Anti-Metalloproteases: Production and Characterization of Polyclonal IgG Anti-F2 Fraction Antibodies Purified from the Venom of the Snake Bitis arietans.

Bitis arietans is a medically important snake found in Sub-Saharan Africa. The envenomation is characterized by local and systemic effects, and the lack of antivenoms aggravates the treatment. This study aimed to identify venom toxins and develop antitoxins. The F2 fraction obtained from Bitis arietans venom (BaV) demonstrated the presence of several proteins in its composition, including metalloproteases. Titration assays carried out together with the immunization of mice demonstrated the development of anti-F2 fraction antibodies by the animals. The determination of the affinity of antibodies against different Bitis venoms was evaluated, revealing that only BaV had peptides recognized by anti-F2 fraction antibodies. In vivo analyses demonstrated the hemorrhagic capacity of the venom and the effectiveness of the antibodies in inhibiting up to 80% of the hemorrhage and 0% of the lethality caused by BaV. Together, the data indicate: (1) the prevalence of proteins that influence hemostasis and envenomation; (2) the effectiveness of antibodies in inhibiting specific activities of BaV; and (3) isolation and characterization of toxins can become crucial steps in the development of new alternative treatments. Thus, the results obtained help in understanding the envenoming mechanism and may be useful for the study of new complementary therapies.

The Current State-of-the-Art Identification of Unknown Proteins Using Mass Spectrometry Exemplified on De Novo Sequencing of a Venom Protease from Bothrops moojeni.

(1) Background: The amino acid sequence elucidation of peptides from the gas phase fragmentation mass spectra, de novo sequencing, is a valuable method for the identification of unknown proteins complementary to Edman sequencing. It is increasingly used in shot-gun mass spectrometry (MS)-based proteomics experiments. We review the current state-of-the-art and use the identification of an unknown snake venom protein targeting the human tissue factor (TF) as an example to describe the analysis process based on manual spectrum interrogation. (2) Methods: The immobilized TF was incubated with a crude B. moojeni venom solution. The potential binding partners were eluted and further purified by gel electrophoresis. Edman degradation was performed to elucidate the N-terminus of the 31 kDa protein of interest. High-resolution MS with collision-induced dissociation was employed to generate peptide fragmentation spectra. Sequence tags were deduced and used for searches in the NCBI and Uniprot databases. Protein matches from the snake species were further validated by target MS/MS. (3) Results: Sequence tag D [K/Q] D [I/L] VDD [K/Q] led to a snake venom serine protease (SVSP) from lancehead B. jararaca (P81824). With target MS/MS, 24% of the SVSP sequence were confirmed; an additional 41% were tentatively assigned by data-independent MS. Edman sequencing provided information for 10 N-terminal amino acid residues, also confirming the match to SVSP. (4) Conclusions: The identification of unknown proteins continues to be a challenge despite major advances in MS instrumentation and bioinformatic tools. The main requirement is the generation of meaningful, high-quality MS peptide fragmentation spectra. These are used to elucidate sufficiently long sequence tags, which can subsequently be submitted to searches in protein databases. This basic method does not require extensive bioinformatics because peptide MS/MS spectra, especially of doubly-charged ions, can be analysed manually. We demonstrated the procedure with the elucidation of SVSP. While de novo sequencing quickly indicates the correct protein group, the validation of the entire protein sequence of amino acid-by-amino acid will take time. Reasons are the need to properly assign isobaric amino acid residues and modifications. With the ongoing efforts in genomics and transcriptomics and the availability of ever more data in public databases, the need for de novo MS sequencing will decrease. Still, not every animal and plant species will be sequenced, so the combination of MS and Edman sequencing will continue to be of importance for the identification of unknown proteins.

In Vitro and In Vivo Neutralizing Activity of Uvaria chamae Leaves Fractions on the Venom of Naja nigricollis in Albino Rat and Bovine Blood.

