GPVI and CLEC-2 in hemostasis and vascular integrity

The glycoprotein VI (GPVI)-FcR gamma-chain complex initiates powerful activation of platelets by the subendothelial matrix proteins collagen and laminin through an immunoreceptor tyrosine-based activation motif (ITAM)-regulated signaling pathway. ITAMs are characterized by two YxxL sequences separated by 6-12 amino acids and are found associated with several classes of immunoglobulin (Ig) and C-type lectin receptors in hematopoietic cells, including Fc receptors. Cross-linking of the Ig GPVI leads to phosphorylation of two conserved tyrosines in the FcR gamma-chain ITAM by Src family tyrosine kinases, followed by binding and activation of the tandem SH2 domain-containing Syk tyrosine kinase and stimulation of a downstream signaling cascade that culminates in activation of phospholipase Cgamma2 (PLCgamma2). In contrast, the C-type lectin receptor CLEC-2 mediates powerful platelet activation through Src and Syk kinases, but regulates Syk through a novel dimerization mechanism via a single YxxL motif known as a hemITAM. CLEC-2 is a receptor for podoplanin, which is expressed at high levels in several tissues, including type 1 lung alveolar cells, lymphatic endothelial cells, kidney podocytes and some tumors, but is absent from vascular endothelial cells and platelets. In this article, we compare the mechanism of platelet activation by GPVI and CLEC-2 and consider their functional roles in hemostasis and other vascular processes, including maintenance of vascular integrity, angiogenesis and lymphogenesis.

Involvement of proteinase-activated receptors 1 and 2 in spreading and phagocytosis by murine adherent peritoneal cells: Modulation by the C-terminal of S100A9 protein

Proteinase-activated receptors (PAR) are widely recognized for their modulatory properties in inflammatory and immune responses; however, their direct role on phagocyte effector functions remains unknown. S100A9, a protein secreted during inflammatory responses, deactivates activated peritoneal macrophages, and its C-terminal portion inhibits spreading and phagocytosis of adherent peritoneal cells. Herein, the effect of PAR1 and PAR2 agonists was investigated on spreading and phagocytosis by adherent peritoneal cells, as well as the ability of murine C-terminal of S100A9 peptide (mS100A9p) to modulate this effect. Adherent peritoneal cells obtained from mouse abdominal cavity were incubated with PAR1 and PAR2 agonists and spreading and phagocytosis of Candida albicans particles were evaluated. PAR1 agonists increased both the spreading and the phagocytic activity, but PAR2 agonists only increased the spreading index. mS100A9p reverted both the increased spreading and phagocytosis induced by PAR1 agonists, but no interference in the increased spreading induced by PAR2 agonists was noticed. The shorter homologue peptide to the C-terminal of mS100A9p, corresponding to the H(92)-E(97) region, also reverted the increased spreading and phagocytosis induced by PAR1 agonists. These findings show that proteinase-activated receptors have an important role for spreading and phagocytosis of adherent peritoneal cells, and that the pepticle corresponding to the C-terminal of S100A9 protein is a remarkable candidate for use as a novel compound to modulate PAR1 function. (C) 2009 Elsevier B.V. All rights reserved.

Purification and preliminary crystallographic analysis of a new Lys49-PLA2 from B-jararacussu

BjVIII is a new myotoxic Lys49-PLA2 isolated from Bothrops jararacussu venom that exhibits atypical effects on human platelet aggregation. To better understand the mode of action of BjVIII, crystallographic studies were initiated. Two crystal forms were obtained, both containing two molecules in the asymmetric unit (ASU). Synchrotron radiation diffraction data were collected to 2.0 angstrom resolution and 1.9 angstrom resolution for crystals belonging to the space group P2(1)2(1)2(1) (a = 48.4 angstrom, b = 65.3 angstrom, c = 84.3 angstrom) and space group P3(1)21 (a = b = 55.7 angstrom, c = 127.9 angstrom), respectively. Refinement is currently in progress and the refined structures are expected to shed light on the unusual platelet aggregation activity observed for BjVIII.

The effect of BMP-2 on the osteoconductive properties of beta-tricalcium phosphate in rat calvaria defects

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystallization and preliminary X-ray diffraction studies of BnSP-6, a myotoxic phospholipase A2 from Bothrops neuwiedi venom

BnSP-6 (myotoxin I) is a phospholipase A2 homologue isolated from Bothrops neuwiedi pauloensis venom. Crystals of BnSP-6 were obtained which diffracted X-rays to 2.5 Angstrom resolution using a synchrotron radiation source at room temperature and belong to space group P3(1)21. The unit cell dimensions are a=b=57.7, c=131.1 Angstrom. The structure was solved by molecular replacement using the coordinates of bothropstoxin I from B. jararacussu venom. There are two molecules in the asymmetric unit.

