Deyanira Fuentes-Silva

Development of a rational nomenclature for naming peptide and protein toxins from sea anemones

Sea anemone toxins are predominantly peptide and proteins that act mainly on sodium and potassium channels, as well as in a variety of target cells causing lysis. Over recent years, the number of sea anemone peptide toxins as well as cytolytic pore-forming proteins and phospholipase A(2) sequences submitted to databases has been rapidly increasing due to the developments in DNA sequencing technology and proteomic approaches. However, the lack of a systematic nomenclature has resulted in multiple names being assigned to the same toxins, toxins from unrelated species being designated by the same name, and ambiguous name designations. Therefore, in this work we propose a systematic nomenclature in which we adopted specific criteria, based on order of discovery and phylogenetic analysis, in order to avoid redundant sea anemone toxin names. Implementation of the nomenclature proposed here not only allowed us to rename the already published 191 anemone toxins without ambiguities, but it can be used to unambiguously name newly discovered toxins whether or not they are related to previously published sea anemone sequences. In the new nomenclature each toxin name contains information about the toxins biological activity, origin and relationship to known isoforms. Ongoing increases in the speed of DNA sequencing will raise significantly the number of sea anemone toxin sequences in the literature. This will represent a constant challenge in their clear identification and logical classification, which could be solved using the proposed nomenclature.

A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring λ6 Light-Chain Fibrillogenesis

Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although lambda chains, particularly those belonging to the lambda6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the lambda6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the V(L) (variable region of the light chain)-V(L) interface. This mutant crystallized in two orthorhombic polymorphs, P2(1)2(1)2(1) and C222(1). In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222(1) lattice showed the establishment of intermolecular beta-beta interactions that involved the N-terminus and beta-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the V(L) interface in lambda6 LCs.

Low molecular weight compounds from Zoanthus sociatus impair insulin secretion via Ca+2 influx blockade and cause glucose intolerance in vivo

Cnidarians comprise a taxon with a high biodiversity of cytolitic, neurotoxic and cardiotoxic compounds, which have not been studied on insulin release. We tested the effect of a crude extract of Zoanthus sociatus (Ellis, 1767) and the low molecular weight fraction of this extract on insulin secretion in isolated rat β-cells and also in a glucose tolerance test in vivo. We observed that the extract inhibited insulin release by reducing the amount secreted by individual β-cells and also by silencing a fraction of the secreting population. This effect coincided with a diminished rise of intracellular Ca(+2) in response to high glucose and high K+ -induced depolarization. Moreover intraperitoneal administration of the low molecular weight fraction produced glucose intolerance in adult rats. The active fraction exhibited molecular weights similar to the neurotoxins described in the phylum. Our results broaden the toxic effects of cnidarian venoms and show evidence of potential modulators of voltage-gated Ca(+2) channels in this group.

Toxins from Physalia physalis (Cnidaria) Raise the Intracellular Ca 2+ of Beta-Cells and Promote Insulin Secretion

Physalia physalis is a marine cnidarian from which high molecular weight toxins with hemolytic and neurotoxic effects have been isolated. In the present work, two novel toxins, PpV9.4 and PpV19.3 were purified from P. physalis by bioactive guideline isolation. It involved two steps of column chromatography, gel filtration and RP-HPLC. The molecular weights were 550.7 and 4720.9 Da for PpV9.4 and PpV19.3, respectively. In the light of the Edman sequencing results, the structure of these toxins included the presence of modified amino acids. Both toxins increased the percentage of insulin secreting beta-cells and induced cytosolic Ca2+ elevation. To date, this is the first report of low molecular weight toxins increasing insulin secretion purified from cnidarians, by constituting a new approach to the study of beta-cells physiology.

Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes.

It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l−1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

Structural analysis of the endogenous glycoallergen Hev b 2 (endo-β-1,3-glucanase) from Hevea brasiliensis and its recognition by human basophils.

Endogenous glycosylated Hev b 2 (endo-β-1,3-glucanase) from Hevea brasiliensis is an important latex allergen that is recognized by IgE antibodies from patients who suffer from latex allergy. The carbohydrate moieties of Hev b 2 constitute a potentially important IgE-binding epitope that could be responsible for its cross-reactivity. Here, the structure of the endogenous isoform II of Hev b 2 that exhibits three post-translational modifications, including an N-terminal pyroglutamate and two glycosylation sites at Asn27 and at Asn314, is reported from two crystal polymorphs. These modifications form a patch on the surface of the molecule that is proposed to be one of the binding sites for IgE. A structure is also proposed for the most important N-glycan present in this protein as determined by digestion with specific enzymes. To analyze the role of the carbohydrate moieties in IgE antibody binding and in human basophil activation, the glycoallergen was enzymatically deglycosylated and evaluated. Time-lapse automated video microscopy of basophils stimulated with glycosylated Hev b 2 revealed basophil activation and degranulation. Immunological studies suggested that carbohydrates on Hev b 2 represent an allergenic IgE epitope. In addition, a dimer was found in each asymmetric unit that may reflect a regulatory mechanism of this plant defence protein.

Crystallization and identification of the glycosylated moieties of two isoforms of the main allergen Hev b 2 and preliminary X-ray analysis of two polymorphs of isoform II.

Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a beta-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content consisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4(1) with unit-cell parameters a = b = 150.17, c = 77.41 A were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 A, beta = 113.6 degrees. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.