BACKGROUND: Snakebite envenomation is a global priority ranked top among other neglected tropical diseases. There is a folkloric claim that Uvaria chamae is beneficial for the management of snakebite and wounds in African ethnobotanical surveys. Besides, there are many registered patents asserting the health benefits of U. chamae. OBJECTIVE: This study aimed to investigate U. chamae's potentials and identify candidates for the development of tools for the treatment and management of N. nigricollis envenomation. METHODS: Freshly collected U. chamae leaves were air-dried, powdered, and extracted in methanol. The median lethal dose of the extract was determined and further fractionated with n-hexane, n-butanol and ethyl acetate. Each fraction was tested for neutralizing effect against venom-induced haemolytic, fibrinolytic, hemorrhagic, and cytotoxic activities. RESULTS: U. chamae fractions significantly (p<0.05) neutralized the haemolytic activity of N. nigricollis venom in n-butanol; 31.40%, n-hexane; 33%, aqueous residue; 39.60% and ethyl acetate; 40.70% at the concentration of 100mg/ml of each fraction against 10mg/ml of the snake venom when compared to the positive control. The fibrinolytic activity of N. nigricollis venom was significantly (p<0.05) neutralized in n-hexane at 73.88%, n-butanol; 72.22% and aqueous residue; 72.22% by the fractions of U. chamae. In addition, haemorrhagic activity of N. nigricollis venom was significantly (p<0.05) neutralized by U. chamae fractions at the concentrations of 100mg/ml, 200mg/ml and 400mg/ml except for n-butanol and aqueous residues at 400 mg/ml. CONCLUSION: U. chamae leaves fractions possess a high level of protection against N. nigricollis venoms-induced lethality and thus validate the pharmacological rationale for its usage in the management of N. nigricollis envenomation.

Preclinical studies of a novel snake venom-derived recombinant disintegrin with antitumor activity: A review.

Snake venoms consist of a complex mixture of many bioactive molecules. Among them are disintegrins, which are peptides without enzymatic activity, but with high binding affinity for integrins, transmembrane receptors that function to connect cells with components of the extracellular matrix. Integrin-mediated cell attachment is critical for cell migration and dissemination, as well as for signal transduction pathways involved in cell growth. During tumor development, integrins play key roles by supporting cancer cell proliferation, angiogenesis, and metastasis. The recognition that snake venom disintegrins can block integrin functions has spawned a number of studies to explore their cancer therapeutic potential. While dozens of different disintegrins have been isolated, none of them as yet has undergone clinical evaluation in cancer patients. Among the best-characterized and preclinically most advanced disintegrins is vicrostatin (VCN), a recombinant disintegrin that was rationally designed by fusing 62 N-terminal amino acids derived from the disintegrin contortrostatin with 6 C-terminal amino acids from echistatin, the disintegrins from another snake species. Bacterially produced VCN was shown to target multiple tumor-associated integrins, achieving potent anti-tumor and anti-angiogenic effects in in vitro and in vivo models in the absence of noticeable toxicity. This review will introduce the field of snake venom disintegrins as potential anticancer agents and illustrate the translational development and cancer-therapeutic potential of VCN as an example.

A catalog for transcripts in the venom gland of the Agkistrodon acutus: identification of the toxins potentially involved in coagulopathy.

Agkistrodon acutus is a special agkistrodon halys, only distributed in Southern China, with a few exceptions in Vietnam. It is a cherished element used in traditional Chinese medicine. In order to produce a global panorama of gene expression in the Agkistrodon acutus venom gland, a non-normalized cDNA library was constructed, and 8696 high quality 5' end expressed sequenced tags (ESTs) were sequenced and analyzed. The initial sequences were assembled into 2855 clusters. Of these clusters, only 45.60% clusters matched known sequence and 54.40% had no match to any known sequence in GenBank. Except for putative cellular proteins (1184 clusters), the remaining 118 clusters (40.16% of all ESTs) corresponded to sequences associated with diverse toxin function. According to expression abundance, the major toxin components were metalloproteinases (32.08%) and C-type lectin (5.22%), and other components including bradykinin-potentiating peptide (0.90%), serine proteases (0.51%), nucleotidase and nuclease (0.41%), phospholipase A2 (0.30%), disintegrin (0.05%), cytokine-like molecules (0.06%), and other proteins (0.63%). The majority of these components are thought to be responsible for coagulopathy after A. acutus bites. We have therefore generated a comprehensive catalog of the A. acutus venom gland described so far. Gene expression from the very specialized secretory tissue, especially for those involved in coagulopathy, can be surveyed and provide important information in finding novel toxins.

