Martha Pedraza-Escalona

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.

Negative-shift activation, current reduction and resurgent currents induced by β-toxins from Centruroides scorpions in sodium channels.

The β-toxins purified from the New World scorpion venoms of the Centruroides species affect several voltage-gated sodium channels (VGSCs) and thus are essential tools not only for the discrimination of different channel sub-types but also for studying the structure-function relationship between channels and toxins. This communication reports the results obtained with four different peptides purified from three species of Centruroides scorpions and assayed on seven distinct isoforms of VGSC (Na(v)1.1-Na(v)1.7) by specific functional analysis conducted through single cell electrophysiology. The toxins studied were CssII from Centruroides suffusus suffusus, Cll1 and Cll2 from Centruroides limpidus limpidus and a novel toxin from Centruroides noxius, which was characterized for the first time here. It has 67 amino acid residues and four disulfide bridges with a molecular mass of 7626 Da. Three different functional features were identified: current reduction of macroscopic conductance, left shift of the voltage-dependent activation and induction of resurgent currents at negative voltages following brief, strong depolarizations. The isoforms which revealed to be more affected resulted to be Na(v)1.6 > 1.1 > 1.2 and, for the first time, a β-toxin is here shown to induce resurgent current also in isoforms different from Na(v)1.6. Additionally, these results were analyzed with molecular modelling. In conclusion, although the four toxins have a high degree of identity, they display tri-modal function, each of which shows selectivity among the different sub-types of Na+ -channels. Thus, they are invaluable as tools for structure-function studies of β-toxins and offer a basis for the design of novel ion channel-specific drugs.

Proteomic characterization of the venom and transcriptomic analysis of the venomous gland from the Mexican centipede Scolopendra viridis.

UNLABELLED This communication reports the results of proteomic, transcriptomic, biochemical and electrophysiological analysis of the soluble venom and venom glands of the Mexican centipede Scolopendra viridis Say (here thereafter abbreviated S. viridis). Separation of the soluble venom permitted to obtain 54 different fractions, from which a mass finger printing analysis permitted the identification of at least 86 components, where 70% of the molecules have low molecular masses. Two-dimensional electrophoretic separation of this venom revealed the presence of about forty proteins with molecular weights ranging from 17 to 58kDa. The novo sequencing of 149 peptides obtained by LC-MS/MS from the 2D-gels showed the presence of proteins with amino acid sequences similar to several enzymes and venom allergens type 3. Furthermore, a total of 180 sequences were obtained from a cDNA library prepared with two venomous glands. From this, 155 sequences correspond to complete genes containing more than 200 base pairs each. Comparative sequence analyses of these sequences indicated the presence of different types of enzymes and toxin-like genes. Two proteins with molecular weights around 37,000 and 42,000Da were shown to contain hyaluronidase activity. Electrophysiological assays performed with soluble venom show that it decreases mammalian sodium channel currents. BIOLOGICAL SIGNIFICANCE Animal venoms of Scolopendra species have been scarcely studied, although they have been reported to contain several bioactive compounds, some of which with potential therapeutic interest. The Mexican centipede S. viridis contains a powerful venom, capable of inflicting immediate effects on their preys. This communication is focused on the identification and description of a proteomic and transcriptomic analysis of the protein components of this venom. Several amino acid sequences similar to reported enzymes are the principal components in the S. viridis venom, but also a low number of toxins were identified. This knowledge should contribute to the understanding of the pharmacological effects caused by bites of this centipede species.

Analysis of B-cell epitopes from the allergen Hev b 6.02 revealed by using blocking antibodies

Hev b 6.02 (hevein), identified as a major allergen from natural rubber latex (NRL), is involved in the latex-fruit syndrome and also acts as a pathogenesis defense-related protein. Its 3D structure has been solved at high resolution, and its linear epitopes have already been reported. However, information about conformational epitopes is still controversial, even though it is relevant for an accurate diagnosis and treatment, as well as for the study of allergen-antibody molecular interactions. We sought to analyze the B-cell epitopes of Hev b 6.02 at a molecular and structural level, using specific recombinant antibodies. We obtained a murine monoclonal antibody (mAb 6E7) and three human single chain fragments (scFvs A6, H8, and G7) anti-Hev b 6.02 that were able to compete for hevein binding with serum IgEs from latex allergic patients. In vitro assays showed that the mAb 6E7 and scFv H8 recognized the area of Hev b 6.02 where the aromatic residues are exposed; while the scFv G7 defined the amino and carboxy-terminal regions that lie close to each other, as a different epitope. The structural modeling of the Hev b 6.02-scFv H8 and Hev b 6.02-scFv G7 complexes revealed the putative regions of two conformational epitopes. In one of these, the aromatic residues, as well as polar side chains are important for the interaction, suggesting that they are part of a dominant conformational epitope also presented on the Hev b 6.02-IgE interactions. Antibodies recognizing this important allergen have potential to be used to diagnose and ultimately treat latex allergy.

