr- and -Lys · Ala-, though it split several other sites of oxidized insulin B-chain, too, indicating that the action pattern of the proteinase is distinguished from those of alkaline proteinases so far reported.     

Extracellular proteinases of the isolate of Botrytis cinerea virulent to apple tissues

B. cinerea produces extracellular acid proteinases: aspartic proteinase and carboxypeptidase, separable on CM-Sepharose CL-6B. Aspartic proteinase showed the maximum activity at pH 2.5-3.0, was inactivated by diazoacetyl-DL-norleucine methyl ester and was unable to hydrolyse carbobenzoxy Glu-Tyr. Carboxypeptidase showed the maximum activity at pH 4.7-5.0, was inactivated by diisopropyl fluorophosphate, and carbobenzoxy-Glu-Tyr served as an efficient enzyme substrate. The isolated aspartic proteinase hydrolysed proteins in the preparations of apple cell walls. Excretion of aspartic proteinase by B. cinerea preceded that of carboxypeptidase.     

Molecular cloning and characterization of procirsin,an active aspartic protease precursor from Cirsium vulgare (Asteraceae)

Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well-known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on other Astereaceae plant species for identification of new vegetable rennets. Here we report on the cloning, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Procirsin also displayed a high specificity towards κ-casein and milk-clotting activity, suggesting it might be an effective vegetable rennet.The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases.     

Properties and specificity of the major metal-chelator-sensitive proteinase in the keratinolytic larvae of the webbing clothes moth.

The metal chelator-sensitive proteinase activity from the larvae of the webbing clothes moth, Tineola bisselliella, was fractionated into two components by chromatography on DEAE-cellulose and the properties of the major fraction investigated. The approximate molecular weight obtained by gel filtration was 24 000. The pH optimum of 9.4 and the high stability between pH 9.0 and 11.5 are consistent with the alkaline conditions known to be present in the larval mid-gut. The enzyme also showed a second region of high stability around pH 2.3. The cleavage specificity against S-carboxy-methyl A and B chains of insulin was quite different to that of the metal chelator-sensitive proteinases from snake venoms and microorganisms. 10 bonds in the A-chain and 8 bonds in the B-chain were cleaved and the tentative rules governing the specificity limitations of this metal-chelator-sensitive proteinase are discussed.     

The identification of a major product of the degradation of insulin by ''insulin proteinase'' (EC 3.4.22.11).

We have studied a major product in the degradation of insulin by insulin proteinase (EC 3.4.22.11). Semisynthetic [[3H]PheB1]insulin and [[3H]GlyA1]insulin were used in the experiments. The structure of the fragment was deduced by observing the chromatographic and electrophoretic migration of the label both before and after further digestion of the fragment with proteinases of known specificity, with and without additional treatment by performic acid. Ambiguities were resolved by studying the behaviour of authentic fragments of known structure, isolated and characterized after digestion of intact insulin by proteinases of known specificity. We conclude that a major product in the degradation of insulin by insulin proteinase consists of a truncated section of the A chain, joined by the disulphide bridge B7-A7 to a truncated section of the B chain. The A-chain fragment consists most probably of residues A1-A13, and the B-chain fragment consists most probably of residues B1-B9. The similarity between this fragment and that found by other workers when insulin is degraded by intact hepatocytes is significant in the light of proposals that insulin proteinase is a possible participant in the physiological degradation of insulin by target cells.     

Properties of soluble fusions between mammalian aspartic proteinases and bacterial maltose-binding protein

The mammalian aspartic proteinases procathepsin D and pepsinogen form insoluble inclusion bodies when expressed in bacteria. They become soluble but nonnative when synthesized as fusions to the carboxy terminus of E. coli maltose-binding protein (MBP). Since these nonnative states of the two aspartic proteinases showed no tendency to form insoluble aggregates, their biophysical properties were analyzed. The MBP portions were properly folded as shown by binding to amylose, but the aspartic proteinase moieties failed to bind pepstatin and lacked enzymatic activity, indicating that they were not correctly folded. When treated with proteinase K, only the MBP portion of the fusions was resistant to proteolysis. The fusion between MBP and cathepsin D had increased hydrophobic surface exposure compared to the two unfused partners, as determined by bis-ANS binding. Ultracentrifugal sedimentation analysis of MBP–procathepsin D and MBP–pepsinogen revealed species with very large and heterogeneous sedimentation values. Refolding of the fusions from 8 M urea generated proteins no larger than dimers. Refolded MBP–pepsinogen was proteolytically active, while only a few percent of renatured MBP–procathepsin D was obtained. The results suggest that MBP–aspartic proteinase fusions can provide a source of soluble but nonnative folding states of the mammalian polypeptides in the absence of aggregation.     

Effects of lysosomal proteinase inhibition on the development of the rat embryo in vitro

Altered lysosomal function in the visceral yolk sac can result in abnormal development. As proteolysis is an important function of the rodent visceral yolk sac during early and mid-gestation, we characterized the lysosomal proteolytic enzyme activity of this extraembryonic membrane and determined the effects of inhibitors of protein degradation on embryonic development. Constituent activities of cysteine and aspartic acid proteinases were measured in rat visceral yolk sac on gestation day 12, and the effects of the cysteine proteinase inhibitors leupeptin, E-64 [trans-epoxysuccinyl-l-leucylamido(4-guanido)butane] and N-ethylmaleimide and the aspartic acid proteinase inhibitor pepstatin were determined in Sprague-Dawley rat embryos cultured in vitro from gestation days 10-12. It was determined that only cysteine proteinases, primarily cathepsins B and L, are active in the mid-gestation visceral yolk sac. The cysteine proteinase inhibitors leupeptin and E-64 both produced a concentration-related decrease in embryonic growth, as measured by crown-rump length, somite number, and embryonic protein content, and a concentration-related increase in incidence of abnormalities. A characteristic pattern of abnormalities was produced which involved a decrease in neural tube volume and the formation of a subectodermal blister opposite the point of attachment of the vitelline vessels. At high concentrations, anophthalmia was also observed. The decreased neural tube volume was associated with increased osmolality of the exocoelomic fluid, the major extraembryonic fluid compartment. It is possible that the osmotic change decreased neural tube volume by causing water to move to the compartment with a higher solute concentration, out of the embryo.(ABSTRACT TRUNCATED AT 250 WORDS)