Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 μg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.     

Primary structure characterization of Bothrops jararacussu snake venom lectin

The complete amino acid sequence of the lectin from Bothrops jararacussu snake venom (BJcuL) is reported. The sequence was determined by Edman degradation and amino acid analysis of the S-carboxymethylated BJcuL derivative (RC-BJcuL) and from its peptides originated from enzymatic digestion. The sequence of amino acid residues showed that this lectin displays the invariant amino acid residues characterized in C-type lectins. Amino acids analysis revealed a high content of acidic amino acids and leucine. These findings suggest that BJcuL, like other snake venom lectins, possesses structural similarities to the carbohydrate recognition domain (CRD) of calcium-dependent animal lectins belonging to the C-type -galactoside binding lectin family.     

广西眼镜王蛇毒碱性磷脂酶A_2(PLA_2)的分离纯化及其性质的研究

广西眼镜王蛇毒用羧甲基纤维素CM-52、磷酸纤维素P-11和Sepharose CL-6B柱层析纯化,得到一个在聚丙烯酰胺凝胶电泳上为单一蛋白带,PLA_2的比活性较原蛇毒提高3.6倍,分子量的为13000,由122个氨基酸组成,_pI为8.9,具有良好的热稳定性。从碱性PLA_2对红细胞影响的电镜观察可见,对人的红细胞膜有明显的作用,而对山羊红细胞作用不明显。PLA_2无论对人还是对山羊、兔和豚鼠红细胞电泳速度都有明显的迟缓作用。     

Cloning, characterization, and structural analysis of a C-type lectin from Bothrops insularis (BiL) venom

Lectins are carbohydrate-binding molecules that mediate a variety of biological processes. In this work, we identify and characterize a lectin from Bothrops insularis venom, with respect to its biochemical properties and theoretical structure. Initially, from a venom gland cDNA library, we cloned and sequenced a cDNA encoding a protein with high identity to snake venom lectins. A lectin molecule was purified to homogeneity from the venom by affinity column and gel filtration. This protein named BiL displayed hemagglutinating activity that was inhibited by galactose, lactose, and EDTA. Mass spectrometry analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that BiL is a disulfide-linked dimeric protein consisting of monomers with 16,206 m/z. The amino acid sequence, deduced from its cDNA sequence, was confirmed by Edman sequencing and by peptide mass fingerprint analysis. BiL shows similarity to other C-type lectin family members. Modeling studies provide insights into BiL dimeric structure and its structural determinants for carbohydrate and calcium binding.     

The calcium-dependent proteolytic system calpain-calpastatin in Drosophila melanogaster.

Ca2+-dependent proteolytic activity was detected at pH 7.5 in head extracts of the fruit fly Drosophila melanogaster. This activity was abolished by iodoacetate, but was unaffected by phenylmethanesulphonyl fluoride. These properties resemble those of the Ca2+-dependent thiol-proteinase calpain. The activity appeared at Mr 280,000 on Sepharose CL-6B gel chromatography. DEAE-cellulose chromatography revealed two activity peaks, with elution positions corresponding to vertebrate calpains I and II. The fly head enzymes were inhibited by a heat-stable and trypsin-sensitive component of the fly head extract, which also inhibited calpains from rat kidney. The inhibitor emerged from Sepharose CL-6B columns at Mr 310,000 and from DEAE-cellulose at a position corresponding to the protein inhibitor calpastatin from other sources. It is concluded that Drosophila heads comprise the Ca2+-dependent calpain-calpastatin proteolytic system.     

Structural and functional properties of Bp-LAAO,a new l-amino acid oxidase isolated from Bothrops pauloensis snake venom

An l-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL-4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65 kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were l-Met, l-Leu, l-Phe and l-Ile and the enzyme showed a strong reduction of its catalytic activity upon l-Met and l-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAO-cDNA of 1548 bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58 kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Bp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents.     

Isolation and characterization of three Ca2+-dependent beta-galactoside-specific lectins from snake venoms.

