A SequenceSpace analysis of Lys49 phospholipases A2: Clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity

'SequenceSpace' analysis is a novel approach which has been used to identify unique amino acids within a subfamily of phospholipases A2 (PLA2) in which the highly conserved active site residue Asp49 is substituted by Lys (Lys49-PLA2s). Although Lys49-PLA2s do not bind the catalytic co-factor Ca2+ and possess extremely low catalytic activity, they demonstrate a Ca2+-independent membrane damaging activity through a poorly understood mechanism, which does not involve lipid hydrolysis. Additionally, Lys49-PLA2s possess combined myotoxic, oedema forming and cardiotoxic pharmacological activities, however the structural basis of these varied functions is largely unknown. Using the 'SequenceSpace' analysis we have identified nine residues highly unique to the Lys49-PLA2 sub-family, which are grouped in three amino acid clusters in the active site, hydrophobic substrate binding channel and homodimer interface regions. These three highly specific residue clusters may have relevance for the Ca2+-independent membrane damaging activity. Of a further 15 less stringently conserved residues, nine are located in two additional clusters which are well isolated from the active site region. The less strictly conserved clusters have been used in predictive sequence searches to correlate amino acid patterns in other venom PLA2s with their pharmacological activities, and motifs for presynaptic and combined toxicities are proposed.

Crystallization and preliminary X-ray analysis of SIII-SPIII, a ahospholipase A2 isolated from the venom of Bothrops jararacussu

Large single crystals have been obtained of SIII-SPIII, a phospholipase A2 from the venom of Bothrops jararacussu. The crystals belong to the orthorhombic system space group C222, and diffract X-rays to a resolution of 1.9 Å. Preliminary analysis reveals the presence of one molecule in the crystallographic asymmetric unit. The crystal structure is currently being determined using molecular replacement techniques.

A theoretical study of the A2 ∑+-X2∏ system of the SiP molecule

The A2∑+ and Z2∏ electronic states of the SiP species have been investigated theoretically at a very high level of correlation treatment (CASSCF/MRSDCI). Very accurate potential energy curves are presented for both states, as well as the associated spectroscopic constants as derived from the vib-rotational energy levels determined by means of the numerical solution of the radial Schrödinger equation. Electronic transition moment function, oscillator strengths, Einstein coefficients for spontaneous emission, and Franck-Condon factors for the A2∑+-X2∏ system have been calculated. Dipole moment functions and radiative lifetimes for both states have also been determined. Spin-orbit coupling constants are also reported. The radiative lifetimes for the A2∑+ state, taking into account the spin-orbit diagonal correction to the X2∏ state, decrease from a value of 138 ms at v′ = 0 to 0.48 ms at v′ = 8, and, for the X2∏ state, from 2.32 s at v″ = 1 to 0.59 s at v″ = 5. Vibrational and rotational transitions are expected to be relatively strong.

Structure of myotoxin II, a catalytically inactive Lys49 phospholipase A2 homologue from Atropoides nummifer venom

Lys49 snake-venom phospholipase A2 (PLA2) homologues are highly myotoxic proteins which, although lacking catalytic activity, possess the ability to disrupt biological membranes, inducing significant muscle-tissue loss and permanent disability in severely envenomed patients. Since the structural basis for their toxic activity is still only partially understood, the structure of myotoxin II, a monomeric Lys49 PLA2 homologue from Atropoides nummifer, has been determined at 2.08 Å resolution and the anion-binding site has been characterized. © 2006 International Union of Crystallography. All rights reserved.

Myotoxic phospholipases A2 isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: Cytotoxic effect on microorganism and tumor cells

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A2 (PLA2s) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing Mr ∼ 14,000 for the monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA2s from snake venoms, MTX-I belonging to Asp49 PLA2 class, enzymatically active, and MTX-II to Lys49 PLA2s, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA2 and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA2s induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA2 proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer. © 2008 Elsevier Inc. All rights reserved.

Crystallographic portrayal of different conformational states of a Lys49 phospholipase A2 homologue: Insights into structural determinants for myotoxicity and dimeric configuration

Catalytically inactive phospholipase A2 (PLA2) homologues play key roles in the pathogenesis induced by snake envenomation, causing extensive tissue damage via a mechanism still unknown. Although, the amino acid residues directly involved in catalysis are conserved, the substitution of Asp49 by Arg/Lys/Gln or Ser prevents the binding of the essential calcium ion and hence these proteins are incapable of hydrolyzing phospholipids. In this work, the crystal structure of a Lys49-PLA2 homologue from Bothrops brazili (MTX-II) was solved in two conformational states: (a) native, with Lys49 singly coordinated by the backbone oxygen atom of Val31 and (b) complexed with tetraethylene glycol (TTEG). Interestingly, the TTEG molecule was observed in two different coordination cages depending on the orientation of the nominal calcium-binding loop and of the residue Lys49. These structural observations indicate a direct role for the residue Lys49 in the functioning of a catalytically inactive PLA2 homologue suggesting a contribution of the active site-like region in the expression of pharmacological effects such as myotoxicity and edema formation. Despite the several crystal structures of Lys49-PLA2 homologues already determined, their biological assembly remains controversial with two possible conformations. The extended dimer with the hydrophobic channel exposed to the solvent and the compact dimer in which the active site-like region is occluded by the dimeric interface. In the MTX-II crystal packing analysis was found only the extended dimer as a possible stable quaternary arrangement. © 2012 Elsevier B.V.