Proteomic Characterization of the Hyaluronidase (EC 3.2.1.35) from the Venom of the Social Wasp Polybia paulista

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structure of a calcium-independent phospholipase-like myotoxic protein from Bothrops asper venom

Myotoxin II, a myotoxic calcium-independent phospholipase-like protein isolated from the venom of Bothrops asper, possesses no detectable phospholipase activity. The crystal structure has been determined and refined at 2.8 Angstrom to an R factor of 16.5% (F>3 sigma) with excellent stereochemistry. Amino-acid differences between catalytically active phospholipases and myotoxin LI in the Ca2+-binding region, specifically the substitutions Tyr28-->Asn, Gly32-->Leu and Asp49-->Lys, result in an altered local conformation. The key difference is that the epsilon-amino group of Lys49 fills the site normally occupied by the calcium ion in catalytically active phospholipases. In contrast to the homologous monomeric Lys49 variant from Agkistrodon piscivorus piscivorus, myotoxin II is present as a dimer both in solution and in the crystalline state. The two molecules in the asymmetric unit are related by a nearly perfect twofold axis, yet the dimer is radically different from the dimer formed by the phospholipase from Crotalus atrox. Whereas in C. atrox the dimer interface occludes the active sites, in myotoxin II they are exposed to solvent.

CRYSTALLIZATION AND PRELIMINARY DIFFRACTION DATA OF 2 MYOTOXINS ISOLATED FROM THE VENOMS OF BOTHROPS-ASPER (TERCIOPELO) AND BOTHROPS-NUMMIFER (JUMPING VIPER)

Two myotoxins isolated from B. asper (myotoxin II) and B. nummifer (myotoxin I) snake venoms have been crystallized and their diffraction properties are described. These myotoxins are phospholipase A2 variants which lack enzymatic activity; B. asper myotoxin II is a lysine-49 phospholipase. Crystals were obtained at room temperature by standard hanging-drop vapour diffusion methods. Crystals diffracted to a resolution of 2.8 and 2.3 angstrom, respectively.

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.

Assignment of the bisulfide bridges in bothropstoxin-I, a Myonecrotic Lys49 PLA2 homolog from bothrops jararacussu snake venom

Bothropstoxin-I (BthTX-I), a Lys49 phospholipase A2 homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys50-Cysl31, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys 123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cysl05 by determination of the crystal structure, showing that BthTX-I disulfide bonds follow the normal pattern of group II PLA2s. © 2001 Plenum Publishing Corporation.

Bothropstoxin-I reduces evoked acetylcholine release from rat motor nerve terminals: Radiochemical and real-time video-microscopy studies

Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA2 homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [3H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals. © 2012 Elsevier Ltd.

Structural and Phylogenetic Studies with MjTX-I Reveal a Multi-Oligomeric Toxin - a Novel Feature in Lys49-PLA2s Protein Class

The mortality caused by snakebites is more damaging than many tropical diseases, such as dengue haemorrhagic fever, cholera, leishmaniasis, schistosomiasis and Chagas disease. For this reason, snakebite envenoming adversely affects health services of tropical and subtropical countries and is recognized as a neglected disease by the World Health Organization. One of the main components of snake venoms is the Lys49-phospholipases A2, which is catalytically inactive but possesses other toxic and pharmacological activities. Preliminary studies with MjTX-I from Bothrops moojeni snake venom revealed intriguing new structural and functional characteristics compared to other bothropic Lys49-PLA2s. We present in this article a comprehensive study with MjTX-I using several techniques, including crystallography, small angle X-ray scattering, analytical size-exclusion chromatography, dynamic light scattering, myographic studies, bioinformatics and molecular phylogenetic analyses.Based in all these experiments we demonstrated that MjTX-I is probably a unique Lys49-PLA2, which may adopt different oligomeric forms depending on the physical-chemical environment. Furthermore, we showed that its myotoxic activity is dramatically low compared to other Lys49-PLA2s, probably due to the novel oligomeric conformations and important mutations in the C-terminal region of the protein. The phylogenetic analysis also showed that this toxin is clearly distinct from other bothropic Lys49-PLA2s, in conformity with the peculiar oligomeric characteristics of MjTX-I and possible emergence of new functionalities inresponse to environmental changes and adaptation to new preys. © 2013 Salvador et al.

O efeito da BMP-2 sobre as propriedades osteocondutoras do beta-tricálcio fosfato em defeitos de calvária de ratos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Alkylation of Histidine Residues of Bothrops jararacussu Venom Proteins and Isolated Phospholipases A(2): A Biotechnological Tool to Improve the Production of Antibodies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da teneurina-2 (Ten-2) nos astrócitos do córtex cerebral de ratos adultos após lesão mecânica. Estudo imuno-histoquímico e de expressão gênica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)