Three lactose-inhibited lectins from the venoms of the snakes Agkistrodon contortrix contortrix (southern copperhead), Ancistrodon piscivorous leukostoma (western cottonmouth moccasin) and Crotalus atrox (western diamondback rattlesnake) have been isolated and newly characterized. The three lectins are similar to thrombolectin, a lectin isolated from the venom of Bothrops atrox (fer-de-lance) (Gartner, Stocker & Williams, 1980), with regard to sugar specificity, Mr, Ca2+ requirements and sensitivity to reducing agents. Each lectin is a dimer (Mr 28 000) consisting of monomers (Mr 14 000) indistinguishable on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Haemagglutination activity is dependent on the presence of Ca2+ and is inhibited by reducing agents. The lectins are not identical and can be distinguished on the basis of relative affinities for inhibiting sugars, isoelectric points and immunoprecipitation assays using anti-(cottonmouth lectin) serum.     

Inhibition of enzymatic and pharmacological activities of some snake venoms and toxins by Mandevilla velutina (Apocynaceae) aqueous extract

Phospholipases A(2) (PLA(2)) are multifunctional proteins which exhibit varied biological activities correlated to the structural diversities of the sub-classes. The crude aqueous extract from subterranean system of Mandevilla velutina, a plant found in Brazilian savanna, was assayed for its ability to inhibit biological activities of several snake venoms and isolated PLA(2)s. The extract induced total inhibition of the phospholipase activity of Crotalus durissus terrificus venom and only partial inhibition of Bothrops venoms. When assayed against purified toxins, the highest efficacy was detected against CB and crotoxin, while almost ineffective against PLA(2)s from the genus Bothrops. Although M. velutina crude extract significantly inhibited the myotoxic activity of C. d. terrificus venom and CB, it produced only partial inhibition of either Bothrops jararacussu venom or its main myotoxins BthTX-I (basic Lys49), BthTX-II (basic Asp49) and BthA-I-PLA(2) (acidic Asp49). The extract exhibited also full inhibition of hemorrhage caused by Bothrops alternatus, Bothrops moojeni and Bothrops pirajai snake venoms, but partial inhibition (90%) of that induced by B. jararacussu venom. The extract was ineffective to inhibit the fibrinogenolytic activity of B. moojeni, B. alternatus and B. pirajai crude venoms, while their caseinolytic activity was only partially inhibited. No inhibition of the anticoagulant activity, although partial reduction of the edema-inducing activity of C. d. terrificus and B. alternatus crude venoms, CB, PrTX-I, BthTX-I and crotoxin was observed. Besides extending survival of mice injected with lethal doses of C. d. terrificus and B. jararacussu venoms, M. velutina extract decreased to 50% the lethality of mice. Extracts of 18 month old micropropagated plants were able to partially neutralize the effect of the crude venoms and toxins.     

Amino acid sequence and characterization of C-type lectin purified from the snake venom of Crotalus ruber

Galactoside-binding lectin was purified from the snake venom of Crotalus ruber by affinity chromatography on a lactose-agarose column, and the complete amino acid sequence was determined. The C. ruber venom lectin (CRL) showed a single band of 28 kDa by SDS-polyacrylamide electrophoresis under non-reducing conditions, but it showed a single band of 15 kDa under reducing conditions, indicating that CRL is a disulfide-linked homodimer of 15 kDa subunit. CRL specifically recognized beta-galactosides such as thiodigalactoside followed by N-acetylgalactosamine when examined with their inhibitory effects on CRL-induced hemagglutination. A CRL subunit was composed of 135 residues containing nine Cys residues and showed a high similarity to other C-type galactoside-binding lectins from snake venoms. C. atrox lectin (CAL) showed almost the same sequence except for eight amino acid residues. Neither CRL nor CAL induced platelet aggregation by itself or inhibited platelet aggregation mediated by von Willebrand factor or fibrinogen with agonists. CRL showed a similar oligomeric form and the sugar specificity as CAL, but it showed different divalent cation sensitivity such as Mn(2+) and Ni(2+). Homology modeling suggested that the amino acid substitution found in CRL does not affect sugar recognition of the lectin but might alter the conformation and influence the sugar binding pocket induced by the metal-ion binding.     

