Two-Dimensional Difference Gel Electrophoresis (DiGE) Analysis of Plasmas from Dengue Fever Patients Lidiane M. Albuquerque,†,§ Monique R. O. Trugilho,†,# Alex Chapeaurouge,†,# Patrı´cia B. Jurgilas,†,# Patrı´cia T. Bozza,‡ Fernando A. Bozza,§ Jonas Perales,†,# and Ana G. C. Neves-Ferreira*,†,# Lab. Toxinologia and Lab. Imunofarmacologia, Pavilha˜o Ozo´rio de Almeida, Instituto Oswaldo Cruz and Instituto de Pesquisa Clı´nica Evandro Chagas, Fiocruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil, and Rede Proteoˆmica do Rio de Janeiro, Brazil Received March 10, 2009
Dengue fever is the world’s 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, R1-antichymotrypsin, vitamin D-binding protein, fibrinogen γ-chain, R1-acid glycoprotein, apolipoprotein J and complement component C3c), while 7 others had decreased expression in the same samples (R-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. Keywords: dengue fever • plasma • proteome • DIGE
Introduction Dengue fever is considered the world’s most important arthropod-born viral disease affecting humans in terms of morbidity and mortality. More than 2.5 billion people are at risk of infection, and more than 100 countries have endemic dengue transmission. Annually, about 50-100 million cases of febrile illnesses occur, including more than 500 000 cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/ DSS), with a case-fatality ratio of 1-5%.1,2 In Brazil, 230 829 suspected dengue fever cases were reported to the Ministry of Health from January to April 2008, including 1069 confirmed dengue hemorrhagic fever cases and 77 deaths.3 Dengue fever can be caused by any of four virus serotypes (DENV-1 to DENV-4) of the Flavivirus genus. They are composed of a single, positive-strand RNA genome surrounded by an icosahedral nucleocapsid that is coated with a lipidic envelope. The genome encodes a single polyprotein that is cleaved into three structural proteins, capsid (C), membrane (M, which is expressed as a precursor protein) and envelop (E), and seven nonstructural proteins, NS1, NS2a, NS2b, NS3, NS4a, * To whom correspondence should be addressed. Phone: +55-21-25621381. Fax: +55-21- 2562-1410. E-mail:
[email protected]. † Lab. Toxinologia, Fiocruz. ‡ Lab. Imunofarmacologia, Fiocruz. § Instituto de Pesquisa Clı´nica Evandro Chagas, Fiocruz. # Rede Proteoˆmica do Rio de Janeiro. 10.1021/pr900236f CCC: $40.75
2009 American Chemical Society
NS4b and NS5. The major biological properties of the viruses are attributed to E protein, while some nonstructural proteins are involved in viral replication.4,5 A spectrum of clinical manifestations is associated with dengue virus infections. According to the World Health Organization (WHO),6 they can be classified as asymptomatic or undifferentiated fever, dengue fever (DF) or dengue hemorrhagic fever. However, the WHO criteria for DHF has failed to identify severe disease cases, including fatal ones, in adult Latin American populations. What constitutes a better system for severity stratification has been a subject of intense controversy.6,7 During dengue infection, the expression of several plasma proteins is altered, which may be related to the pathogenesis of the disease, but the profiles associated with dengue fever evolution and prognosis are not well-established. In a recent study using a multiplex immunofluorescent bead array system, we identified that IL-1β, IFN-γ, IL-4, IL-6, IL-13, IL-7 and GMCSF were significantly increased in patients with severe dengue fever.8 Additionally, MIP-β was characterized as a good prognostic marker in contrast to IFN-γ, which was associated with disease severity.8 Honsawek et al.9 showed an increased level of hyaluronic acid in the sera of patients with dengue infection, which was more pronounced in severe cases. Serum transaminase levels were also higher in DHF/DSS than in DF patients, suggesting the involvement of the liver in the pathogenesis of dengue infection.10 The secretome of the hepatic cell line Journal of Proteome Research 2009, 8, 5431–5441 5431 Published on Web 10/21/2009
research articles HepG2 infected with dengue virus serotype 2 was recently characterized.11 With the use of a qualitative proteomic analysis, the authors compared secreted protein profiles from mockinfected and virus-infected cells. One of the most striking differences observed was R-enolase, which was detected in much higher amounts in the secretome of infected cells. This multifunctional enzyme has been recurrently identified as being differentially expressed in several studies, regardless of the experiment, tissue or species, and could represent a common cellular stress response.12 The systematic characterization of circulating proteins in human plasma during health and disease may lead to the development of new biomarkers for diagnosis, prognosis and treatment follow-up in several pathologies, ranging from infectious diseases to cancer.13,14 Proteomics is a valuable approach to find patterns of protein markers for a given disease,15 and great investments have been made in this field in the past few years. However, despite all efforts, only a few successful cases are known, and several papers analyzing the pitfalls of proteomic strategies have been published.16-18 The present study aims to identify changes in the circulating protein content during acute dengue fever comparing the bidimensional electrophoretic profiles of plasmas from patients with severe dengue fever and from healthy donors. To increase the chances of finding lower-abundance proteins with high confidence, we used plasmas depleted of the six most abundant proteins,19 analyzed by two-dimensional difference gel electrophoresis (2D-DIGE).20 This multiplex technology allows for the detection and quantitation of differences between samples resolved on the same gel, or across multiple gels, when linked by an internal standard.21 2D-DIGE circumvents the main drawbacks associated with conventional bidimensional polyacrylamide gel electrophoresis (2D-PAGE), such as low sensitivity, reduced dynamic range, and gel-to-gel variability, enabling more accurate and sensitive quantitative proteomics studies.22 This proteomic approach applied to the study of dengue fever may ultimately contribute to a better understanding of the virus’s pathology, unraveling key proteins for the cellular processes involved in this endemic viral infection.
Experimental Section Patients and Controls. Thirteen patients with confirmed severe dengue fever recently admitted (less than 48 h) to the Hospital das Clı´nicas de Nitero´i, Nitero´i, Rio de Janeiro State, Brazil, were prospectively included (8 females, 5 males, age range 18-72 years). These patients were hospitalized between 4 and 10 days after disease onset with serious forms of the illness during the months of March and April 2002, which was an epidemic period in the region caused by the dengue virus type 3 (DENV3). The dengue virus infection was confirmed either by antidengue enzyme-linked immunoabsorbent assay (ELISA)-IgM, serotype-specific reverse transcription-polymerase chain reaction (RT-PCR) or virus isolation.23-25 As previously reported,7,26 we were often unable to characterize the severe disease forms based on WHO criteria [http:// www.who.int/csr/resources/publications/dengue/Denguepublication/en/]. Therefore, we classified severity as described by Harris et al.27 According to these criteria, we considered severe dengue fever as confirmed dengue cases with severe thrombocytopenia (
