Timoteo Olamendi-Portugal

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.

Two new scorpion toxins that target voltage-gated Ca2+ and Na+ channels.

This report describes the isolation, primary structure determination, and functional characterization of two similar toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively). KLII from P. granulatus is identical to kurtoxin from Parabuthus transvaalicus (a 63 amino-acid long toxin) whereas KLI is a new peptide containing 62 amino acid residues closely packed by four disulfide bridges with a molecular mass of 7244. Functional assays showed that both toxins, KLI and kurtoxin from P. granulatus, potently inhibit native voltage-gated T-type Ca(2+) channel activity in mouse male germ cells. In addition, KLI was shown to significantly affect the gating mechanisms of recombinant Na(+) channels and weakly block alpha(1)3.3Ca(V) channels expressed in Xenopus oocytes. KLI and kurtoxin from P. granulatus represent new probes to study the role of ion channels in germ cells, as well as in cardiac and neural tissue.

A strategy for the generation of specific human antibodies by directed evolution and phage display. An example of a single-chain antibody fragment that neutralizes a major component of scorpion venom.

This study describes the construction of a library of single‐chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 × 108 recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446‐fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD50 of Cn2 toxin when a 1 : 10 molar ratio of toxin‐to‐antibody fragment was used. It was also able to neutralize 2 LD50 of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.

Scorpion toxins that block T-type Ca2+ channels in spermatogenic cells inhibit the sperm acrosome reaction

The acrosome reaction (AR) is a Ca(2+)-dependent event required for sperm to fertilize the egg. The activation of T-type voltage-gated Ca(2+) channels plays a key role in the induction of this process. This report describes the actions of two toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively) on sperm Ca(2+) channels. Both toxins decrease T-type Ca(2+) channel activity in mouse spermatogenic cells and inhibit the AR in mature sperm. Saturating concentrations of the toxins inhibited at most approximately 70% of the whole-cell Ca(2+) current, suggesting the presence of a toxin-resistant component. In addition, both toxins inhibited approximately 60% of the AR, which is consistent with the participation of T-type Ca(2+) channels in the sperm AR.

Two Novel Toxins from the Venom of the Scorpion Pandinus imperator Show that the N-terminal Amino Acid Sequence is Important for their Affinities towards Shaker B K+ Channels

Abstract. Two novel peptides were purified from the venom of the scorpion Pandinus imperator, and were named Pi2 and Pi3. Their complete primary structures were determined and their blocking effects on Shaker B K+ channels were studied. Both peptides contain 35 amino acids residues, compacted by three disulfide bridges, and reversibly block the Shaker B K+ channels. They have only one amino acid changed in their sequence, at position 7 (a proline for a glutamic acid). Whereas peptide Pi2, containing the Pro7, binds the Shaker B K+ channels with a Kd of 8.2 nm, peptide Pi3 containing the Glu7 residue has a much lower affinity of 140 nm. Both peptides are capable of displacing the binding of 125I-noxiustoxin to brain synaptosome membranes. Since these two novel peptides are about 50% identical to noxiustoxin, the present results support previous data published by our group showing that the amino-terminal region of noxiustoxin, and also the amino-terminal sequence of the newly purified homologues: Pi2, and Pi3, are important for the recognition of potassium channels.

Structural characterization and phylogenetic relationships of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a Lys49 phospholipase A2 homologue

In order to analyze its structure–function relationships, the complete amino acid sequence of myotoxin II from Atropoides (Bothrops) nummifer from Costa Rica was determined. This toxin is a Lys49-type phospholipase A2 (PLA2) homologue, devoid of catalytic activity, structurally belonging to class IIA. In addition to the Asp49→Lys change in the (inactive) catalytic center, substitutions in the calcium-binding loop suggest that its lack of enzymatic activity is due to the loss of ability to bind Ca2+. The toxin occurs as a homodimer of basic subunits of 121 residues. Its sequence has highest similarity to Lys49 PLA2s from Cerrophidion, Trimeresurus, Bothrops and Agkistrodon species, which form a subfamily of proteins that diverged early from Asp49 PLA2s present in the same species, as shown by phylogenetic analysis. The tertiary structure of the toxin was modeled, based on the coordinates of Cerrophidion godmani myotoxin II. Its exposed C-terminal region 115–129 shows several differences in comparison to the homologous sequences of other Lys49 PLA2s, i.e. from Agkistrodon p. piscivorus and Bothrops asper. Region 115–129 of the latter two proteins has been implicated in myotoxic activity, on the basis of the direct membrane-damaging of their corresponding synthetic peptides. However, peptide 115–129 of A. nummifer myotoxin II did not exert toxicity upon cultured skeletal muscle cells or mature muscle in vivo. Differences in several amino acid residues, either critical for toxicity, or influencing the conformation of free peptide 115–129 from A. nummifer myotoxin II, may account for its lack of direct membrane-damaging properties.

Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment

We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591–2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD50 of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.

Isolation and characterization of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a new Lys49 phospholipase A2 homologue

Myotoxic phospholipases A2 of class II are commonly found in the venoms of crotalid snakes. As an approach to understanding their structure-activity relationship, diverse natural variants have been characterized biochemically and pharmacologically. This study describes a new myotoxic phospholipase A2 homologue, isolated from the venom of Atropoides (Bothrops) nummifer from Costa Rica. A. nummifer myotoxin 1 is a basic protein, with an apparent subunit molecular mass of 16 kDa, which migrates as a dimer in sodium dodecylsulfate-polyacrylamide gel electrophoresis under nonreducing conditions. It is strongly recognized by antibodies generated against Bothrops asper myotoxin II, by enzyme-immunoassay. The toxin induces rapid myonecrosis upon intramuscular injection in mice (evidenced by an early increase in plasma creatine kinase activity), and significant edema in the footpad assay. It also displays cytolytic activity upon cultured murine endothelial cells. The toxin (up to 50 microg) has no detectable phospholipase A2 activity on egg yolk phospholipids, and does not show an anticoagulant effect on sheep platelet-poor plasma in vitro. N-terminal sequence determination (53 amino acid residues) demonstrated that A. nummifer myotoxin II is a new Lys49 variant of the family of myotoxic, class II phospholipases A2. Sequence comparison with other phospholipases A2 revealed Asn53 as a novel substitution. In addition, this myotoxin is the first Lys49 variant presenting Asn in its N-terminus. Consequently, these findings suggest that neither Ser1 or Lys53, usually found in this family of proteins, are essential amino acid residues for their myotoxic, cytolytic, or edema-inducing effects.

The use of synthetic peptides can be a misleading approach to generate vaccines against scorpion toxins

Seven peptides corresponding to the amino acid sequence of toxin 2 from the scorpion Centruroides noxius were chemically synthesized, purified and assayed in mice for their putative neutralizing properties against scorpion toxins. All the peptides were immunogenic and some produced neutralizing antibodies, as verified by injecting the antisera with toxin into naive animals. However, direct challenge of pre-immunized mice (with the longest synthetic peptides of 27 and 57 amino acid residues) revealed an unexpected sensitization phenomena: the animals did not resist injection of one LD50 of purified toxin 2 (5% survival), but pre-immunization of mice with native toxin protected 100% of the animals. These findings suggest that vaccine preparations with synthetic peptides corresponding to the amino acid sequence of certain toxins should be analyzed cautiously.

A subfamily of acidic alpha-K+ toxins

Three homologous acidic peptides have been isolated from the venom of three different Parabuthus scorpion species, P. transvaalicus, P. villosus, and P. granulatus. Analysis of the primary sequences reveals that they structurally belong to subfamily 11 of short chain α-K+-blocking peptides (Tytgat, J., Chandy, K. G., Garcia, M. L., Gutman, G. A., Martin-Eauclaire, M. F., van der Walt, J. J., and Possani, L. D. (1999) Trends Pharmacol. Sci. 20, 444–447). These toxins are 36–37 amino acids in length and have six aligned cysteine residues, but they differ substantially from the other α-K+ toxins because of the absence of the critical Lys27 and their total overall negative charge. Parabutoxin 1 (PBTx1), which has been expressed by recombinant methods, has been submitted to functional characterization. Despite the lack of the Lys27, this toxin blocks several Kv1-type channels heterologously expressed in Xenopus oocytes but with low affinities (micromolar range). Because a relationship between the biological activity and the acidic residue substitutions may exist, we set out to elucidate the relative impact of the acidic character of the toxin and the lack of the critical Lys27 on the weak activity of PBTx1 toward Kv1 channels. To achieve this, a specific mutant named rPBTx1 T24F/V26K was made recombinantly and fully characterized on Kv1-type channels heterologously expressed in Xenopus oocytes. Analysis of rPBTx1 T24F/V26K displaying an affinity toward Kv1.2 and Kv1.3 channels in the nanomolar range shows the importance of the functional dyad above the acidic character of this toxin.