A single amino acid substitution on the surface of a natural hevein isoform (Hev b 6.0202), confers different IgE recognition

Decreased immune reactivity of isoforms of major allergens has been reported. However, such claims have always been based on experiments with recombinant proteins. This work describes the molecular and physicochemical characterization of a hevein (Hev b 6.0201) natural isoform (Hev b 6.0202), which is present in rubber latex from Hevea brasiliensis. The isoallergen has a single substitution Asn14Asp, which gives rise to local differences in the surface potential, as observed from the crystal structure presented here. Besides, ELISA inhibition using serum pools of adult and pediatric patients showed reduced IgE‐binding capacity (∼27%) with the isoallergen. Overall, these results are relevant to delineate crucial residues involved in this dominant discontinuous epitope.

Crystal structure of Cu / Zn superoxide dismutase from Taenia solium reveals metal-mediated self-assembly

Taenia solium is the cestode responsible for porcine and human cysticercosis. The ability of this parasite to establish itself in the host is related to its evasion of the immune response and its antioxidant defence system. The latter includes enzymes such as cytosolic Cu/Zn superoxide dismutase. In this article, we describe the crystal structure of a recombinant T. solium Cu/Zn superoxide dismutase, representing the first structure of a protein from this organism. This enzyme shows a different charge distribution at the entrance of the active channel when compared with human Cu/Zn superoxide dismutase, giving it interesting properties that may allow the design of specific inhibitors against this cestode. The overall topology is similar to other superoxide dismutase structures; however, there are several His and Glu residues on the surface of the protein that coordinate metal ions both intra‐ and intermolecularly. Interestingly, one of these ions, located on the β2 strand, establishes a metal‐mediated intermolecular β–β interaction, including a symmetry‐related molecule. The factors responsible for the abnormal protein–protein interactions that lead to oligomerization are still unknown; however, high metal levels have been implicated in these phenomena, but exactly how they are involved remains unclear. The present results suggest that this structure could be useful as a model to explain an alternative mechanism of protein aggregation commonly observed in insoluble fibrillar deposits.

Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: Neutralization capacity versus thermodynamic stability

The single-chain antibody fragment (scFv) 6009F, obtained by directed evolution, neutralizes the effects of the Cn2 toxin, which is the major toxic component of Centruroides noxius scorpion venom. In this work we compared the neutralization capacity and the thermodynamic stability of scFv 6009F with those of two other derived formats: Fab 6009F and diabody 6009F. Additionally, the affinity constants to Cn2 toxin of the three recombinant antibody fragments were determined by means of BIAcore. We found a correlation between the thermodynamic stability of these antibody fragments with their neutralization capacity. The order of thermodynamic stability determined was Fab≫scFv>diabody. The Fab and scFv were capable of neutralizing the toxic effects of Cn2 and whole venom but the diabody was unable to fully neutralize intoxication. In silico analysis of the diabody format indicates that the reduction of stability and neutralization capacity could be explained by a less cooperative interface between the heavy and the light variable domains.

Proteomic and transcriptomic analysis of saliva components from the hematophagous reduviid Triatoma pallidipennis

Species belonging to the Triatominae subfamily are commonly associated with Chagas disease, as they are potential vectors of the parasite Trypanosoma cruzi. However, their saliva contains a cocktail of diverse anti-hemostatic proteins that prevent blood coagulation, vasodilation and platelet aggregation of blood; components with indisputable therapeutic potential. We performed a transcriptomic and proteomic analyses of salivary glands and protein spots from 2DE gels of milked saliva, respectively, from the Mexican Triatoma pallidipennis. Massive sequencing techniques were used to reveal this protein diversity. A total of 78 out of 233 transcripts were identified as proteins in the saliva, divided among 43 of 55 spots from 2DE gels of saliva, identified by LC-MS/MS analysis. Some of the annotated transcripts putatively code for anti-hemostatic proteins, which share sequence similarities with proteins previously described for South American triatomines. The most abundant as well as diverse transcripts and proteins in the saliva were the anti-hemostatic triabins. For the first time, a transcriptomic analysis uncovered other unrelated but relevant components in triatomines, including antimicrobial and thrombolytic polypeptides. Likewise, unique proteins such as the angiotensin-converting enzyme were identified not just in the salivary gland transcriptome but also at saliva proteome of this North American bloodsucking insect. BIOLOGICAL SIGNIFICANCE This manuscript is the first report of the correlation between proteome and transcriptome of Triatoma pallidipennis, which shows for the first time the presence of proteins in this insect that have not been characterized in other species of this family. This information contributes to a better understanding of the multiple host defense mechanisms that are being affected at the moment of blood ingestion by the insect. Furthermore, this report gives a repertoire of possible therapeutic proteins.

