Cloning and preliminary characterization of a novel cuticular antigen from the filarial parasite Dirofilaria immitis

We have described here the cloning and partial characterization of a cDNA encoding a cuticular antigen of Dirofilaria immitis. A 48-h third-stage larval D. immitis cDNA library was immunoscreened with sera raised in mice against third-stage larval cuticles (mouse anti-L3 cuticle antisera). A strongly immunoreactive clone (L3MC4) was isolated. Sequence analysis of L3MC4 showed that it was a partial length cDNA. The missing 5′ end of the clone was amplified by PCR from D. immitis adult female first-strand cDNA using the nematode 22-base splice leader sequence and a L3MC4-specific antisense primer. The composite cDNA sequence comprised 616 bases (nDiL3MC4) encoding a full-length protein of 146 amino acids (DiL3MC4). GenBank analysis showed that DiL3MC4 shared some homology to an unknown C. elegans gene product (31%) at the amino acid level. However, there were no related filarial expressed sequence tags in the current GenBank™ database. Antibodies to recombinant DiL3MC4 (rDiL3MC4) identified a 19-kDa native antigen in the adults and in the L3 and L4 larval stages of D. immitis. In addition, the antibodies bound to the cortical layers of the L3 cuticle, as revealed by immuno-gold electron microscopy. The native protein was not detected in larval and adult excretory–secretory products. Immunoblot analysis showed that serum from a rabbit that was repeatedly injected with a small number of D. immitis third stage larvae reacted with rDiL3MC4. Thus, DiL3MC4 is a novel cuticular antigen of a filarial parasite.     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

Purification and partial characterization of transglutaminase from Physarum polycephalum.

An intracellular form of calcium ion-dependent transglutaminase (R-glutaminylpeptide:amine gamma-glutaminyltransferase, EC 2.3.2.13) was purified 818-fold to apparent homogeneity from acetone powder preparations of spherules of the acellular slime mold Physarum polycephalum. The enzyme was purified by combined methods of precipitation with 15% (wt/vol) polyethylene glycol, DEAE-cellulose chromatography, and isoelectric focusing in a pH 5 to 7 gradient. The isoelectric point of the enzyme was 6.1. The molecular mass of the denatured enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 39.6 kDa. A molecular weight of 77,000 was found by gel filtration of the native enzyme on a Superose 12 fast protein liquid chromatography column, indicating that the native functional protein is a dimer. The purified transglutaminase catalyzed the incorporation of [14C]putrescine into protein substrates including casein, N,N'-dimethylcasein, actin purified from P. polycephalum, and actin purified from bovine muscle. Actin was the preferred substrate for the enzyme, both as a purified protein and in crude extracts prepared from P. polycephalum. With N,N'-dimethylcasein as the amine acceptor substrate, [14C]putrescine, [14C]spermidine, and [14C]spermine were all effective amine donor substrates with Km values of 49, 21.4, and 31.7 microM, respectively. All three of these polyamines demonstrated strong substrate inhibition of the enzyme activity between 100 and 200 microM. Upon starvation induced by depletion of a carbon source for growth, the specific activity of this enzyme increased sixfold during the differentiation of P. polycephalum microplasmodia to spherules. This suggests a role for transglutaminase in the construction of spherules, which have the capacity to survive starvation and dessication.     

Post-translational modification of glutamine and lysine residues of HIV-1 aspartyl protease by transglutaminase increases its catalytic activity

The human immunodeficiency virus type 1 aspartyl protease (HIV-1 PR) is a homodimeric aspartyl endopeptidase that is required for virus replication. HIV-1 PR was shown to act invitro as acyl-donor and -acceptor for both guinea pig liver transglutaminase (TG, EC 2.3.2.13) and human Factor XIIIa. These preliminary evidences suggested that the HIV-1 PR contains at least three TG-reactive glutaminyl and one lysyl residues. We report here that the incubation of HIV-1 PR with TG increases its catalytic activity. This increase is dependent upon the time of incubation, the concentration of TG and the presence of Ca²⁺. Identification of ε-(γ-glutamyl)lysine in the proteolytic digest of the TG-modified HIV-1 PR suggested intramolecular covalent cross-linking of this protease which may promote a non-covalent dimerization and subsequent activation of this enzyme via a conformational change. This hypothesis is supported by the observation that the TG-catalyzed activation of HIV-1 PR was completely abolished by spermidine (SPD) which acts as a competitive inhibitor of ε-(γ-glutamyl)lysine formation. Indeed, in the presence of 1 mM SPD the formation of the isopeptide was decreased of about 80%. The main products of the TG-catalyzed modification of HIV-1 PR in the presence of SPD were N₁-mono(γ-glutamyl)SPD and N₈-mono(γ-glutamyl)SPD. Negligible amount of N₁,N₈-bis(γ-glutamyl)SPD were found. The significance of these results is discussed with respect to the activation of the protease by post-translational modification and design of potential inhibitors.     

