Biophysical characterization of recombinant subunits of Antigen B (AgB) from Echinococcus granulosus.

SILVA, VALERIA; CÓRSICO, B.

BUENOS AIRES

Congreso; XXXV Congreso de la Sociedad Argentina de Biofísica; 2011

SAB

Antigen B (AgB) is an abundant and immunogenic lipoprotein produced by the larval stage of Echinococcus granulosus, which structure and function have not been completely elucidated. Its apolipoprotein component shares homology with the group of hydrophobic ligand binding proteins present exclusively in cestode organisms, and consists of different isoforms of an 8-kDa protein coded by a multigene and polymorphic family that comprise five subfamilies (EgAgB1 to EgAgB5). Some of these subunits have been cloned, allowing their expression in Escherichia coli, which represent important tools for the study of AgB structure and function. Since E. granulosus have a very limited lipid metabolism, being unable to synthesise most of their own lipids de novo, it is generally thought that AgB could participate in their acquisition from the host organism, having an important role in the biology of the parasite. The aim of this work is to contribute to the understanding of AgB structural organization and function, through the study of protein-lipid interactions that would take place. In this regard, we are investigating the binding of AgB subunits to different hydrophobic ligands and the interaction between AgB subunits and model membranes. Three recombinant subunits, AgB8/1, AgB8/2 and AgB8/3, were over-expressed as GST-fusion proteins and purified using a glutathione affinity column. Delipidation of these subunits was successfully achieved by reverse phase–HPLC, using an increasing acetonitrile gradient. Ligand binding properties of AgB subunits are under analysis by using anthroyloxy-fatty acids (AOFA), suggesting that they could bind these fatty acid analogues with high affinity. In addition, employing a fluorescence energy transfer assay, we have analyzed the rates of AOFA transfer from AgB to phospholipids membranes containing an energy transfer acceptor of the anthroyloxy group donor. The results suggest that these proteins are able to transfer fatty acids to acceptor membranes.  Further analysis of the mechanism involved in ligand transfer will be directed to evaluate the factors that modulate the interactions between AgB and membranes. The understanding of these interactions between AgB and lipids will contribute to the understanding of AgB functions in E. granulosus biology.