Ana Gisele C. Neves-Ferreira

Bothrops insularis venomics: A proteomic analysis supported by transcriptomic-generated sequence data

A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A(2) and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and M(r) of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies.

Isolation and characterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupialis serum.

From Didelphis marsupialis serum, two antihemorrhagic proteins were isolated by DEAE-Sephacel, Phenyl-Sepharose and Superdex 200 and characterized. Their masses by mass spectrometry were 40318 AMU for DM40 and 42373 and 43010 AMU for DM43, indicating the presence of isoforms for the last. Molecular masses of 44.8 and 47.3 were obtained by SDS-PAGE, respectively for DM40 and DM43. Both inhibitors showed isoelectric points lower than 3.5 and glycosylation percentages varying from 20.5 to 29.0%, as estimated by chemical deglycosylation and amino acid analysis. N-terminal sequences of the first 17 residues of DM40 and DM43 were identical except for the exchange of R9 for P9. Both were homologous to oprin, a similar inhibitor from Didelphis virginiana serum. No evidence of complex formation between DM40 and DM43 was observed either by native PAGE or gel filtration chromatography. In addition to the antihemorrhagic activity, DM40 and DM43 inhibited the hydrolysis of casein, fibrinogen and fibronectin by Bothrops jararaca venom. DM43 also showed antilethal, antiedematogenic and antihyperalgesic activities. None of the inhibitors showed enzymatic activity on casein. Both proteins formed stable complexes with jararhagin and inhibited its hemorrhagic effect as well as the enzymatic activity of this toxin on fluorogenic substrate.

Two-dimensional difference gel electrophoresis (DiGE) analysis of plasmas from dengue fever patients.

Dengue fever is the worlds most important arthropod-born viral disease affecting humans. To contribute to a better understanding of its pathogenesis, this study aims to identify proteins differentially expressed in plasmas from severe dengue fever patients relative to healthy donors. The use of 2-D Fluorescence Difference Gel Electrophoresis to analyze plasmas depleted of six high-abundance proteins (albumin, IgG, antitrypsin, IgA, transferrin and haptoglobin) allowed for the detection of 73 differentially expressed protein spots (n = 13, p < 0.01), of which 37 could be identified by mass spectrometry. These 37 spots comprised a total of 14 proteins, as follows: 7 had increased expression in plasmas from dengue fever patients (C1 inhibitor, alpha1-antichymotrypsin, vitamin D-binding protein, fibrinogen gamma-chain, alpha1-acid glycoprotein, apolipoprotein J and complement component C3c), while 7 others had decreased expression in the same samples (alpha-2 macroglobulin, prothrombin, histidine-rich glycoprotein, apolipoproteins A-IV and A-I, transthyretin and complement component C3b). The possible involvement of these proteins in the inflammatory process triggered by dengue virus infection and in the repair mechanisms of vascular damage occurring in this pathology is discussed in this study.

Insularinase A, a prothrombin activator from Bothrops insularis venom, is a metalloprotease derived from a gene encoding protease and disintegrin domains

Abstract The first low-molecular-mass metalloprotease presenting prothrombin activating activity was purified from Bothrops insularis venom and named insularinase A. It is a single-chain protease with a molecular mass of 22 639 Da. cDNA sequence analysis revealed that the disintegrin domain of the precursor protein is post-translationally processed, producing the mature insularinase A. Analysis of its deduced amino acid sequence showed a high similarity with several fibrin(ogen)olytic metalloproteases and only a moderate similarity with prothrombin activators. However, SDS-PAGE of prothrombin after activation by insularinase A showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin independently of the prothrombinase complex. In addition, insularinase A activates factor X and hydrolyses fibrinogen and fibrin. Chelating agents fully inhibit all insularinase A activities. Insularinase A induced neither detachment nor apoptosis of human endothelial cells and was also not able to trigger an endothelial proinflammatory cell response. Nitric oxide and prostacyclin levels released by endothelial cells were significantly increased after treatment with insularinase A. Our results show that, although its primary structure is related to class P-I fibrin(ogen)olytic metalloproteases, insularinase A is functionally similar to group A prothrombin activators.