Activation of transglutaminase in mu-calpain null erythrocytes

Intracellular transglutaminases (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) are calcium-dependent thiol enzymes that catalyze the covalent cross-linking of proteins, including those in the erythrocyte membrane. Several studies suggest that the activation of some transglutaminases is positively regulated by the calcium-dependent cysteine protease, mu-calpain. Using mu-calpain null (Capn1(-/-)) mouse erythrocytes, we demonstrate that the activation of soluble as well as membrane-bound forms of transglutaminase (TG2) in mouse erythrocytes was independent of mu-calpain. Also, the absence of mu-calpain or any detectable cysteine protease did not affect the transglutaminase activity in the erythrocyte lysate. Our studies also identify physiological substrates of mu-calpain in the erythrocyte membrane and show that their cleavage has no discernible effect on the transglutaminase mediated cross-linking of membrane proteins. Taken together, these data suggest the existence of a calpain-independent mechanism for the activation of transglutaminase 2 by calcium ions in the mouse erythrocytes and presumably also in non-erythroid cells.     

Synthesis and evaluation of peptidic irreversible inhibitors of tissue transglutaminase

Herein we report the synthesis and the evaluation of eight novel compounds as irreversible inhibitors of transglutaminase (TGase). These compounds are based on a minimal peptidic scaffold shown previously [Chem. Biol. 2005, 12, 469–475] to confer affinity for the TGase active site and bear electrophilic groups such as ,β-unsaturated amide, chloroacetamide or maleimide; their general structure being Cbz-Phe-spacer-electrophile. The affinity conferred by the Cbz-Phe scaffold was determined by comparison to N-propylacrylamide and the length of the spacer was also varied to evaluate its importance. The inhibitory efficiencies (k_inact/K_I) of these compounds vary up to 10⁵ M⁻¹ min⁻¹, among the highest reported for derivatives based on this simple Cbz-Phe peptidic scaffold.     

Physicochemical characterization of an aspin (rBm-33) from a filarial parasite Brugia malayi against the important human aspartic proteases

The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable ‘enzyme-product’ complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (K_b) values between 10.23?×?10³ and 6.52?×?10³ M^?1. The binding reactions were enthalpy driven with ΔH_b values between ?50.99 and ?46.07 kJ mol^?1. From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.     

Purification and partial characterization of a thermostable laccase from an unidentified basidiomycete

A thermostable laccase was isolated from an unidentified fungal isolate [Enz. Microb. Technol. 33 (2003) 212], and tentatively named UD4. This work indicates that the enzyme has unique properties other than its thermostability. Investigation into the kinetic parameters of the thermostable laccase yielded an unusually high affinity for ABTS as a substrate (low K_m) when compared with available published data for other laccase isozymes. The specificity constant (k_cat/K_m) was found to be considerably higher than laccase from other sources and is comparable to “white” laccase from Pleurotus ostreatus (POXA1). However, POXA1 isozyme exhibits a large turnover number (k_cat) that contributes to its high specificity constant whereas the high specificity constant for UD4 laccase is achieved by having a high substrate affinity. The UD4 thermostable laccase, like most other laccases, is able to utilize guaiacol as a substrate, whereas POXA1 is unable to oxidize guaiacol, indicating a broader substrate range for the thermostable laccase from UD4. The thermostable laccase is inhibited by sodium azide through non-competitive inhibition, and by thioglycolic acid and hydroxylamine through competitive inhibition. The high specificity constant, substrate affinity and broader substrate range of the thermostable laccase from UD4 indicates that it is a highly favourable candidate enzyme for industrial application.     

