Cesar V.F. Batista

Two novel toxins from the Amazonian scorpion Tityus cambridgei that block Kv1.3 and Shaker B K+-channels with distinctly different affinities

Two novel toxic peptides (Tc30 and Tc32) were isolated and characterized from the venom of the Brazilian scorpion Tityus cambridgei. The first have 37 and the second 35 amino acid residues, with molecular masses of 3,871.8 and 3,521.5, respectively. Both contain three disulfide bridges but share only 27% identity. They are relatively potent inhibitors of K(+)-currents in human T lymphocytes with K(d) values of 10 nM for Tc32 and 16 nM for Tc30, but they are less potent or quite poor blockers of Shaker B K(+)-channels, with respective K(d) values of 74 nM and 4.7 microM. Tc30 has a lysine in position 27 and a tyrosine at position 36 identical to those of charybdotoxin. These two positions conform the dyad considered essential for activity. On the contrary, Tc32 has a serine in the position equivalent to lysine 27 of charybdotoxin and does not contain any aromatic amino acid. Due to its unique primary sequence and to its distinctive preference for K(+)-channels of T lymphocytes, it was classified as the first example of a new subfamily of K(+)-channel-specific peptides (alpha-KT x 18.1). Tc30 is a member of the Tityus toxin II-9 subfamily and was given the number alpha-KT x 4.4.

A novel heterodimeric antimicrobial peptide from the tree-frog Phyllomedusa distincta

We present here the purification and the analysis of the structural and functional properties of distinctin, a 5.4 kDa heterodimeric peptide with antimicrobial activity from the tree‐frog Phyllomedusa distincta. This peptide was isolated from the crude extract of skin granular glands by different chromatographic steps. Its minimal inhibitory concentration was determined against pathogenic Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa strains. Amino acid sequencing and mass spectrometric investigations demonstrated that distinctin is constituted of two different polypeptide chains connected by an intermolecular disulphide bridge. Circular dichroism and Fourier‐transformed infrared spectroscopy studies showed that this molecule adopts, in water, a structure containing a significant percentage of anti‐parallel β‐sheet. A conformational variation was observed under experimental conditions mimicking a membrane‐like environment. Database searches did not show sequence similarities with any known antimicrobial peptides. In the light of these results, we can consider distinctin as the first example of a new class of antimicrobial heterodimeric peptides from frog skin.

Proteomic analysis of the venom from the fish eating coral snake Micrurus surinamensis: Novel toxins, their function and phylogeny

The protein composition of the soluble venom from the South American fish‐eating coral snake Micrurus surinamensis surinamensis, here abbreviated M. surinamensis, was separated by RP‐HPLC and 2‐DE, and their components were analyzed by automatic Edman degradation, MALDI‐TOF and ESI‐MS/MS. Approximately 100 different molecules were identified. Sixty‐two components possess molecular masses between 6 and 8 kDa, are basically charged molecules, among which are cytotoxins and neurotoxins lethal to fish (Brachidanios rerio). Six new toxins (abbreviated Ms1–Ms5 and Ms11) were fully sequenced. Amino acid sequences similar to the enzymes phospholipase A2 and amino acid oxidase were identified. Over 20 additional peptides were identified by sequencing minor components of the HPLC separation and from 2‐DE gels. A functional assessment of the physiological activity of the six toxins was also performed by patch clamp using muscular nicotinic acetylcholine receptor assays. Variable degrees of blockade were observed, most of them reversible. The structural and functional data obtained were used for phylogenetic analysis, providing information on some evolutionary aspects of the venom components of this snake. This contribution increases by a factor of two the total number of α‐neurotoxins sequenced from the Micrurus genus in currently available literature.

Tc1, from Tityus cambridgei, is the first member of a new subfamily of scorpion toxin that blocks K+-channels

A new peptide, Tc1, containing only 23 amino acids closely packed by three disulfide bridges was isolated from the Amazonian scorpion Tityus cambridgei. It blocks reversibly the Shaker B K+‐channels with a K d of 65 nM and displaces binding of noxiustoxin to mouse brain synaptosome membranes. It is the shortest known peptide from scorpion venom that recognizes K+‐channels and constitutes a new structural subfamily of toxin, classified as alphaKTx 13.1.

Antimicrobial peptides from the Brazilian frog Phyllomedusa distincta1

Different peptides were purified by chromatographic procedures from the skin-secretory glands of the frog Phyllomedusa distincta. These are the first peptides reported from this frog species. Their primary structure was determined by a combination of automated Edman degradation and mass spectrometry. Peptide Q2 contains 25 amino acid residues, peptide Q1 and L have 28 each, peptide M contains 31, and peptide K has 33 amino acid residues. They all showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria, presenting minimal inhibitory concentrations from 0.6 to 40 microM, when tested against Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Peptides K, L, and Q1 were chemically synthesized and shown to be active.

Novel α-conotoxins from Conus spurius and the α-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors

α‐Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7‐type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [γ15E]SrIB) were compared to those exerted by the α4/7‐conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch‐clamp technique in mammalian cells expressing α1β1γδ, α4β2 and α3β4 nicotinic acetylcholine receptors. At high concentrations (10 µm), the peptides SrIA, SrIB and [γ15E]SrIB showed weak blocking effects only on α4β2 and α1β1γδ subtypes, but EI also strongly blocked α3β4 receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of α1β1γδ and α4β2 nicotinic acetylcholine receptors if briefly (2–15 s) applied at concentrations several orders of magnitude lower (EC50, 1.78 and 0.37 nm, respectively). These results suggest not only that the novel α‐conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both α1β1γδ and α4β2 nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin–receptor interaction, as well as models with which to design highly specific therapeutic drugs.

