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Lipid-free antigen B subunits from echinococcus granulosus: oligomerization, ligand binding, and membrane interaction properties.

Silva-Álvarez V, Franchini GR, Pórfido JL, Kennedy MW, Ferreira AM, Córsico B - PLoS Negl Trop Dis (2015)

Bottom Line: Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues.We 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.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP) (UNLP-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de La Plata (UNLP), La Plata, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina.

ABSTRACT

Background: The 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 findings: Lipid-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/significance: We 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.

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Related in: MedlinePlus

Removal of co-purifying hydrophobic ligands from rEgAgB8 by RP-HPLC.The lipid composition of rEgAgB8 subunits prior to delipidation (Pre), as well as rEgAgB8 subjected to RP-HPLC (Post) were analysed by TLC, in parallel with standards for neutral and polar lipids. Lipid bands were visualised using CuSO4/H3PO4 and identified by comparison with the standards. (A) The lipids extracted from E. coli grown under the same culture conditions is shown for comparison. TLCs from neutral and polar lipids were undertaken separately. (B) The lipid moiety of recombinant subunits were analysed by TLC using double development. The lipid fraction of rEgAgB8 pre-HPLC contained mainly polar lipids (PE and CL), which were successfully removed by the RP-HPLC method. PC: phosphatidylcholine; PS: phosphatidylserine; PI: phosphatidylinositol; CL: cardiolipin; PE: phosphatidylethanolamine; Cho: cholesterol; FA: free fatty acids; DAG: diacylglycerols; TAG: triacylglycerols; SE: sterol esters.
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pntd.0003552.g002: Removal of co-purifying hydrophobic ligands from rEgAgB8 by RP-HPLC.The lipid composition of rEgAgB8 subunits prior to delipidation (Pre), as well as rEgAgB8 subjected to RP-HPLC (Post) were analysed by TLC, in parallel with standards for neutral and polar lipids. Lipid bands were visualised using CuSO4/H3PO4 and identified by comparison with the standards. (A) The lipids extracted from E. coli grown under the same culture conditions is shown for comparison. TLCs from neutral and polar lipids were undertaken separately. (B) The lipid moiety of recombinant subunits were analysed by TLC using double development. The lipid fraction of rEgAgB8 pre-HPLC contained mainly polar lipids (PE and CL), which were successfully removed by the RP-HPLC method. PC: phosphatidylcholine; PS: phosphatidylserine; PI: phosphatidylinositol; CL: cardiolipin; PE: phosphatidylethanolamine; Cho: cholesterol; FA: free fatty acids; DAG: diacylglycerols; TAG: triacylglycerols; SE: sterol esters.

Mentions: We purified lipid-free EgAgB8 subunits in order to analyse their capacity to oligomerize, their lipid binding properties, as well as their ability to transfer fatty acids to membrane vesicles. Among the five distinct EgAgB8 subfamilies, EgAgB8/2 and EgAgB8/3 were chosen because they represent the two distinct subfamilies within EgAgB family as mentioned above (Fig. 1). The recombinant subunits rEgAgB8/2 and rEgAgB8/3 were purified as GST fusion proteins from E. coli and recovered after thrombin treatment, as previously described [38]. We anticipated that rEgAgB8/2 and rEgAgB8/3 subunits would bind lipids during their synthesis in E. coli. We therefore analysed by TLC the lipids recovered by Folch extraction from the purified recombinant subunits and compared them with those from E.coli. Fig. 2A shows that fatty acids and phospholipids, mainly phosphatidylethanolamine (PE) and cardiolipin (CL), are the major lipids present in E. coli under our extraction conditions. Extractions from both rEgAgB8 subunits yielded mainly PE and CL (Fig. 2B). The absence of phosphatidylcholine, the main phospholipid found in native EgAgB [15], may be due to the lack of this phospholipid in E. coli (Fig. 2A).


Lipid-free antigen B subunits from echinococcus granulosus: oligomerization, ligand binding, and membrane interaction properties.

Silva-Álvarez V, Franchini GR, Pórfido JL, Kennedy MW, Ferreira AM, Córsico B - PLoS Negl Trop Dis (2015)

Removal of co-purifying hydrophobic ligands from rEgAgB8 by RP-HPLC.The lipid composition of rEgAgB8 subunits prior to delipidation (Pre), as well as rEgAgB8 subjected to RP-HPLC (Post) were analysed by TLC, in parallel with standards for neutral and polar lipids. Lipid bands were visualised using CuSO4/H3PO4 and identified by comparison with the standards. (A) The lipids extracted from E. coli grown under the same culture conditions is shown for comparison. TLCs from neutral and polar lipids were undertaken separately. (B) The lipid moiety of recombinant subunits were analysed by TLC using double development. The lipid fraction of rEgAgB8 pre-HPLC contained mainly polar lipids (PE and CL), which were successfully removed by the RP-HPLC method. PC: phosphatidylcholine; PS: phosphatidylserine; PI: phosphatidylinositol; CL: cardiolipin; PE: phosphatidylethanolamine; Cho: cholesterol; FA: free fatty acids; DAG: diacylglycerols; TAG: triacylglycerols; SE: sterol esters.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358968&req=5

pntd.0003552.g002: Removal of co-purifying hydrophobic ligands from rEgAgB8 by RP-HPLC.The lipid composition of rEgAgB8 subunits prior to delipidation (Pre), as well as rEgAgB8 subjected to RP-HPLC (Post) were analysed by TLC, in parallel with standards for neutral and polar lipids. Lipid bands were visualised using CuSO4/H3PO4 and identified by comparison with the standards. (A) The lipids extracted from E. coli grown under the same culture conditions is shown for comparison. TLCs from neutral and polar lipids were undertaken separately. (B) The lipid moiety of recombinant subunits were analysed by TLC using double development. The lipid fraction of rEgAgB8 pre-HPLC contained mainly polar lipids (PE and CL), which were successfully removed by the RP-HPLC method. PC: phosphatidylcholine; PS: phosphatidylserine; PI: phosphatidylinositol; CL: cardiolipin; PE: phosphatidylethanolamine; Cho: cholesterol; FA: free fatty acids; DAG: diacylglycerols; TAG: triacylglycerols; SE: sterol esters.
Mentions: We purified lipid-free EgAgB8 subunits in order to analyse their capacity to oligomerize, their lipid binding properties, as well as their ability to transfer fatty acids to membrane vesicles. Among the five distinct EgAgB8 subfamilies, EgAgB8/2 and EgAgB8/3 were chosen because they represent the two distinct subfamilies within EgAgB family as mentioned above (Fig. 1). The recombinant subunits rEgAgB8/2 and rEgAgB8/3 were purified as GST fusion proteins from E. coli and recovered after thrombin treatment, as previously described [38]. We anticipated that rEgAgB8/2 and rEgAgB8/3 subunits would bind lipids during their synthesis in E. coli. We therefore analysed by TLC the lipids recovered by Folch extraction from the purified recombinant subunits and compared them with those from E.coli. Fig. 2A shows that fatty acids and phospholipids, mainly phosphatidylethanolamine (PE) and cardiolipin (CL), are the major lipids present in E. coli under our extraction conditions. Extractions from both rEgAgB8 subunits yielded mainly PE and CL (Fig. 2B). The absence of phosphatidylcholine, the main phospholipid found in native EgAgB [15], may be due to the lack of this phospholipid in E. coli (Fig. 2A).

Bottom Line: Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues.We 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.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP) (UNLP-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de La Plata (UNLP), La Plata, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina.

ABSTRACT

Background: The 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 findings: Lipid-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/significance: We 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.

Show MeSH
Related in: MedlinePlus