Purification and partial characterization of exopolygalacturonase I from Penicillium frequentans

A polygalacturonase with a molecular mass of 74 kDa, an isoelectric point around pH 4.2 and pH – and temperature optima of 3.9 and 50°C, respectively, was purified from a culture fluid of Penicillium frequentans. The enzyme was characterized as an exo-α-1,4-polygalacturonase (exo-PG I). K_m and V_max for sodium polypectate hydrolysis were 0.68 g/l and 596.8 U × mg⁻¹, respectively. The enzyme, a glycoprotein with a carbohydrate content of 81%, is probably the main pectinase of Penicillium frequentans responsible for cleaving monomer units from the non-reducing end of pectin.     

Combination of DEC plus aspirin induced mitochondrial mediated apoptosis in filarial parasite Setaria cervi

Diethylcarbamazine (DEC) is the main drug used against lymphatic filariasis but it is only microfilaricidal. Hence there is an urgent need for adulticidal drug. Aspirin is known nonsteroidal anti-inflammatory drugs which can inhibit prostaglandin H synthase and also induces apoptosis. Studies presented in this paper demonstrated that exposure of worms to the combination of DEC plus aspirin (DEC + A) at 100 μM concentration irreversibly paralyzed adult worms as well as microfilariae within 2 h. Some of the apoptosis markers viz; DNA fragmentation with accompanying ladder formation, upregulation of Bax expression and decrease in Bcl-2 have suggested that the parasite may be killed due to mitochondrial mediated apoptosis. The levels of several apoptosis regulating proteins and enzymes have also shown to be altered. DEC + A treated worms showed significant decrease in prostaglandin H synthase activity (PGHS) and increase in the level of nitric oxide (NO) and cysteine proteases while glutathione (GSH) and peroxidase level was found to be decreased. NO is known inducer of mitochondrial mediated apoptosis and acts by increasing the permeability of mitochondrial membrane through Bax and allowing cytochrome c to release in cytosol, inducing caspases leading to apoptosis. The DEC + A concentration used in this study is much lower than recommended dose so its intake is safe. Here we report for the first time that combination of DEC and aspirin is more effective and could be used as an adulticidal for control of human filarial infections.     

Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization

The immobilization of a glutamate dehydrogenase from Thermus thermophilus (GDH) on glyoxyl agarose beads at pH 7 has permitted to perform the immobilization, purification and stabilization of this interesting enzyme. It was cloned in Escherichia coli and a first thermal shock of the crude preparation destroyed most mesophilic multimeric proteins. Glyoxyl agarose can only immobilize enzymes via a multipoint and simultaneous attachment. Therefore, only proteins having several terminal amino groups in a position that permits their interaction with a flat surface can be immobilized. GDH became rapidly immobilized at pH 7 and its multimeric structure became stabilized as evidenced by SDS-PAGE. This derivative was stable at acidic pH value while the non-stabilized enzyme was very unstable under these conditions due to subunit dissociation. After immobilization, a further incubation at pH 10 improved enzyme stability under any inactivating conditions by increasing the enzyme–support bonds. In fact, GDH immobilized at pH 7 and incubated at pH 10 preserved more activity than GDH directly immobilized at pH 10 (50% versus 15% after 24 h of incubation) and was also more stable (1.5- to 3-fold, depending on the conditions).This method could be extended to any other multimeric enzyme expressed in mesophilic hosts.     

Purification and partial characterization of azoreductase from Enterobacter agglomerans

Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100mg/L of MR. The enzyme was purified approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35 degrees C. This activity was NADH dependent. The K(m) values for both NADH and MR were 58.9 and 29.4 microM, respectively. The maximal velocity (V(max)) was 9.2 micromol of NADH min(-1)mg(-1). The purified enzyme is inhibited by several metal ions including Fe(2+) and Cd(2+).     

Purification and partial characterization of manganese peroxidase from immobilized Phanerochaete chrysosporium

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45 000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN₃, ascorbic acid, β-mercaptoethanol and dithreitol. The K_m values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.     