Structure, function, and chemical synthesis of Vaejovis mexicanus peptide 24: a novel potent blocker of Kv1.3 potassium channels of human T lymphocytes.

Animal venoms are rich sources of ligands for studying ion channels and other pharmacological targets. Proteomic analyses of the soluble venom from the Mexican scorpion Vaejovis mexicanus smithi showed that it contains more than 200 different components. Among them, a 36-residue peptide with a molecular mass of 3864 Da (named Vm24) was shown to be a potent blocker of Kv1.3 of human lymphocytes (K(d) ∼ 3 pM). The three-dimensional solution structure of Vm24 was determined by nuclear magnetic resonance, showing the peptide folds into a distorted cystine-stabilized α/β motif consisting of a single-turn α-helix and a three-stranded antiparallel β-sheet, stabilized by four disulfide bridges. The disulfide pairs are formed between Cys6 and Cys26, Cys12 and Cys31, Cys16 and Cys33, and Cys21 and Cys36. Sequence analyses identified Vm24 as the first example of a new subfamily of α-type K(+) channel blockers (systematic number α-KTx 23.1). Comparison with other Kv1.3 blockers isolated from scorpions suggests a number of structural features that could explain the remarkable affinity and specificity of Vm24 toward Kv1.3 channels of lymphocytes.

Scorpion toxins from Tityus cambridgei that affect Na+-channels

By means of high performance liquid chromatography (HPLC) the soluble venom of the Amazonian scorpion Tityus cambridgei was fractionated into over 50 different components. Four toxic and/or lethal peptides to mice were obtained in pure form and sequenced. Mass spectrometry analysis showed molecular weights of 7310, 7151, 7259 and 7405, respectively, for toxins Tc48a, Tc49a, Tc54 and Tc49b. The N-terminal amino acid sequence was obtained for the three first toxins mentioned, whereas the full primary structure was determined for Tc49b. It contains 64 amino acid residues, closely packed by four disulfide bridges. Sequence comparison analysis showed similarities around 50% with other toxins from scorpions of the genus Tityus of Brazil. It is lethal to mice at doses of 20 microg per 20 g mouse. The toxin was shown to affect the Na(+)-currents permeability of rat cerebellum granular cells in culture. Almost a complete elimination of current was observed with 100 nM toxin concentration. This effect was partially reversible. Furthermore, this toxin does not modify the function of the Shaker B K(+)-channels expressed on Sf9 cells, nor does it modify the Na(+)-channel function in a similar manner as those reported for the alpha-scorpion toxins purified from other scorpions.

Disulfide bridges and blockage of Shaker B K(+)-channels by another butantoxin peptide purified from the Argentinean scorpion Tityus trivittatus.

A peptide was isolated from the venom of the scorpion Tityus trivittatus. It is an isoform of the toxin TsTX-IV earlier described [Toxicon 37 (1999) 651] and identical to butantoxin [Arch. Biochem. Biophys. 379 (2000) 18], both isolated from the Brazilian scorpion Tityus serrulatus. This newly characterized peptide contains 40 amino acid residues with a molecular mass of [M+H(+)] 4507.0, cross-linked by four disulfide bridges, made between the cysteine pairs: Cys2-Cys5, Cys10-Cys31, Cys16-Cys36 and Cys20-Cys38. It blocks in a completely reversible manner the Shaker B K(+)-channels, with a K(d) around 660nM. It belongs to the sub-family 12 and it is now being classified as alpha-KTx 12.2.

Eastern coral snake Micrurus fulvius venom toxicity in mice is mainly determined by neurotoxic phospholipases A2.

UNLABELLED Here we show for the first time that the venom from an elapid (Micrurus fulvius) contains three finger toxin (3FTxs) peptides with low toxicity but high content of lethal phospholipases A2 (PLA2). The intravenous venom LD50 in mice was 0.3μg/g. Fractionation on a C18 column yielded 22 fractions; in terms of abundance, 58.3% of them were components of 13-14kDa and 24.9% were molecules of 6-7kDa. Two fractions with PLA2 activity represented 33.4% of the whole venom and were the most lethal fractions. Fractions with low molecular mass (<7000Da) partially and reversibly blocked the nicotinic acetylcholine receptor (nAChR), with the exception of one that blocked it completely. The fraction that blocked 100% contained two protein species whose dose-response was determined; the IC50s were 13±1 and 9.5±0.3nM. Despite the apparent effect on nAChR none of the low molecular mass fractions were lethal in mice, at concentrations of 1μg/g. From 2D-PAGE and LC-MS/MS, we identified fourteen species of PLA2, four protein species of C-type lectin, three zinc metalloproteinases, one phosphodiesterase and one 3FTx. The N-terminal amino acid sequence of fractions with biological interest was obtained. BIOLOGICAL SIGNIFICANCE In contrast with coral snake venoms from South America, M. fulvius has minor amounts of low molecular mass components, but high content of PLA2, which is responsible for the venom lethality of this species. The results reported here contribute to better understanding of envenomation development and to improve antivenom design and production. These findings break from the paradigm that neurotoxicity caused by Micrurus venoms is mainly attributable to 3FTx neurotoxins and encourage future studies on Micrurus evolution and venom specialization. This article is part of a Special Issue entitled Non-model organisms.