Isolation and characterization of a mucin-glycoprotein from rat submandibular glands

A blood group A+ mucin-glycoprotein was purified from aqueous extracts of rat submandibular glands by sequential chromatography on columns of Sepharose CL-6B and Sephacryl S-300 in urea-containing buffers. Final purification was facilitated by reductive methylation which appeared to release contaminating (hydrophobic) peptides. Homogeneity of the purified mucin was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at varying concentrations of acrylamide, lectin affinity chromatography, and Western blot analysis. In contrast to previously described preparations, the purified mucin contained only trace amounts of N-acetylglucosamine and aromatic amino acids. In addition, only low levels of basic amino acids were present.     

Ophioluxin,a convulxin-like C-type lectin from Ophiophagus hannah (King cobra) is a powerful platelet activator via glycoprotein VI

Ophioluxin, a potent platelet agonist, was purified from the venom of Ophiophagus hannah (King cobra). Under nonreducing conditions it has a mass of 85 kDa, similar to convulxin, and on reduction gives two subunits with masses of 16 and 17 kDa, slightly larger than those of convulxin. The N-terminal sequences of both subunits are very similar to those of convulxin and other C-type lectins. Ophioluxin induces a pattern of tyrosine-phosphorylated proteins in platelets like that caused by convulxin, when using appropriate concentrations based on aggregation response, because it is about 2-4 times more powerful as agonist than the latter. Ophioluxin and convulxin induce [Ca(2+)](i) elevation both in platelets and in Dami megakaryocytic cells, and each of these C-type lectins desensitizes responses to the other. Convulxin agglutinates fixed platelets at 2 microg/ml, whereas ophioluxin does not, even at 80 microg/ml. Ophioluxin resembles convulxin more than echicetin or alboaggregin B because polyclonal anti-ophioluxin antibodies recognize both ophioluxin and convulxin, but not echicetin, and platelets adhere to and spread on ophioluxin- or convulxin-precoated surfaces in the same way that is clearly different from their behavior on an alboaggregin B surface. Immobilized ophioluxin was used to isolate the glycoprotein VI-Fcgamma complex from resting platelets, which also contained Fyn, Lyn, Syk, LAT, and SLP76. Ophioluxin is the first multiheterodimeric, convulxin-like snake C-type lectin, as well as the first platelet agonist, to be described from the Elapidae snake family.     

Purification and characterization of jararassin-I, A thrombin-like enzyme from Bothrops jararaca snake venom

Snake venoms of the Viperidae family contain a numberof proteins that cause hemostatic disturbances. Enveno-mation of this family is characterized by hemorrhage,edema, local tissue damage, myonecrosis, fibrinolytic andkinin releasing activities [1]. In southeastern Brazil, theviper Bothrops jararaca (Viperidae) is responsible for 90%of snakebite accidents [2]. The enzymes that have proteolytic, coagulate andhemorraghic activities can activate or interfere withthe process of coagulation, and…     

Isolation and functional characterization of proinflammatory acidic phospholipase A2 from Bothrops leucurus snake venom

In the present study, an acidic PLA(2), designated Bl-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000Da and pI was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-α, IL-1β and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA(2) induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism.     

Expression of isolated C-type carbohydrate recognition domains

A galactose-binding lectin from the venom of the snake Trimeresurus stejnegeri consists of isolated carbohydrate recognition domains, belonging to group VII of the C-type animal lectins. As a first step toward determining the tertiary structure of the galactose-specific lectin, we produced the lectin in Escherichia coli. By in vitro refolding and affinity chromatography, modest amounts (8 mg/liter) of active recombinant proteins were obtained. The recombinant protein was homogeneous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. Its amino acid sequence without the initiated methionine at the N-terminus was also characterized by mass spectrometry. The data of hemagglutination and enzyme-linked lectin binding assays demonstrated that the recombinant lectin showed similar sugar-binding activity as the native protein. In addition, fluorescence spectroscopy and circular dichroism also showed obviously their structural similarity.     

