New insights into the complex mixture of latex cysteine peptidases in Calotropis procera.

The latex of Calotropis procera is a rich source of proteolytic activity. This latex is known to contain two distinct cysteine peptidases: procerain and procerain B. In this study, new cysteine peptidases were purified from C. procera latex. The enzymes were purified by two sequential ion-exchange chromatography steps (CM-Sepharose plus Resource S(®)) at pH 5.0 and 6.0. The purified enzymes had molecular mass spectra corresponding to CpCP-1=26,213, CpCP-2=26,133 and CpCP-3=25,086 Da. These enzymes exhibited discrete differences in terms of enzymatic activity at a broad range of pH and temperature conditions and contained identical N-terminal amino acid sequences. In these respects, these three new proteins are distinct from those previously studied (procerain and procerain B). Circular dichroism analysis revealed that the new peptidases contain extensive secondary structures, α(15-20%) and ß(26-30%), that were stabilized by disulfide bonds. The purified enzymes exhibited plasma-clotting activity mediated by a thrombin-like mechanism. The set of results suggest the three isolated polypeptides correspond to different post-translationally processed forms of the same protein.

Physico-chemical and antifungal properties of protease inhibitors from Acacia plumosa.

This study was aimed at investigating the purification, biological activity, and some structural properties of three serine protease inhibitors isoforms, denoted ApTIA, ApTIB, and ApTIC from Acacia plumosa Lowe seeds. They were purified from the saline extract of the seeds, using Superdex-75 gel filtration and Mono-S ion exchange chromatography. They were further investigated by mass spectrometry, spectroscopic measurements, surface plasmon resonance, and inhibition assays with proteases and phytopathogenic fungi. The molecular mass of each isoform was estimated at ca. 20 kDa. Each contained two polypeptide chains linked by a disulfide bridge, with different isoelectric points that are acidic in nature. The N-terminal sequences of both chains indicated that they were Kunitz-type inhibitors. Circular dichroism (CD) analyses suggested the predominance of both disordered and beta-strands on ApTI isoforms secondary structure, as expected for beta-II proteins. In addition, it was observed that the proteins were very stable, even at either extreme pH values or at high temperature, with denaturation midpoints close to 75 degrees C. The isoinhibitors could delay, up to 10 times, the blood coagulation time in vitro and inhibited action of trypsin (Ki 1.8 nM), alpha-chymotrypsin (Ki 10.3 nM) and kallikrein (Ki 0.58 microM). The binding of ApTIA, ApTIB, and ApTIC to trypsin and alpha-chymotrypsin, was investigated by surface plasmon resonance (SPR), this giving dissociation constants of 0.39, 0.56 and 0.56 nM with trypsin and 7.5, 6.9 and 3.5 nM with alpha-chymotrypsin, respectively. The growth profiles of Aspergillus niger, Thielaviopsis paradoxa and Colletotrichum sp. P10 were also inhibited by each isoforms. These three potent inhibitors from A. plumosa may therefore be of great interest as specific inhibitors to regulate proteolytic processes.

SBTX, a new toxic protein distinct from soyatoxin and other toxic soybean [Glycine max] proteins, and its inhibitory effect on Cercospora sojina growth.

SBTX, a novel toxin from soybean, was purified by ammonium sulfate fractionation followed by chromatographic steps DEAE-Cellulose, CM-Sepharose and Superdex 200 HR fast-protein liquid chromatography (FPLC). Lethality of SBTX to mice (LD(50) 5.6 mg/kg) was used as parameter in the purification steps. SBTX is a 44-kDa basic glycoprotein composed of two polypeptide chains (27 and 17 kDa) linked by a disulfide bond. The N-terminal sequences of the 44 and 27kDa chains were identical (ADPTFGFTPLGLSEKANLQIMKAYD), differing from that of 17 kDa (PNPKVFFDMTIGGQSAGRIVMEEYA). SBTX contains high levels of Glx, Ala, Asx, Gly and Lys and showed maximum absorption at 280 nm, epsilon(1cm)(1%) of 6.3, and fluorescence emission in the 290-450 nm range upon excitation at 280nm. The secondary structure content was 35% alpha-helix, 13% beta-strand and beta-sheet, 27% beta-turn, 25% unordered, and 1% aromatic residues. Immunological assays showed that SBTX was related to other toxic proteins, such as soyatoxin and canatoxin, and cross-reacted weekly with soybean trypsin inhibitor and agglutinin, but it was devoid of protease-inhibitory and hemagglutinating activities. The inhibitory effect of SBTX on growth of Cercospora sojina, fungus causing frogeye leaf spot in soybeans, was observed at 50 microg/ml, concentration 112 times lesser than that found to be lethal to mice. This effect on phytopathogenic fungus is a potential attribute for the development of transgenic plants with enhanced resistance to pathogens.

Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins / Reconocimiento por anticuerpos de péptidos sintéticos que imitan regiones inmunodominantes de las proteínas p24 y p17 de VIH-1

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant a-helical structure and perform important functions throughout thevirallife-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration.In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic a-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzymeimmunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short a-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C- terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accesibility to the minimal epitopes byspecific antibodies, in solution.

El gen gag del VIH-1 codifica una región de 55kDA que contiene tres subdominios: matriz (p17), cápside (p24) y nucleocápside (p15). Las proteínas p24 y p17 tienen una estructura predominante helicoidal y cumplen un rol importante en el ciclo de vida del virus. En este trabajo presentamos los resultados de inmunorreactividad de péptidos sintéticos que imitan regiones helicoidales de p24 y p17. Utilizando enzimoinmunoensayos se evaluó la influencia de modificaciones en las secuencias nativas sobre la capacidad de reconocimiento de anticuerpos específicos en solución y en fase sólida, incluyendo el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes mínimos. La conformación de los péptidos se determinó por dicroísmo circular y los resultados se correlacionaron con los de inmunorreactividad. Se observó que la capacidad de reconocimiento de anticuerpos por péptidos pequeños que imitan estructuras helicoidales de p24 y p17 mejoró con el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes. Estas modificaciones mejoran la inmovilización sobre las superficies sólidas y permiten una mayor accesibilidad de los anticuerpos a los epitopes mínimos en solución.

Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins.

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant alpha-helical structure and perform important functions throughout the viral life-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration. In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic alpha-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzyme immunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short alpha-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C-terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accessibility to the minimal epitopes by specific antibodies, in solution.

Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins.

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant alpha-helical structure and perform important functions throughout the viral life-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration. In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic alpha-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzyme immunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short alpha-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C-terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accessibility to the minimal epitopes by specific antibodies, in solution.

A sugarcane cystatin: recombinant expression, purification, and antifungal activity.

Plants possess several defense mechanisms against pathogenic attack. One of these defenses is the use of protease inhibitor proteins, which interfere in the development and growth of pathogens. Sugarcane productivity can be impacted by the plant's susceptibility to fungal diseases that result in production losses. A relevant line of investigation, therefore, is into the plant's natural defense mechanisms for the control of phytopathogens using cystatins-proteins that specifically inhibit cysteine proteases. In this paper, we discuss the expression, in Escherichia coli, of a sugarcane cystatin, its purification, antifungal activity, and circular dichroism to monitor correct folding. These studies revealed a secondary structure similar to that of the oryzacystatin I of rice. Moreover, the purified protein proved capable of inhibiting the growth of the filamentous fungus Trichoderma reesei, suggesting that it can also be employed to inhibit the growth of pathogenic sugarcane fungi.

Unfolding and refolding studies of frutalin, a tetrameric D-galactose binding lectin.

Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of frutalin, a tetrameric lectin that presents structural homology with jacalin but shows a more marked biological activity. The initial state in our refolding puzzle was that proteins were unfolded after thermal denaturation or denaturation induced by guanidine hydrochloride, and under both conditions, frutalin was refolded. The denaturation curves, measured by fluorescence emission, gave values of conformational stability of 17.12 kJ.mol-1 and 12.34 kJ.mol-1, in the presence and absence of d-galactose, respectively. Native, unfolded, refolded frutalin and a distinct molecular form denoted misfolded, were separated by size-exclusion chromatography (SEC) on Superdex 75. The native and unfolded samples together with the fractions separated by SEC were also analyzed for heamagglutination activity by CD and fluorescence spectroscopy. The secondary structure content of refolded frutalin estimated from the CD spectra was found to be close to that of the native molecule. All the results obtained confirmed the successful refolding of the protein and suggested a nucleation-condensation mechanism, whereby the sugar-binding site acts as a nucleus to initiate the refolding process. The refolded monomers, after adopting their native three-dimensional structures, spontaneously assemble to form tetramers.

Preliminary crystallographic studies of EcTI, a serine proteinase inhibitor from Enterolobium contortisiliquum seeds.

Enterolobium contortisiliquum trypsin inhibitor (EcTI) belongs to the Kunitz family of plant inhibitors, which are widely distributed in nature, especially in plant seeds. EcTI is composed of two polypeptide chains with a total of 174 residues, homologous to other inhibitors from the same family. EcTI crystals, which were obtained with the acupuncture-gel technique, diffract to 2.0 A resolution and belong to space group P2(1), with unit-cell parameters a = 37.12, b = 38.42, c = 54.08 A, beta = 98.08 degrees. Molecular-replacement techniques using Erythrina caffra trypsin inhibitor (PDB code 1tie) as the search model indicate one monomer in the asymmetric unit. The secondary-structure content of EcTI was determined by circular dichroism spectroscopy, yielding values compatible with the expected topology.

