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)
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