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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

Cross-linking of rEgAgB subunits with EDC.Covalent cross-linking of proteins with EDC was carried out for 30 minutes at 25°C in PBS and separated on 15% SDS-PAGE followed by silver staining. Controls without added EDC were undertaken under the same conditions. (A) Cross-linking of lipid-free EgAgB8/2 and EgAgB8/3 subunits. (B) Cross-linking of non-delipidated EgAgB8/2 and EgAgB8/3 subunits. (C) Cross-linking of native EgAgB. (D) Control with lipid-free and non-delipidated ABA-1-A1, a recombinant single unit of the helix-rich LBP of the nematode parasite Ascaris suum, which is monomeric at the concentration used. Molecular masses of the standard proteins are indicated in each panel.
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pntd.0003552.g005: Cross-linking of rEgAgB subunits with EDC.Covalent cross-linking of proteins with EDC was carried out for 30 minutes at 25°C in PBS and separated on 15% SDS-PAGE followed by silver staining. Controls without added EDC were undertaken under the same conditions. (A) Cross-linking of lipid-free EgAgB8/2 and EgAgB8/3 subunits. (B) Cross-linking of non-delipidated EgAgB8/2 and EgAgB8/3 subunits. (C) Cross-linking of native EgAgB. (D) Control with lipid-free and non-delipidated ABA-1-A1, a recombinant single unit of the helix-rich LBP of the nematode parasite Ascaris suum, which is monomeric at the concentration used. Molecular masses of the standard proteins are indicated in each panel.

Mentions: In order to further examine the self-assembly of EgAgB8 apolipoproteins, we performed covalent cross-linking experiments analysing the formation of oligomers by SDS-PAGE under reducing conditions (Fig. 5A). We found that in the absence of EDC both subunits were predominantly present as monomers, and EDC addition led to the formation of different cross-linked products. In the range of 5 mM to 20 mM EDC, lipid-free rEgAgB8/2 formed mainly a ∼45 kDa oligomer, whereas lipid-free rEgAgB8/3 formed a heterogeneous array of oligomers (a ladder-like pattern from around 16 to more than 97 kDa). Cross-linked products of higher molecular mass were found for both apolipoproteins. These results suggest that rEgAgB8 subunits have an intrinsic ability to self-assemble, but with differences in the structural organization acquired by rEgAgB8/2 and rEgAgB8/3 in aqueous solution, since the oligomers that they form have different apparent molecular weights according to SEC and EDC cross-linking analysis. For comparison, we also analysed the cross-linked products of non-delipidated rEgAgB8 subunits to assess the role of lipids in EgAgB oligomerization. This showed that non-delipidated proteins form even larger oligomers of apparent MW around 60–90 kDa and higher than 97 kDa (Fig. 5B). These results agree with those previously described by Monteiro and collaborators, obtained using glutaraldehyde as cross-linker [33], and indicate that, whilst lipids are not absolutely required, they may participate in the formation of larger complexes. In parallel, the cross-linking of natural, parasite-derived EgAgB led to the formation of oligomers of high molecular mass, showing a similar pattern to non-delipidated recombinant subunits (Fig. 5C), also in agreement with previous reports [34]. Both, non-delipidated EgAgB subunits and parasite-derived EgAgB, presented larger complexes even in the absence of cross-linker (Fig. 5B and 5C), in contrast to lipid-free EgAgB subunits (Fig. 5A). This suggests that lipids are involved in the oligomerization of EgAgB that is commonly observed in SDS-PAGE analysis under reducing conditions [32]. As a control for cross-linking experiments, we used lipid-free ABA-1-A1 (a helix-rich small LBP from the nematode parasite Ascaris suum), a protein which is not expected to oligomerize even at high concentrations [39]. Both lipid-free and non-delipidated ABA-1-A1 were treated under the same conditions and oligomers were not detected (Fig. 5D).


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)

Cross-linking of rEgAgB subunits with EDC.Covalent cross-linking of proteins with EDC was carried out for 30 minutes at 25°C in PBS and separated on 15% SDS-PAGE followed by silver staining. Controls without added EDC were undertaken under the same conditions. (A) Cross-linking of lipid-free EgAgB8/2 and EgAgB8/3 subunits. (B) Cross-linking of non-delipidated EgAgB8/2 and EgAgB8/3 subunits. (C) Cross-linking of native EgAgB. (D) Control with lipid-free and non-delipidated ABA-1-A1, a recombinant single unit of the helix-rich LBP of the nematode parasite Ascaris suum, which is monomeric at the concentration used. Molecular masses of the standard proteins are indicated in each panel.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003552.g005: Cross-linking of rEgAgB subunits with EDC.Covalent cross-linking of proteins with EDC was carried out for 30 minutes at 25°C in PBS and separated on 15% SDS-PAGE followed by silver staining. Controls without added EDC were undertaken under the same conditions. (A) Cross-linking of lipid-free EgAgB8/2 and EgAgB8/3 subunits. (B) Cross-linking of non-delipidated EgAgB8/2 and EgAgB8/3 subunits. (C) Cross-linking of native EgAgB. (D) Control with lipid-free and non-delipidated ABA-1-A1, a recombinant single unit of the helix-rich LBP of the nematode parasite Ascaris suum, which is monomeric at the concentration used. Molecular masses of the standard proteins are indicated in each panel.
Mentions: In order to further examine the self-assembly of EgAgB8 apolipoproteins, we performed covalent cross-linking experiments analysing the formation of oligomers by SDS-PAGE under reducing conditions (Fig. 5A). We found that in the absence of EDC both subunits were predominantly present as monomers, and EDC addition led to the formation of different cross-linked products. In the range of 5 mM to 20 mM EDC, lipid-free rEgAgB8/2 formed mainly a ∼45 kDa oligomer, whereas lipid-free rEgAgB8/3 formed a heterogeneous array of oligomers (a ladder-like pattern from around 16 to more than 97 kDa). Cross-linked products of higher molecular mass were found for both apolipoproteins. These results suggest that rEgAgB8 subunits have an intrinsic ability to self-assemble, but with differences in the structural organization acquired by rEgAgB8/2 and rEgAgB8/3 in aqueous solution, since the oligomers that they form have different apparent molecular weights according to SEC and EDC cross-linking analysis. For comparison, we also analysed the cross-linked products of non-delipidated rEgAgB8 subunits to assess the role of lipids in EgAgB oligomerization. This showed that non-delipidated proteins form even larger oligomers of apparent MW around 60–90 kDa and higher than 97 kDa (Fig. 5B). These results agree with those previously described by Monteiro and collaborators, obtained using glutaraldehyde as cross-linker [33], and indicate that, whilst lipids are not absolutely required, they may participate in the formation of larger complexes. In parallel, the cross-linking of natural, parasite-derived EgAgB led to the formation of oligomers of high molecular mass, showing a similar pattern to non-delipidated recombinant subunits (Fig. 5C), also in agreement with previous reports [34]. Both, non-delipidated EgAgB subunits and parasite-derived EgAgB, presented larger complexes even in the absence of cross-linker (Fig. 5B and 5C), in contrast to lipid-free EgAgB subunits (Fig. 5A). This suggests that lipids are involved in the oligomerization of EgAgB that is commonly observed in SDS-PAGE analysis under reducing conditions [32]. As a control for cross-linking experiments, we used lipid-free ABA-1-A1 (a helix-rich small LBP from the nematode parasite Ascaris suum), a protein which is not expected to oligomerize even at high concentrations [39]. Both lipid-free and non-delipidated ABA-1-A1 were treated under the same conditions and oligomers were not detected (Fig. 5D).

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