Lipid-Free Antigen B Subunits from Echinococcus granulosus: Oligomerization, Ligand Binding, and Membrane Interaction Properties

Abstract

BackgroundThe hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied.Methodology/Principal FindingsLipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles.Conclusions/SignificanceWe show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy.

Antigen B from Echinococcus granulosus is a novel ligand for C‐reactive protein

“…Lipid‐free recombinant EgAgB8 apolipoproteins, including rEgAgB8/1, rEgAgB8/2 and rEgAgB8/3, were obtained as previously described . Competition binding assays were performed immobilizing native EgAgB (0.1 μg/well) on 96‐well ELISA plates and then incubating with a solution of a nonsaturated concentration of hCRP (4 × 10 −9 mol/L), which was preincubated for 45 minutes with rEgAgB8/1, rEgAgB8/2 or rEgAgB8/3 (10 μg/mL), or binding buffer as a control.…”

Parasite molecules and host responses in cystic echinococcosis

“…In support of this hypothesis, the lipid content of native EgAgB includes lipids that are likely host‐derived . Furthermore, lipid‐free subunits can bind fatty acids and transfer them to phospholipid membranes in vitro , and experimental data support the involvement of T. solium HLBPs in the fatty acid transport across the larval wall . The differential expression of EgAgB genes in parasite stages/tissues may be associated with the need to contact different host cell types as sources of lipids, or different parasite tissues for lipid delivery.…”

“…After removal of their ER‐targeting leader peptides, the EgAg protein products are α‐helix‐rich 8 kDa polypeptides without known glycosylation. All of these polypeptides share the ability to oligomerize and are thus referred to as EgAgB subunits . Oligomerization is disulphide‐independent but partly resistant to denaturation, so that native EgAgB gives a ladder pattern upon SDS‐PAGE .…”

“…Oligomerization is disulphide‐independent but partly resistant to denaturation, so that native EgAgB gives a ladder pattern upon SDS‐PAGE . The electrostatically driven oligomerization is predicted by structure modelling .…”

Antigen B from Echinococcus granulosus enters mammalian cells by endocytic pathways

“…The lipid moiety associated with AgB was analyzed and different 82 lipids were identified, with cholesterol, phospholipids and triacylglycerides being the most 83 prominent [13]. Moreover, delipidated recombinant AgB8/2 and AgB8/3 subunits were 84 capable of transferring fatty acids analogues to artificial phospholipid membranes [14]. E. 85 granulosus genome lacks sequences for several key enzymes for fatty acid and 86 cholesterol synthesis, thus the parasite is incapable of synthesizing these compounds de 87 novo [15,16].…”