Expression, refolding, and activity of a recombinant nonhemorrhagic snake venom metalloprotease.

Snake venoms are rich sources of proteases that strongly affect the vascular system, by promoting blood coagulation, hemorrhage, and fibrinolysis. Hemorrhagic activity is mostly due to the enzymatic action of metalloproteases on capillary basement membrane components, such as collagen IV, laminin, and fibronectin. A few low-molecular-weight snake venom metalloproteases (svMP) have been described as being devoid of hemorrhagic activity, but they have strong direct-acting fibrinolytic activity that could be very helpful in thrombosis therapy. We have developed an expression system for production of a recombinant svMP from a cDNA (ACLPREF) coding for a small metalloprotease (ACLF) with three disulfide bonds from an Agkistrodon contortrix laticinctus (broad-banded copperhead) venom gland cDNA library. The mature protein-coding region was amplified by PCR and subcloned into the pET28a vector, and the resulting plasmid was used to transform BL21(DE3) Escherichia coli cells. Culture of the transformants at either 37 or 20 degrees C led to the overexpression of an insoluble and inactive 30-kDa protein after 1.0 mM IPTG induction. The expressed protein (rACLF) was recovered from inclusion bodies with 6 M buffered urea solution and purified on a nickel-Sepharose column followed by gel filtration chromatography, both under denaturing conditions. After treatment with dithiothreitol, protein refolding was performed by gradual removal of the denaturing agent by dialysis. The refolded recombinant protein was active in fibrin-agarose plates. The purified protein achieved a conformation similar to that of the native enzyme as judged by circular dichroism analysis.

Influence of the histidine tail on the structure and activity of recombinant chlorocatechol 1,2-dioxygenase.

We present two efficient expression systems for the chlorocatechol 1, 2-dioxygenase (CCD) from Pseudomonas putida. In the first, CCD (encoded by the clcA gene) was expressed in the pETCLCA vector with the addition of an N-terminal histidine tail. After purification, the enzyme (CCD 6xHis) was proteolytically cleaved with thrombin to remove the His tail. The CD spectra of the cleaved and uncleaved enzymes present only minor differences, indicative of correct protein folding. However, the activity of CCD 6xHis, over a wide range of pH, was typically five times lower. This may be the result of steric hindrance caused by the histidine tail. These data are consistent with results obtained using an alternative construct employing a vector which produces a protein product devoid of the His tail. These results suggest that the His tail may induce subtle effects close to the active site which compromise the recovery of full biological activity.

Structural and thermodynamic studies of KM+, a d-mannose binding lectin from Artocarpus integrifolia seeds.

The KM+ lectin exhibits a novel and unusual circular dichroism (CD) spectrum that could be explained by a high proline content that would be inducing deformation of the beta-structure and/or unusual turns. KM+ was shown to be a very rigid lectin, which was very stable under a broad variety of conditions (urea, guanidine, hydrolysis, pH, etc.). Only incubation for 60 min at 333-338 K and extreme basic pH were able to induce conformational changes which could be observed by CD and fluorescence measurements. Data from CD are typical for protein denaturing associated with changes in the overall secondary structure. Data from high-performance size exclusion chromatography (SEC) showed that the denatured forms produced at pH 12.0 are eluted in clusters that co-elute with the native forms. A significant contribution from the tyrosines to the fluorescence emission upon denaturation was observed above 328 K. In fact at 328 K some broadening of the emission spectrum takes place followed by the appearance of a shoulder (approx. 305 nm) at 333 K and above. The sensitivity of tryptophan fluorescence to the addition of sugar suggests a close proximity of the tryptophan residues to the sugar binding site, K(a)=(2.9+/-0.6)x10(3) M(-1). The fraction of chromophore accessible to the quencher obtained is f(a)=0.43+/-0.08, suggesting that approximately 50% of the tryptophan residues are not accessible to quenching by d-mannose. KM+ thermal denaturation was found to be irreversible and was analyzed using a two-state model (N-->D). The results obtained for the activation energy and transition temperature from the equilibrium CD studies were: activation energy, E(a)=134+/-11 kJ/mol and transition temperature, T(m)=339+/-1 K, and from the fluorescence data: E(a)=179+/-18 kJ/mol and T(m)=337+/-1 K. Kinetic studies gave the following values: E(a)=108+/-18 kJ/mol and E(a)=167+/-12 kJ/mol for CD and fluorescence data, respectively.

Isolation and partial characterization of a lectin from Artocarpus incisa L. seeds.