Deciphering the proteomic profile of Mycobacterium leprae cell envelope

The complete sequence of the Mycobacterium leprae genome, an obligate intracellular pathogen, shows a dramatic reduction of functional genes, with a coding capacity of less than 50%. Despite this massive gene decay, the leprosy bacillus has managed to preserve a minimal gene set, most of it shared with Mycobacterium tuberculosis, allowing its survival in the host with ensuing pathological manifestations. Thus, the identification of proteins that are actually expressed in vivo by M. leprae is of high significance in understanding obligate, intracellular mycobacterial pathogenesis. In this study, a high‐throughput proteomic approach was undertaken resulting in the identification of 218 new M. leprae proteins. Of these, 60 were in the soluble/cytosol fraction, 98 in the membrane and 104 in the cell wall. Although several proteins were identified in more than one subcellular fraction, the majority were unique to one. As expected, a high percentage of these included enzymes responsible for lipid biosynthesis and degradation, biosynthesis of the major components of the mycobacterial cell envelope, proteins involved in transportation across lipid barriers, and lipoproteins and transmembrane proteins with unknown functions. The data presented in this study contribute to our understanding of the in vivo composition and physiology of the mycobacterial cell envelope, a compartment known to play a major role in bacterial pathogenesis.

Crotalid snake venom subproteomes unraveled by the antiophidic protein DM43.

Snake venoms are mixtures of proteins and peptides with different biological activities, many of which are very toxic. Several animals, including the opossum Didelphis aurita, are resistant to snake venoms due to the presence of neutralizing factors in their blood. An antihemorrhagic protein named DM43 was isolated from opossum serum. It inhibits snake venom metalloproteinases through noncovalent complex formation with these enzymes. In this study, we have used DM43 and proteomic techniques to explore snake venom subproteomes. Four crotalid venoms were chromatographed through an affinity column containing immobilized DM43. Bound fractions were analyzed by one- and two-dimensional gel electrophoresis, followed by identification by MALDI-TOF/TOF mass spectrometry. With this approach, we could easily visualize and compare the metalloproteinase compositions of Bothrops atrox, Bothrops jararaca, Bothrops insularis, and Crotalus atrox snake venoms. The important contribution of proteolytic processing to the complexity of this particular subproteome was demonstrated. Fractions not bound to DM43 column were similarly analyzed and were composed mainly of serine proteinases, C-type lectins, C-type lectin-like proteins, l-amino acid oxidases, nerve growth factor, cysteine-rich secretory protein, a few metalloproteinases (and their fragments), and some unidentified spots. Although very few toxin families were represented in the crotalid venoms analyzed, the number of protein spots detected was in the hundreds, indicating an important protein variability in these natural secretions. DM43 affinity chromatography and associated proteomic techniques proved to be useful tools to separate and identify proteins from snake venoms, contributing to a better comprehension of venom heterogeneity.

Differential proteomics of the plasma of individuals with sepsis caused by Acinetobacter baumannii

This study examines alterations in the plasma proteome in ten adults affected by sepsis caused by Acinetobacter baumannii as compared to paired healthy controls. 2-DE profiles of plasma from patients and paired healthy donors, depleted of the six most abundant proteins, were analysed by the DIGE technique. Protein spot detection and quantification were performed with the Differential In-gel Analysis and Biological Variation Analysis modules of the DeCyder() software. Differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) after colloidal Coomassie blue staining. Almost 900 spots were detected on a unique 2-D gel by the DIGE technique. A total of 269 protein spots of differential abundance were shown to be statistically significant (2.5-fold) with p values of p< or =0.01 (135 spots) and p< or =0.05 (134 spots) as determined by the t test. Seventy-one spots were submitted to mass spectrometry and about 30% could be successfully identified. This multiplex approach significantly reduced experimental variability, allowing for the confident detection of small differences in protein levels. Results include differentially expressed lipoproteins as well as proteins belonging to inflammatory/coagulation pathways and the kallikrein-kinin system. These data improves the knowledge for future developments in sepsis diagnosis, staging and therapy.