Simultaneous isolation of protein C activator,fibrin clot promoting enzyme (fiprozyme) and phospholipase A2 from the venom of the southern copperhead snake

The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal–chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A₂.     

Salt-soluble lectins of corn grain

Lectins extracted from corn (Zea mays L.) kernel with Tris-HCl buffer pH 7.5 were isolated from the crude extract by affinity chromatography on Sepharose 6B-N-acetyl-d-galactosamine and Sepharose 6B-methylα-d-mannoside, and also by lectin affinity chromatography using concanavalin A and Lens culinaris lectin as ligands. According to preferential monosaccharide specificity, salt-soluble lectins of corn seed comprise at least two distinct types: N-acetyl-d-galactosamine-interactive and mannose-interactive lectins. The extracted lectins are unstable, with a tendency to form aggregates during storage.     

Snake venomics of the Brazilian pitvipers Bothrops cotiara and Bothrops fonsecai. Identification of taxonomy markers

We report the proteomic characterization of venom of the pitvipers Bothrops cotiara and Bothrops fonsecai. Crude venoms were fractionated by reverse-phase HPLC, followed by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and CID-MS/MS. Each venom contained around 30 proteins in the range of 7-110 kDa belonging to only 8 (B. cotiara) and 9 (B. fonsecai) families which may target the hemostatic system, albeit distinctly distributed among the two species. B. cotiara and B. fonsecai share medium-sized disintegrins, disintegrin-like/cysteine-rich (DC) fragments, snake venom vascular endothelial growth factor, cysteine-rich secretory proteins, serine proteinases, C-type lectins, l-amino acid oxidase, and Zn(2+)-dependent metalloproteinases. In addition, B. fonsecai expresses a high abundance PLA(2) molecule (13,890 Da), whereas PLA(2) molecules were not detected in B. cotiara's venom. This striking finding is in line with previous biochemical analyses showing the absence of phospholipasic activity in the venom of B. cotiara. The potential adaptive significance of the lack of PLA(2) molecules is enigmatic, and alternative explanations are discussed. B. fonsecai is morphologically extremely similar to B. cotiara. Our comparative proteomic analysis shows that compositional differences between their venoms can be employed as a taxonomy signature for unambiguous species identification independently of geographic origin and morphological characteristics.     

Isolation and properties of a mannan-binding lectin from the coelomic fluid of the holothurian Cucumaria japonica

A new 44-kD, C-type mannan-binding lectin (MBL-C) consisting of two identical subunits was isolated from the coelomic fluid of the holothurian Cucumaria japonica. In the direct hemagglutination assay, the lectin was effectively inhibited by highly branched mannans similar in structure to yeast mannans and composed of alpha-(1-->2)- and alpha-(1-->6)-bound D-mannopyranose residues. Hemagglutination was not inhibited by mannosaccharides, common constituents of the hydrocarbon chains of "normal" glycoproteins. The lectin reaction depends on Ca2+ concentration: maximum activity of MBL-C is observed at 10 mM Ca2+. The activity of MBL-C increases in the pH range from 5 to 7 and reaches maximum at pH 7.0. The lectin is sensitive to temperature. Heating of the lectin solution at temperatures above 40 degrees C decreases activity, while incubation at 90 degrees C for 1 h leads to complete irreversible inactivation. Carbohydrate specificity, Ca2+-dependence, and amino acid composition indicate that MBL-C belongs to the C-type mannan-binding lectins. Polyclonal antibodies against MBL-C revealed its immunochemical similarity to a mannan-binding lectin from another holothurian species, Stichopus japonicus; this provides evidence for structural homology between these proteins.