A lectin was isolated from the saline extract of Artocarpus incisa seed by affinity chromatography on cross-linked Adenanthera pavonina galactomannan in 0.15 M NaCl. The lectin was also retained in a D-gal-agarose resin and had no requirements for divalent metal cations (Ca2+ and Mn2+) for activity. The lectin contains 2.1% of carbohydrate and is characterized by high contents of acidic and hydroxylated amino acids. The lectin presented two protein bands in SDS-PAGE, with M(r) 15.5 and 12 kDa, respectively, and contains no alpha-helix, 64% antiparallel beta-sheet and 21% parallel beta-sheet/beta-turn. When submitted to gel filtration in Superose 12 R (FPLC) and Superdex 75 HR 5/5 (HPLC) columns, the lectin showed an M(r) of 48-49 kDa, suggesting a tetrameric structure.

Crystallization and preliminary crystallographic data of a neutrophil migration-inducing lectin (KM+) extracted from the seed of Artocarpus integrifolia.

The tetrameric KM+ lectin from the seeds of Artocarpus integrifolia has, when compared to other plant lectins, the singular property of directly inducing neutrophil migration into the peritoneal cavity or into the air pouch of rats. This protein crystals have been grown and they belong to the orthorhombic system with space group C222(1). The unit cell parameters are a = 54.4 A, b = 127.9 A and c = 99.8 A. A native diffraction dataset to 2.8 A was collected and an analysis of the self-rotation function has shown the presence of only one independent non-crystallographic 2-fold axis orthogonal to the crystal b-axis, compatible with a dimer in the asymmetric unit.

A neutrophil migration-inducing lectin from Artocarpus integrifolia.

A neutrophil migration-inducing protein has been isolated from the saline extract of Artocarpus integrifolia seeds by successive sugar affinity chromatography steps during which the protein was not absorbed by D-galactose resin, and then was absorbed to and eluted from D-mannose resin by 0.1 M D-mannose. Gel filtration on Superdex 75 HR indicated a molecular mass of 52 kDa when 0.1 M D-mannose was present in the elution buffer. A single band of apparent molecular mass of 13 kDa was demonstrable by SDS-PAGE only after heating, both in the presence and absence of reducing agent, suggesting that the molecule is a tetramer formed by the noncovalent association of 13 kDa chains. Isoelectric forms corresponding to isoelectric points of 4.0, 4.2, 5.0, and 5.2 were demonstrable by isoelectric focusing-PAGE, and four active forms having the same isoelectric points were separated by chromatofocusing. The minimal m.w. calculated from amino acid analysis data was 13,193. The protein, denoted KM+, stimulated neutrophil migration in the rat peritoneal cavity assay in a dose-related manner in the range of 1 to 300 micrograms per rat. The dose-response curve of the in vitro chemotactic activity of KM+ was bell shaped and its ascending limb was dose dependent in the range of 1 ng to 10 micrograms/well. D-Mannose (0.1 M) inhibited the in vitro (80%) and in vivo (60%) neutrophil migration-inducing activities of KM+ and also its hemmaglutinating activity. The chemotactic activity was shown to be caused by haptotaxis rather than chemokinesis. The physical and biologic properties of KM+ suggest that this lectin may attract neutrophils by a mechanism involving a haptotactic gradient as has been proposed for IL-8. KM+ might be used as tool to study protein-carbohydrate interactions during neutrophil migration through the extracellular matrix.

Canine liver releases neuropeptide Y during sympathetic nerve stimulation.

To determine whether the liver or gut releases neuropeptide Y (NPY) from their sympathetic nerves, we performed bilateral thoracic sympathetic nerve stimulation (BTSNS) in halothane-anesthetized dogs and calculated gut and liver NPY spillover. BTSNS markedly increased hepatic NPY spillover (delta = +32 +/- 8 ng/min) and arterial NPY concentration (delta = +220 +/- 56 pg/ml), despite no effect on gut NPY spillover (delta = +8 +/- 7 ng/min). To determine the liver's contribution to this increase of circulating NPY, hepatic nerves were selectively stimulated (HNS). Liver NPY spillover increased markedly (delta = +114 +/- 42 ng/min, P < 0.025) during HNS, causing a large increase of arterial NPY (delta = +586 +/- 237 pg/ml, P < 0.025). Using this ratio of liver spillover to arterial increments of NPY, we calculated that the liver makes a major contribution (70%) to circulating NPY levels during BTSNS. The predominant form of canine NPY coeluted with synthetic [Met17]NPY and the minor form of canine NPY coeluted with the oxidized form of [Met17]NPY on high-performance liquid chromatography. We therefore conclude that dog NPY is likely [Met17]NPY and that the liver, rather than the gut, is a major source of circulating NPY during sympathetic nerve stimulation and perhaps stress.