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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

Amino acid sequence analysis of EgAgB8 subunits.Analysis of EgAgB8 subunits were undertaken using Biology Workbench 3.2 (University of Illinois, National Center for Supercomputing Applications, USA). (A) Alignment of amino acid sequences of the mature peptides of EgAgB8/1, EgAgB8/2, EgAgB8/3, EgAgB8/4 and EgAgB8/5, using ClustalW from Biology Workbench 3.2. Absolutely conserved residues, partially residues and similar residues are shown in black, dark grey and light grey, respectively. Comparison between EgAgB8 mature peptides shows that EgAgB8/1, EgAgB8/3 and EgAgB8/5 are more closely related to each other than to EgAgB8/2 and EgAgB8/4, as shown in the alignment (B) and in the tree (C) obtained employing DrawTree from Biology Workbench 3.2. (D) Biochemical data for EgAgB8/2 and EgAgB8/3 (from sequences squared in A). The theoretical isoelectric point (pI) and the theoretical extinction coefficient were also estimated using Biology Workbench 3.2 software. Accession numbers: EgAgB8/1: AAD38373, EgAgB8/2: AAC47169, EgAgB8/3: AAK64236, EgAgB8/4: AAQ74958 and EgAgB8/5: BAE94835.
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pntd.0003552.g001: Amino acid sequence analysis of EgAgB8 subunits.Analysis of EgAgB8 subunits were undertaken using Biology Workbench 3.2 (University of Illinois, National Center for Supercomputing Applications, USA). (A) Alignment of amino acid sequences of the mature peptides of EgAgB8/1, EgAgB8/2, EgAgB8/3, EgAgB8/4 and EgAgB8/5, using ClustalW from Biology Workbench 3.2. Absolutely conserved residues, partially residues and similar residues are shown in black, dark grey and light grey, respectively. Comparison between EgAgB8 mature peptides shows that EgAgB8/1, EgAgB8/3 and EgAgB8/5 are more closely related to each other than to EgAgB8/2 and EgAgB8/4, as shown in the alignment (B) and in the tree (C) obtained employing DrawTree from Biology Workbench 3.2. (D) Biochemical data for EgAgB8/2 and EgAgB8/3 (from sequences squared in A). The theoretical isoelectric point (pI) and the theoretical extinction coefficient were also estimated using Biology Workbench 3.2 software. Accession numbers: EgAgB8/1: AAD38373, EgAgB8/2: AAC47169, EgAgB8/3: AAK64236, EgAgB8/4: AAQ74958 and EgAgB8/5: BAE94835.

Mentions: EgAgB is an alpha helix-rich 230 kDa lipoprotein [15], which has been considered to be the most specific Echinococcus-genus antigen for human serodiagnosis of hydatid disease [19–21] as well as being an immunomodulatory parasite component [22,23]. It belongs to a cestode-specific family of proteins that bind hydrophobic ligands, referred to as hydrophobic ligand binding proteins (HLBPs). The ligands present in the EgAgB complex account for approximately 40–50% of the total mass of the native antigen and consist of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids [15]. At the protein level, native EgAgB contains around a dozen apolipoproteins or subunits [15], which are encoded by a polymorphic multigene family comprising five clades named EgAgB1 to EgAgB5 [24–29]. Interestingly, EgAgB isoforms are expressed differentially during the life-cycle stages of the parasite, as well as within distinct tissues of a given developmental stage; EgAgB1 to EgAgB4 are expressed in the metacestode stage whereas EgAgB5 seems to be expressed in the adult stage. Furthermore, in the metacestode, EgAgB1 to EgAgB4 are expressed in the germinal layer, but EgAgB3 seems to be the most abundantly expressed in protoscoleces [29]. Similar evidence of differential expression of antigen B subunits has also been obtained for the closely related species E. multilocularis [30]. The proteins encoded by EgAgB genes are each approximately 8 kDa in mass, and the different isoforms designated EgAgB8/1 to EgAgB8/5. Comparison of their amino acid sequences shows that EgAgB8/1, EgAgB8/3 and EgAgB8/5 are more similar to each other than to EgAgB8/2 and EgAgB8/4 (Fig. 1).