Structural and functional studies with mytoxin II from Bothrops moojeni reveal remarkable similarities and differences compared to other catalytically inactive phospholipases A2-like

Lys49-phospholipases A2 (Lys49-PLA2s) are proteins found in bothropic snake venoms (Viperidae family) and belong to a class of proteins which presents a phospholipase A2 scaffold but are catalytically inactive. These proteins (also known as PLA2s-like toxins) exert a pronounced local myotoxic effect and are not neutralized by antivenom, being their study relevant in terms of medical and scientific interest. Despite of the several studies reported in the literature for this class of proteins only a partial consensus has been achieved concerning their functional-structural relationships. In this work, we present a comprehensive structural and functional study with the MjTX-II, a dimeric Lys49-PLA2 from Bothrops moojeni venom which includes: (i) high-resolution crystal structure; (ii) dynamic light scattering and bioinformatics studies in order to confirm its biological assembly; (iii) myographic and electrophysiological studies and, (iv) comparative studies with other Lys49-PLA2s. These comparative analyses let us to get important insights into the role of Lys122 amino acid, previously indicated as responsible for Lys49-PLA2s catalytic inactivity and added important elements to establish the correct biological assembly for this class of proteins. Furthermore, we show two unique sequential features of MjTX-II (an amino acid insertion and a mutation) in comparison to all bothropic Lys49-PLA2s that lead to a distinct way of ligand binding at the toxin's hydrophobic channel and also, allowed the presence of an additional ligand molecule in this region. These facts suggest a possible particular mode of binding for long-chain ligands that interacts with MjTX-II hydrophobic channel, a feature that may directly affect the design of structure-based ligands for Lys49-PLA2s. © 2013 Elsevier Ltd.

Biochemical, functional, structural and phylogenetic studies on Intercro, a new isoform phospholipase A2 from Crotalus durissus terrificus snake venom

Crotoxin is a neurotoxin from Crotalus durissus terrificus venom that shows immunomodulatory, anti-inflammatory, antimicrobial, antitumor and analgesic activities. Structurally, this toxin is a heterodimeric complex composed by a toxic basic PLA2 (Crotoxin B or CB) non-covalently linked to an atoxic non-enzymatic and acidic component (Crotapotin, Crotoxin A or CA). Several CA and CB isoforms have been isolated and characterized, showing that the crotoxin venom fraction is, in fact, a mixture of different molecules derived from the combination of distinct subunit isoforms. Intercro (IC) is a protein from the same snake venom which presents high similarity in primary structure to CB, indicating that it could be an another isoform of this toxin. In this work, we compare IC to the crotoxin complex (CA/CB) and/or CB in order to understand its functional aspects. The experiments with IC revealed that it is a new toxin with different biological activities from CB, keeping its catalytic activity but presenting low myotoxicity and absence of neurotoxic activity. The results also indicated that IC is structurally similar to CB isoforms, but probably it is not able to form a neurotoxic active complex with crotoxin A as observed for CB. Moreover, structural and phylogenetic data suggest that IC is a new toxin with possible toxic effects not related to the typical CB neurotoxin. © 2013.

Efeito α-tomatina na proliferação celular, apoptose e expressão de RNAm dos genes APC, Ciclina A2, Catenina, CASP9, BAK, BAX e BCL-XL em células HT29

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização molecular e expressão heteróloga do alérgeno fosfolipase A1 do veneno de Polybia paulista (Hymenoptera; Vespidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudos estruturais com fosfolipases A2 e fosfolipases A2 homólogas isoladas no veneno de Bothrops moojeni

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ação do extrato bruto da Tabernaemontana catharinensis e de seu alcalóide isolado sobre as atividades de fosfolipases A2 ofídicas em preparação neuromuscular de camundongos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudos estruturais com fosfolipases A2 homólogas de veneno botrópicos em presença de íons com importância funcional

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expressão em Escherichia coli de uma proteína recombinante do gene A2 de Leishmania chagasi e utilização desta proteína no diagnóstico da leishmaniose visceral canina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Participação do grupamento catecolaminérgico A2 do núcleo do trato solitário comissural nos ajustes cardiovasculares e do equilíbrio hidroeletrolitíco induzidos por alterações da osmolaridade ou volume plasmático

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)