Purification, properties, and cellular localization of Euglena ferredoxin-NADP reductase

Ferredoxin-NADP reductase from Euglena gracilis Klebs var. Bacillaris Cori purified to apparent homogeneity, yields a typical 36 kDa and an unusual 15 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibits a typical flavoprotein spectrum, contains FAD, and catalyzes NADPH-dependent iodonitrotetrazolium-violet diaphorase, NADPH-specific ferredoxin-dependent cytochrome-c-550 reductase and NADPH-NAD transhydrogenase activities. Rabbit antibody to the purified FNR blocks these activities specifically and also blocks the iodonitrotetrazolium-violet diaphorase activity of Euglena chloroplasts completely. The low iodonitrotetrazolium-violet diaphorase activity in the plastidless mutant, W₁₀BSmL, is mitochondrial and is not specifically blocked by the ferredoxin-NADP reductase antibody. Dark-grown non-dividing (resting) wild-type Euglena cells show a 4-fold increase in ferredoxin-NADP reductase activity during greening at 970 lx. Half of the low ferredoxin-NADP reductase activity in dark-grown cells is initially soluble, but by the end of chloroplast development nearly all of the enzyme is membrane-bound. The binding of ferredoxin-NADP reductase on exposure to light correlates with the extent of thylakoid membrane formation. Immunoblots of wild-type extracts during greening indicate that the 15 kDa polypeptide increases in the same manner as the extent of reductase binding to thylakoid membranes.     

Cloning, production, purification and preliminary crystallographic analysis of a glycosidase from the food lactic acid bacterium Lactobacillus plantarum CECT 748

In recent years, the exquisite stereoselectivity and high efficiency of carbohydrate-processing enzymes have been exploited for many biotechnological applications, including flavor enhancement in foods. In particular, much attention has been focused on the use of β-glucosidases for the enzymatic hydrolysis of flavorless glycoconjugates present in juices and wine beverages for the release aroma volatiles. With the aim to analyze a novel glycosidase with potential applications food industry we have produced and structurally characterized the Bgl glycosidase from the food lactic acid bacterium Lactobacillus plantarum. For that purpose, we have cloned and heterologously expressed the bgl gene (lp_3629) in Escherichia coli. The recombinant protein containing an amino terminal His₆ tag (Bgl) has been produced in a soluble form. Purified recombinant enzyme shows galactosidase activity against 4-nitrophenyl β-d-galactopyranoside but not glucosidase activity. Analytical size-exclusion gel filtration chromatography reveals that Bgl behaves in solution as a mixture of monomeric and a high-molecular weight assembly. Purified Bgl has been crystallized by the hanging-drop vapor-diffusion method at 18 °C. Diffraction data have been collected at ESRF to a resolution of 2.4 Å. The crystals belong to the space group C2 with unit-cell parameters a = 196.7, b = 191.7, c = 105.9, β = 102.7°. The structure refinement is in progress.     

Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley

Proteolytic enzymes hydrolyze cereal seed storage proteins into small peptides and amino acids, which are very important for seed germination and the malting process. A cysteine-class endopeptidase was purified from 4-d-germinated barley (Hordeum vulgare L. cv. Morex). Four purification steps were used, carboxymethyl cellulose cation-exchange chromatography, chromatofocusing, size-exclusion chromatography, and electroelution from a polyacrylamide gel. The endopeptidase was most active at pH 4.5. It's isoelectric point (pI) was 4.4, as determined by isoelectric focusing, and it's SDS-PAGE molecular size was 31 kDa. The enzyme specifically hydrolyzed peptide bonds when the S₂ site contained relatively large hydrophobic amino acids. The N-terminal amino acid sequence residues (1–9) of the 31-kDa endopeptidase had high homology to those of the EP-A and EP-B cysteine proteinases reported previously. The 31-kDa endopeptidase had a hydrolytic specificity similar to that of the Morex green malt 30-kDa endopeptidase we characterized previously, and also reacted with the antibody raised against the purified 30-kDa proteinase, but the two had different mobilities on non-denaturing PAGE. The hydrolytic specificities of both 30- and 31-kDa endopeptidases are such that both would very quickly cleave hordein (barley storage) proteins to small glutamine- and proline-rich peptides that could be quickly degraded to amino acids by barley exopeptidases.Abbreviations CMC carboxymethyl cellulose - E-64 transepoxysuccinyl-l-leucylamido-(4-guanidino)butane - EMI N-ethylmaleimide - IEF isoelectricfocusing - Phen 1,10-phenanthroline - PI isoelectric point - PMSF phenylmethylsulfonyl fluoride We thank the American Malting Barley Association for partially funding this work. Germinated barley seeds were kindly prepared by Eddie D. Goplin. Special thanks to Laurie Marinac for her excellent technical assistance.     

