Limits...
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.

Show MeSH

Related in: MedlinePlus

SEC analysis of lipid-free EgAgB subunits.Analysis of EgAgB lipid-free subunits by SEC was undertaken on a Superdex 200 column employing a flow rate of 0.5 mL/min. Elution profiles of lipid-free EgAgB8/2 and EgAgB8/3 were followed at 215 nm. Both subunits showed a well-defined peak, with a molecular weight estimation of 62 kDa for EgAgB8/2 and 38 kDa for EgAgB8/3 according to standard proteins analysed under the same conditions (a: BSA, 66 kDa; b:carbonic anhydrase, 29 kDa; and c: cytochrome c, 12 kDa).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4358968&req=5

pntd.0003552.g004: SEC analysis of lipid-free EgAgB subunits.Analysis of EgAgB lipid-free subunits by SEC was undertaken on a Superdex 200 column employing a flow rate of 0.5 mL/min. Elution profiles of lipid-free EgAgB8/2 and EgAgB8/3 were followed at 215 nm. Both subunits showed a well-defined peak, with a molecular weight estimation of 62 kDa for EgAgB8/2 and 38 kDa for EgAgB8/3 according to standard proteins analysed under the same conditions (a: BSA, 66 kDa; b:carbonic anhydrase, 29 kDa; and c: cytochrome c, 12 kDa).

Mentions: As said, since previous oligomerization studies were performed with non-delipidated proteins, the involvement of lipids in the oligomerization of rEgAgB subunits cannot be ruled out [33,34]. In order to examine whether lipid-free forms are capable of self-associating, we analysed rEgAgB8 subunits by SEC in aqueous solutions. This showed that lipid-free rEgAgB8/2 and rEgAgB8/3 were eluted in defined peaks that were not commensurate with monomeric forms, according to the elution of the standard (Fig. 4). The apparent molecular weight of these subunits indicated the presence of oligomers of 62 and 39 kDa for rEgAgB8/2 and rEgAgB8/3, equivalent to the formation of oligomers of 7–8 subunits and of 4–5 subunits, for lipid-free rEgAgB8/2 and rEgAgB8/3, respectively. Previous reports using non-delipidated recombinant proteins revealed the presence of larger complexes of around 164 kDa for EgAgB8/2 and 113 kDa for EgAgB8/3, as well as secondary peaks of higher molecular mass for both proteins [33]. Nevertheless, our results suggest that oligomerization of EgAgB subunits is not absolutely dependent on the presence of lipids.


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)

SEC analysis of lipid-free EgAgB subunits.Analysis of EgAgB lipid-free subunits by SEC was undertaken on a Superdex 200 column employing a flow rate of 0.5 mL/min. Elution profiles of lipid-free EgAgB8/2 and EgAgB8/3 were followed at 215 nm. Both subunits showed a well-defined peak, with a molecular weight estimation of 62 kDa for EgAgB8/2 and 38 kDa for EgAgB8/3 according to standard proteins analysed under the same conditions (a: BSA, 66 kDa; b:carbonic anhydrase, 29 kDa; and c: cytochrome c, 12 kDa).
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003552.g004: SEC analysis of lipid-free EgAgB subunits.Analysis of EgAgB lipid-free subunits by SEC was undertaken on a Superdex 200 column employing a flow rate of 0.5 mL/min. Elution profiles of lipid-free EgAgB8/2 and EgAgB8/3 were followed at 215 nm. Both subunits showed a well-defined peak, with a molecular weight estimation of 62 kDa for EgAgB8/2 and 38 kDa for EgAgB8/3 according to standard proteins analysed under the same conditions (a: BSA, 66 kDa; b:carbonic anhydrase, 29 kDa; and c: cytochrome c, 12 kDa).
Mentions: As said, since previous oligomerization studies were performed with non-delipidated proteins, the involvement of lipids in the oligomerization of rEgAgB subunits cannot be ruled out [33,34]. In order to examine whether lipid-free forms are capable of self-associating, we analysed rEgAgB8 subunits by SEC in aqueous solutions. This showed that lipid-free rEgAgB8/2 and rEgAgB8/3 were eluted in defined peaks that were not commensurate with monomeric forms, according to the elution of the standard (Fig. 4). The apparent molecular weight of these subunits indicated the presence of oligomers of 62 and 39 kDa for rEgAgB8/2 and rEgAgB8/3, equivalent to the formation of oligomers of 7–8 subunits and of 4–5 subunits, for lipid-free rEgAgB8/2 and rEgAgB8/3, respectively. Previous reports using non-delipidated recombinant proteins revealed the presence of larger complexes of around 164 kDa for EgAgB8/2 and 113 kDa for EgAgB8/3, as well as secondary peaks of higher molecular mass for both proteins [33]. Nevertheless, our results suggest that oligomerization of EgAgB subunits is not absolutely dependent on the presence of lipids.

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