XXXV Reunião Anual da SBBqResumoID:8318


Structural characterization and subunit aggregation dynamics of Echinococcus granulosus antigen B
Monteiro, K. M.1; Cardoso, M. B.2; Silveira, N. P.2; Zaha, A.1; Ferreira, H. B.1

1Laboratório de Biologia Molecular de Cestódeos, Centro de Biotecnologia, UFRGS, RS; 2 Laboratório de Instrumentação e Dinâmica Molecular, Instituto de Química, UFRGS, RS

Echinococcus granulosus antigen B (AgB) is one of the major antigenic components of the metacestode hydatid fluid. It is highly immunogenic for cystic hydatid disease patients and has a high diagnostic value. AgB is an oligomeric lipoprotein of 120-160 kDa, composed by related subunits of 8 kDa each. However, how these subunits interact in solution to form the oligomeric structure of the native protein remains unclear. Therefore, the aim of this work is to structurally characterize the AgB and obtain information on the aggregation dynamics of its subunits. Our laboratory has cloned and characterized three E. granulosus AgB subunits, namely AgB8/1, AgB8/2 and e AgB8/3. These proteins were expressed in Escherichia coli as fusions with glutathione-S-transferase, purified by affinity chromatography on immobilized glutathione and recovered by thrombin cleavage. The recombinant subunits have been analyzed by dynamic and static light scattering spectroscopies, to access their hydrodynamic radius, behavior in different temperatures and aggregation dynamics. These analyses have showed that the AgB8/2 and AgB8/3 subunits aggregate in response to increase in temperature (from 23 to 37°C), forming homoaggregates with hydrodynamic radii of 150-250 nm and larger than 2 µm. In our experimental conditions, no aggregation of AgB8/1 has been observed. Next, the possible formation of heteroaggregates from mixtures of different recombinant AgB subunits will be analyzed by light scattering and immunoprecipitation. Furthermore, models for each of the AgB subunits with known sequence are being built by molecular modeling in attempt to explain their aggregation mechanism, and possible changes in secondary structures of AgB subunits during the aggregation process will be assessed by circular dichroism experiments. (CABBIO/CNPq, FAPERGS, RTPD Network-SIDA/SAREC)