Adenylylation and catalytic properties of Mycobacterium tuberculosis glutamine synthetase expressed in Escherichia coli versus mycobacteria.

Bacterial glutamine synthetases (GSs) are complex dodecameric oligomers that play a critical role in nitrogen metabolism, converting ammonia and glutamate to glutamine. Recently published reports suggest that GS from Mycobacterium tuberculosis (MTb) may be a therapeutic target (Harth, G., and Horwitz, M. A. (2003) Infect. Immun. 71, 456-464). In some bacteria, GS is regulated via adenylylation of some or all of the subunits within the aggregate; catalytic activity is inversely proportional to the extent of adenylylation. The adenylylation and deadenylylation of GS are catalyzed by adenylyl transferase (ATase). Here, we demonstrate via electrospray ionization mass spectrometry that GS from pathogenic M. tuberculosis is adenylylated by the Escherichia coli ATase. The adenylyl group can be hydrolyzed by snake venom phosphodiesterase to afford the unmodified enzyme. The site of adenylylation of MTb GS by the E. coli ATase is Tyr-406, as indicated by the lack of adenylylation of the Y406F mutant, and, as expected, is based on amino acid sequence alignments. Using electrospray ionization mass spectroscopy methodology, we found that GS is not adenylylated when obtained directly from MTb cultures that are not supplemented with glutamine. Under these conditions, the highly related but non-pathogenic Mycobacterium bovis BCG yields partially ( approximately 25%) adenylylated enzyme. Upon the addition of glutamine to the cultures, the MTb GS becomes significantly adenylylated ( approximately 30%), whereas the adenylylation of M. bovis BCG GS does not change. Collectively, the results demonstrate that MTb GS is a substrate for E. coli ATase, but only low adenylylation states are accessible. This parallels the low adenylylation states observed for GS from mycobacteria and suggests the intriguing possibility that adenylylation in the pathogenic versus non-pathogenic mycobacteria is differentially regulated.

Wide distribution of cysteine-rich secretory proteins in snake venoms: isolation and cloning of novel snake venom cysteine-rich secretory proteins.

Cysteine-rich secretory proteins (CRISPs) are found in epididymis and granules of mammals, and they are thought to function in sperm maturation and in the immune system. Recently, we isolated and obtained clones for novel snake venom proteins that are classified as CRISP family proteins. To elucidate the distribution of snake venom CRISP family proteins, we evaluated a wide range of venoms for immuno-cross-reactivity. Then we isolated, characterized, and cloned genes for three novel CRISP family proteins (piscivorin, ophanin, and catrin) from the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus), king cobra (Ophiophagus hannah), and western diamondback rattlesnake (Crotalus atrox). Our results show the wide distribution of snake venom CRISP family proteins among Viperidae and Elapidae from different continents, indicating that CRISP family proteins compose a new group of snake venom proteins.

Proteins from Mucuna pruriens and enzymes from Echis carinatus venom: characterization and cross-reactions.

Mucuna pruriens seeds have been widely used against snakebite in traditional medicine. The antivenin property of a water extract of seeds was assessed in vivo in mice. The serum of mice treated with extract was tested for its immunological properties. Two proteins of Echis carinatus venom with apparent molecular masses of 25 and 16 kDa were detected by Western blot analysis carried out using IgG of mice immunized with extract or its partially purified protein fractions. By enzymatic in-gel digestion and electrospray ionization-mass spectrometry/mass spectrometry analysis of immunoreactive venom proteins, phospholipase A(2,) the most toxic enzyme of snake venom, was identified. These results demonstrate that the observed antivenin activity has an immune mechanism. Antibodies of mice treated with non-lethal doses of venom reacted against some proteins of M. pruriens extract. Proteins of E. carinatus venom and M. pruriens extract have at least one epitope in common as confirmed by immunodiffusion assay.