Cloning and biochemical characterization of blisterase,a subtilisin-like convertase from the filarial parasite,Onchocerca volvulus

Blisterase is a subtilisin-like proprotein convertase of nematodes. The enzyme is named after the blistered cuticle found in Caenorhabditis elegans with the bli-4 e937 mutation. The critical role of the enzyme in cuticle production makes it a potential drug target for parasitic nematodes. We have cloned and expressed blisterase from the parasitic nematode Onchocerca volvulus, a major cause of blindness in Africa. The catalytic domain of the protease exhibits 84% identity with the corresponding domain of its closest homologue, C. elegans blisterase. O. volvulus blisterase expressed in insect cells has maximal activity in 1 mm calcium at neutral pH. The protease is inhibited by EDTA, the suicide substrate decanoyl-RVKR-chloromethylketone, alpha1-antitrypsin Portland and by its own propeptide. Substrate assays with fluorescent peptides show that O. volvulus blisterase requires a P4 arginine and a basic amino acid at P1 for cleavage. The kcat of blisterase on the peptide substrate, t-butyloxycarbonyl-RVRR-4-methylcoumaryl-7-amide was determined to be 0.018 s-1. In vitro cleavage studies with the nematode polyprotein antigen demonstrated that blisterase cleaved at tetrabasic (RRKR) but not at dibasic (KR) sites. This report describes the first biochemical characterization of the nematode specific protease, blisterase.     

Cloning and characterization of a gene cluster from Bacillus stearothermophilus comprising infC, rpmI and rplT

Summary Using two synthetic deoxyribonucleotide probes encoding segments of the primary structure of initiation factor IF3 from Bacillus stearothermophilus, we identified and cloned a segment of DNA which carries the infC gene. As in Escherichia coli, the infC gene begins with the unusual initiation triplet AUU, and is followed by the structural genes for ribosomal proteins L35 and L20 (rpmI and rplT, respectively).     

Purification and partial characterization of a protease associated with photosystem II particles

A protease was extracted with 1 M NaCl from spinach ( Spinacia oleracea L.) photosystem II (PSII) particles and purified through gel filtration and anion-exchange chromatography. SDS-polyacrylamide gel electrophoresis of the protease revealed a polypeptide with a molecular mass of 43 kDa. The activity of the purified protease was assayed using a 24 kDa water-soluble protein as substrate, visualized through SDS-PAGE. The protease even remained active in the presence of 0.1 and 0.2 M NaCl, although the degradation pattern changed, which indicated that the protease was different from that reported earlier by another group. The presence of 0.3 M NaCl was shown to be inhibitory. The protease was inhibited by 1,10-phenanthroline and EGTA-NaOH (pH 7.0), indicating that the metal ions are essential for activity and that the enzyme is a metal-protease. FTIR spectroscopy was used to examine the conformationally sensitive amide I' bands of the protease. The protease was observed to undergo spectroscopic changes that reflect the conformational changes that take place when Ca²⁺ is bound, which further confirms that the protease is a metal-protease.     

GTP is required to stabilize and display transamidation activity of transglutaminase 2

Transglutaminase 2 (TGase 2) is a bifunctional enzyme that catalyzes calcium-dependent transamidation and GTP binding/hydrolysis. The transamidation activity is proposed to be associated with several neurodegenerative disorders such as Alzheimer's and Hungtinton's disease. However, the regulation mechanism by which TGase 2 causes neurodegeneration is unknown. In this study, we show that two activities of TGase 2 have a differential stability; transamidation activity is less stable than GTP hydrolytic activity, and that GTP was required to stabilize and to display transamidation activity. Moreover, GTP binding-defective mutant of TGase 2 did not show any transamidation activity in transfection experiments. These results indicate that GTP binding is crucial for transamidation activity of TGase 2, suggesting that protein cross-linking by TGase 2 might be associated with G-protein coupled receptor signaling system. Thus, our data could contribute to understand the regulation of TGase 2 activity and TGase 2-associated pathogenesis.