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)

Amino acid sequence analysis of EgAgB8 subunits.Analysis of EgAgB8 subunits were undertaken using Biology Workbench 3.2 (University of Illinois, National Center for Supercomputing Applications, USA). (A) Alignment of amino acid sequences of the mature peptides of EgAgB8/1, EgAgB8/2, EgAgB8/3, EgAgB8/4 and EgAgB8/5, using ClustalW from Biology Workbench 3.2. Absolutely conserved residues, partially residues and similar residues are shown in black, dark grey and light grey, respectively. Comparison between EgAgB8 mature peptides shows that EgAgB8/1, EgAgB8/3 and EgAgB8/5 are more closely related to each other than to EgAgB8/2 and EgAgB8/4, as shown in the alignment (B) and in the tree (C) obtained employing DrawTree from Biology Workbench 3.2. (D) Biochemical data for EgAgB8/2 and EgAgB8/3 (from sequences squared in A). The theoretical isoelectric point (pI) and the theoretical extinction coefficient were also estimated using Biology Workbench 3.2 software. Accession numbers: EgAgB8/1: AAD38373, EgAgB8/2: AAC47169, EgAgB8/3: AAK64236, EgAgB8/4: AAQ74958 and EgAgB8/5: BAE94835.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003552.g001: Amino acid sequence analysis of EgAgB8 subunits.Analysis of EgAgB8 subunits were undertaken using Biology Workbench 3.2 (University of Illinois, National Center for Supercomputing Applications, USA). (A) Alignment of amino acid sequences of the mature peptides of EgAgB8/1, EgAgB8/2, EgAgB8/3, EgAgB8/4 and EgAgB8/5, using ClustalW from Biology Workbench 3.2. Absolutely conserved residues, partially residues and similar residues are shown in black, dark grey and light grey, respectively. Comparison between EgAgB8 mature peptides shows that EgAgB8/1, EgAgB8/3 and EgAgB8/5 are more closely related to each other than to EgAgB8/2 and EgAgB8/4, as shown in the alignment (B) and in the tree (C) obtained employing DrawTree from Biology Workbench 3.2. (D) Biochemical data for EgAgB8/2 and EgAgB8/3 (from sequences squared in A). The theoretical isoelectric point (pI) and the theoretical extinction coefficient were also estimated using Biology Workbench 3.2 software. Accession numbers: EgAgB8/1: AAD38373, EgAgB8/2: AAC47169, EgAgB8/3: AAK64236, EgAgB8/4: AAQ74958 and EgAgB8/5: BAE94835.
Mentions: EgAgB is an alpha helix-rich 230 kDa lipoprotein [15], which has been considered to be the most specific Echinococcus-genus antigen for human serodiagnosis of hydatid disease [19–21] as well as being an immunomodulatory parasite component [22,23]. It belongs to a cestode-specific family of proteins that bind hydrophobic ligands, referred to as hydrophobic ligand binding proteins (HLBPs). The ligands present in the EgAgB complex account for approximately 40–50% of the total mass of the native antigen and consist of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids [15]. At the protein level, native EgAgB contains around a dozen apolipoproteins or subunits [15], which are encoded by a polymorphic multigene family comprising five clades named EgAgB1 to EgAgB5 [24–29]. Interestingly, EgAgB isoforms are expressed differentially during the life-cycle stages of the parasite, as well as within distinct tissues of a given developmental stage; EgAgB1 to EgAgB4 are expressed in the metacestode stage whereas EgAgB5 seems to be expressed in the adult stage. Furthermore, in the metacestode, EgAgB1 to EgAgB4 are expressed in the germinal layer, but EgAgB3 seems to be the most abundantly expressed in protoscoleces [29]. Similar evidence of differential expression of antigen B subunits has also been obtained for the closely related species E. multilocularis [30]. The proteins encoded by EgAgB genes are each approximately 8 kDa in mass, and the different isoforms designated EgAgB8/1 to EgAgB8/5. Comparison of their amino acid sequences shows that EgAgB8/1, EgAgB8/3 and EgAgB8/5 are more similar to each other than to EgAgB8/2 and EgAgB8/4 (Fig. 1).

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