An in-depth snake venom proteopeptidome characterization: Benchmarking Bothrops jararaca

A large-scale proteomic approach was devised to advance the understanding of venom composition. Bothrops jararaca venom was fractionated by OFFGEL followed by chromatography, generating peptidic and proteic fractions. The latter was submitted to trypsin digestion. Both fractions were separately analyzed by reversed-phase nanochromatography coupled to high resolution mass spectrometry. This strategy allowed deeper and joint characterizations of the peptidome and proteome (proteopeptidome) of this venom. Our results lead to the identification of 46 protein classes (with several uniquely assigned proteins per class) comprising eight high-abundance bona fide venom components, and 38 additional classes in smaller quantities. This last category included previously described B. jararaca venom proteins, common Elapidae venom constituents (cobra venom factor and three-finger toxin), and proteins typically encountered in lysosomes, cellular membranes and blood plasma. Furthermore, this report is the most complete snake venom peptidome described so far, both in number of peptides and in variety of unique proteins that could have originated them. It is hypothesized that such diversity could enclose cryptides, whose bioactivities would contribute to envenomation in yet undetermined ways. Finally, we propose that the broad range screening of B. jararaca peptidome will facilitate the discovery of bioactive molecules, eventually leading to valuable therapeutical agents. BIOLOGICAL SIGNIFICANCE Our proteopeptidomic strategy yielded unprecedented insights into the remarkable diversity of B. jararaca venom composition, both at the peptide and protein levels. These results bring a substantial contribution to the actual pursuit of large-scale protein-level assignment in snake venomics. The detection of typical elapidic venom components, in a Viperidae venom, reinforces our view that the use of this approach (hand-in-hand with transcriptomic and genomic data) for venom proteomic analysis, at the specimen-level, can greatly contribute for venom toxin evolution studies. Furthermore, data were generated in support of a previous hypothesis that venom gland secretory vesicles are specialized forms of lysosomes. Two testable hypotheses also emerge from the results of this work. The first is that a nucleobindin-2-derived protein could lead to prey disorientation during envenomation, aiding in its capture by the snake. The other being that the venoms peptidome might contain a population of cryptides, whose biological activities could lead to the development of new therapeutical agents.

SIM-XL: A powerful and user-friendly tool for peptide cross-linking analysis.

Chemical cross-linking has emerged as a powerful approach for the structural characterization of proteins and protein complexes. However, the correct identification of covalently linked (cross-linked or XL) peptides analyzed by tandem mass spectrometry is still an open challenge. Here we present SIM-XL, a software tool that can analyze data generated through commonly used cross-linkers (e.g., BS3/DSS). Our software introduces a new paradigm for search-space reduction, which ultimately accounts for its increase in speed and sensitivity. Moreover, our search engine is the first to capitalize on reporter ions for selecting tandem mass spectra derived from cross-linked peptides. It also makes available a 2D interaction map and a spectrum-annotation tool unmatched by any of its kind. We show SIM-XL to be more sensitive and faster than a competing tool when analyzing a data set obtained from the human HSP90. The software is freely available for academic use at http://patternlabforproteomics.org/sim-xl. A video demonstrating the tool is available at http://patternlabforproteomics.org/sim-xl/video. SIM-XL is the first tool to support XL data in the mzIdentML format; all data are thus available from the ProteomeXchange consortium (identifier PXD001677). This article is part of a Special Issue entitled: Computational Proteomics.

Trypsin Inhibitors in Passion Fruit (Passiflora f. edulis flavicarpa) Leaves : Accumulation in Response to Methyl Jasmonate, Mechanical Wounding, and Herbivory

This work investigates the effect of methyl jasmonte (MeJa), mechanical wounding, and herbivory caused by larval feeding of a specialist insect ( Agraulis vanillae vanillae) upon trypsin inhibitory activity in passion fruit leaves. Despite the fact that all treatments caused accumulation of trypsin inhibitors (TIs), higher levels were observed in MeJa treated leaves when plants were assayed 24 and 48 h after stimulus. Concerning both mechanically injured plants and attacked ones, a systemic induction was observed. Partially purified inhibitors from MeJa exposed plants were further characterized by X-ray film contact print technique and N-terminal sequence. Such analysis indicated that the TIs identified belong to the Kunitz family. Moreover, the partially purified inhibitors strongly inhibited trypsin-like digestive enzymes from sugar cane stalk borer ( Diatraea saccharalis) in vitro. Our results further support the protective function of wound-inducible trypsin inhibitors and their potential as tools to improve important crop species against insect predation through genetic engineering.