Effects of aqueous extract of Casearia sylvestris (Flacourtiaceae) on actions of snake and bee venoms and on activity of phospholipases A2.

The crude aqueous extract from the leaves of Casearia sylvestris, a plant found in Brazilian open pastures, was assayed for its ability to inhibit phospholipase A2 (PLA2) activity and some biological activities of bee and several snake venoms, and of a number of isolated PLA2s. The extract induced partial inhibition of the PLA2 activity of venoms containing class I, II and III PLA2s. When tested against the purified toxins, it showed the highest efficacy against class II PLA2s from viperid venoms, being relatively ineffective against the class I PLA2 pseudexin. In addition, C. sylvestris extract significantly inhibited the myotoxic activity of four Bothrops crude venoms and nine purified myotoxic PLA2s, including Lys-49 and Asp-49 variants. The extract was able to inhibit the anticoagulant activity of several isolated PLA2s, with the exception of pseudexin. Moreover, it partially reduced the edema-inducing activity of B. moojeni and B. jararacussu venoms, as well as of myotoxins MjTX-II and BthTX-I. The extract also prolonged the survival time of mice injected with lethal doses of several snake venoms and neutralized the lethal effect induced by several purified PLA2 myotoxins. It is concluded that C. sylvestris constitutes a rich source of PLA2 inhibitors.

Cyanide formation from histidine in Chlorella. A general reaction of aromatic amino acids catalyzed by amino acid oxidase systems.

The formation of HCN from D-histidine in Chlorella vulgaris extracts is shown to be due to the combined action of a soluble protein and a particulate component. Either horse-radish peroxidase (EC 1.11.1.7) or a metal ion with redox properties can be substituted for the particulate component. Ions of manganese and vanadium are especially effective, as are o-phenanthroline complexes of iron. Cobalt ions are less active. The D-amino acid oxidase (EC 1.4.3.3) from kidney and the L-amino acid oxidase (EC 1.4.3.2) from snake venom likewise cause HCN production from histidine when supplemented with the particulate preparation from Chlorella or with peroxidase or with a redox metal ion. The stereospecificity of the amino acid oxidase determines which of the two stereoisomers of histidine is active as an HCN precursor. Though histidine is the best substrate for HCN production, other naturally occurring aromatic amino acids (viz. tyrosine, phenylalanine and tryptophan) can also serve as HCN precursors with these enzyme systems. The relative effectiveness of each substrate varies with the amino acid oxidase enzyme and with the supplement. With respect to this latter property, the particulate preparation from Chlorella behaves more like a metal ion than like peroxidase.

A D-amino acid oxidase from Chlorella vulgaris.

A procedure has been developed for the partial purification from Chlorella vulgaris of an enzyme which catalyzes the formation of HCN from D-histidine when supplemented with peroxidase of a metal with redox properties. Some properties of the enzyme are described. Evidence is presented that the catalytic activity for HCN formation is associated with a capacity for catalyzing the oxidation of a wide variety of D-amino acids. With D-leucine, the best substrate for O2 consumption, 1 mol of ammonia is formed for half a mol of O2 consumed in the presence of catalase. An inactive apoenzyme can be obtained by acid ammonium sulfate precipitation, and reactivated by added FAD. On the basis of these criteria, the Chlorella enzyme can be classified as a D-amino acid oxidase (EC 1.4.3.3). Kidney D-amino acid oxidase and snake venom L-amino acid oxidase, which likewise form HCN from histidine on supplementation with peroxidase, have been compared with the Chlorella D-amino acid oxidase. The capacity of these enzymes for causing HCN formation from histidine is about proportional to their ability to catalyze the oxidation of histidine.