ABSTRACT
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 venom's peptidome might contain a population of cryptides, whose biological activities could lead to the development of new therapeutical agents.
ABSTRACT
O dengue é uma das principais arboviroses humanas e sua patogênese ainda não é bem compreendida. O estudo desta patologia através da abordagem proteômica poderá contribuir para o melhor entendimento da doença, revelando proteínas críticas para os processos celulares envolvidos na infecção pelo vírus Dengue. O objetivo desse trabalho foi comparar os perfis eletroforéticos bidimensionais dos plasmas de pacientes com dengue grave e de doadores saudáveis, identificando proteínas diferencialmente expressas entre estes 2 grupos. Os plasmas foram coletados no Hospital de Clínicas de Niterói (RJ) durante a epidemia de dengue no Estado do Rio de Janeiro em 2001-2002. Na Fase I do trabalho...Na Fase II...Estas foram...como cadeia beta da fibrina, proteína de ligação de vitamina D, vitronectina, componente C3 do complemento, apolipoproteína A-I, haptoglobina, transferrina e hemopexina. Após a combinação dos resultados de 14 géis (7 pacientes e 7 controles), apenas a apolipoproteína A-I apresentou uma diferença estatisticamente significativa, diminuindo de expressão nos plasmas dos pacientes com dengue grave. Na Fase III do estudo, passamos a retirar as 6 proteínas mais abundantes do plasma utilizando a Coluna de Remoção de Múltipla Afinidade e as amostras foram analisadas através da moderna técnica de eletroforese de fluorescência diferencial em gel 2D (DIGE). As imagens dos géis foram comparadas utilizando o software DeCyder Differential Analysis (GE Healthcare) e diversas manchas com expressão diferencial entre os 2 grupos experimentais foram detectadas. Alguns géis de DIGE foram também revelados com Coomassie coloidal e as manchas de interesse foram identificadas como descrito anteriormente. As proteínas que tenderam a aumentar nos plasmas dos pacientes com dengue foram a (alfa)1-glicoproteina ácida, a proteína de ligação de vitamina D, a (alfa)1-antiquimiotripsina, o componente C3 do complemento e o inibidor do componente C1 do complemento; as proteínas que tenderam...