Autophagy impairment by caspase-1-dependent inflammation mediates memory loss in response to β-Amyloid peptide accumulation

β‐Amyloid peptide accumulation in the cortex and in the hippocampus results in neurodegeneration and memory loss. Recently, it became evident that the inflammatory response triggered by β‐Amyloid peptides promotes neuronal cell death and degeneration. In addition to inflammation, β‐Amyloid peptides also induce alterations in neuronal autophagy, eventually leading to neuronal cell death. Thus, here we evaluated whether the inflammatory response induced by the β‐Amyloid peptides impairs memory via disrupting the autophagic flux. We show that male mice overexpressing β‐Amyloid peptides (5XFAD) but lacking caspase‐1, presented reduced β‐Amyloid plaques in the cortex and in the hippocampus; restored brain autophagic flux and improved learning and memory capacity. At the molecular level, inhibition of the inflammatory response in the 5XFAD mice restored LC3‐II levels and prevented the accumulation of oligomeric p62 and ubiquitylated proteins. Furthermore, caspase‐1 deficiency reinstates activation of the AMPK/Raptor pathway while down‐regulating AKT/mTOR pathway. Consistent with this, we found an inverse correlation between the increase of autophagolysosomes in the cortex of 5XFAD mice lacking caspase‐1 and the presence of mitochondria with altered morphology. Together our results indicate that β‐Amyloid peptide‐induced caspase‐1 activation, disrupts autophagy in the cortex and in the hippocampus resulting in neurodegeneration and memory loss.

New insights into the proteomic characterization of the coral snake Micrurus pyrrhocryptus venom

ABSTRACT A proteomic analysis of the soluble venom of the coral snake Micrurus pyrrhocryptus is reported in this work. The whole soluble venom was separated by RP‐HPLC and the molecular weights of its components (over 100) were determined by mass spectrometry. Three main sets of components were identified, corresponding to peptides with molecular masses from 5 to 8kDa, proteins from 12 to 16kDa and proteins from 20 to 30kDa. Two components were fully sequenced: one &agr;‐neurotoxic peptide of 7210Da with slight blocking activity of the nicotinic acetylcholine receptor (nAChR) and a phospholipase A2 (PLA2) with molecular weight 13517Da and no effect on the nAChR. PLA2 activity was evaluated for all RP‐HPLC components. In addition, N‐terminal sequence was obtained for eleven components using Edman degradation. Among these, three were similar to known PLA2s, six to three‐finger toxins (3FTx) and one to Kunitz‐type serine protease inhibitors. Two‐dimensional gel electrophoresis of the venom allowed the separation of about thirty spots with components of molecular weights from 25 to 70kDa. Seventeen spots were recovered from the gel, digested with trypsin and the corresponding peptides (85) were sequenced by MS/MS allowing identification of amino acid sequences with similarities to snake venom metalloproteases (SVMP), PLA2s, L‐amino acid oxidases (LAAO), acetylcholinesterases (AChE) and serine proteases (SP). In addition, LC‐MS analysis of peptides obtained from tryptic digestion of whole soluble venom allowed the identification of 695 peptides, whose amino acid sequence could correspond to at least 355 components found in other snake venoms, where C‐type lectins, vespryns, zinc finger proteins, and waprins were found, among others. These results show the complexity of the venom and provide important knowledge for future work on identification and activity determination of venom components from this coral snake. HIGHLIGHTSComponents of this coral snake were separated by HPLC and two‐dimensional gel electrophoresis.A complete finger‐printing of soluble venom was obtained by mass spectrometry after HPLC separation.Edman degradation and mass spectrometry analyses allowed the identification of 355 distinct components.Three main types of components were identified: three‐finger peptides, phospholipases and hydrolytic enzymes.Neurotoxic three‐finger peptides predominates in this venom, followed by phospholipases A2.