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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

Fluorimetric titration of 12-AS with EgAgB8 subunits.Changes in relative 12-AS fluorescence were monitored from 400 to 500 nm after excitation at 383 nm upon incremental additions of EgAgB8/2 or EgAgB8/3 to a cuvette initially containing 2 mL of 0.5 μM 12-AS in TBS buffer. (A) Emission spectra of 12AS in TBS or upon adding EgAgB8/2 (0.5 μM). (B) Changes in relative 12-AS fluorescence at 440 nm were used to build the binding isotherm of the complex EgAgB8/2–12AS. (C) Emission spectra of 12AS in TBS or upon adding EgAgB8/3 (0.7 μM). (D) Changes in relative 12-AS fluorescence at 440 nm were used to build the binding isotherm of the complex EgAgB8/2–12AS. For both proteins, 12-AS spectra showed a blue shift in emission spectrum that accompanies a strong increase in fluorescence emission. The data were consistent with one binding site per monomer unit of protein and Kd values of 0.16 ± 0.09 μM for EgAgB8/2 and 0.34 ± 0.02 μM for EgAgB8/3 were obtained using SigmaPlot software. The solid line is the theoretical binding curve for complex formation. One representative experiment of three is shown for both EgAgB8 subunits.
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pntd.0003552.g006: Fluorimetric titration of 12-AS with EgAgB8 subunits.Changes in relative 12-AS fluorescence were monitored from 400 to 500 nm after excitation at 383 nm upon incremental additions of EgAgB8/2 or EgAgB8/3 to a cuvette initially containing 2 mL of 0.5 μM 12-AS in TBS buffer. (A) Emission spectra of 12AS in TBS or upon adding EgAgB8/2 (0.5 μM). (B) Changes in relative 12-AS fluorescence at 440 nm were used to build the binding isotherm of the complex EgAgB8/2–12AS. (C) Emission spectra of 12AS in TBS or upon adding EgAgB8/3 (0.7 μM). (D) Changes in relative 12-AS fluorescence at 440 nm were used to build the binding isotherm of the complex EgAgB8/2–12AS. For both proteins, 12-AS spectra showed a blue shift in emission spectrum that accompanies a strong increase in fluorescence emission. The data were consistent with one binding site per monomer unit of protein and Kd values of 0.16 ± 0.09 μM for EgAgB8/2 and 0.34 ± 0.02 μM for EgAgB8/3 were obtained using SigmaPlot software. The solid line is the theoretical binding curve for complex formation. One representative experiment of three is shown for both EgAgB8 subunits.

Mentions: We found that 12-AS showed a significant increase in its fluorescence emission, accompanied by a blue shift from 456 nm to 446 nm when lipid-free rEgAgB8/2 or rEgAgB8/3 subunits were added to the probe solution (Fig. 6A and 6C). For both subunits, titration experiments described curves that reached saturation, in accordance with a ligand binding stoichiometry consistent with 1:1 binding per monomer (Fig. 6B and 6D), with a Kd of 0.16 ± 0.09 μM for rEgAgB8/2 (r = 0.9976) and 0.34 ± 0.02 μM for rEgAgB8/3 (r = 0.9927). In contrast, negligible enhancement of fluorescent emission was observed for the fatty acid analogue DAUDA upon adding lipid-free rEgAgB8/2 or rEgAgB8/3 (S1 Fig.). Lipidex-treated recombinant subunits EgAgB8/1 and EgAgB8/2 behaved similarly in previous studies since they bound the fatty acid anthroyloxy derivative, 16-AP, but not DAUDA [35]. The Kd values determined for the binding of these probes by our delipidated subunits and by Lipidex-treated subunits were similar, but the latter exhibited a lower value for binding sites for monomer (n value of approximately 0.3), further emphasising the distinction between methods employed to remove bacterial lipids bound to the recombinant subunits. These findings suggest that the hydrophobic Lipidex resin method is not appropriate to achieve adequate delipidation.


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)

Fluorimetric titration of 12-AS with EgAgB8 subunits.Changes in relative 12-AS fluorescence were monitored from 400 to 500 nm after excitation at 383 nm upon incremental additions of EgAgB8/2 or EgAgB8/3 to a cuvette initially containing 2 mL of 0.5 μM 12-AS in TBS buffer. (A) Emission spectra of 12AS in TBS or upon adding EgAgB8/2 (0.5 μM). (B) Changes in relative 12-AS fluorescence at 440 nm were used to build the binding isotherm of the complex EgAgB8/2–12AS. (C) Emission spectra of 12AS in TBS or upon adding EgAgB8/3 (0.7 μM). (D) Changes in relative 12-AS fluorescence at 440 nm were used to build the binding isotherm of the complex EgAgB8/2–12AS. For both proteins, 12-AS spectra showed a blue shift in emission spectrum that accompanies a strong increase in fluorescence emission. The data were consistent with one binding site per monomer unit of protein and Kd values of 0.16 ± 0.09 μM for EgAgB8/2 and 0.34 ± 0.02 μM for EgAgB8/3 were obtained using SigmaPlot software. The solid line is the theoretical binding curve for complex formation. One representative experiment of three is shown for both EgAgB8 subunits.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003552.g006: Fluorimetric titration of 12-AS with EgAgB8 subunits.Changes in relative 12-AS fluorescence were monitored from 400 to 500 nm after excitation at 383 nm upon incremental additions of EgAgB8/2 or EgAgB8/3 to a cuvette initially containing 2 mL of 0.5 μM 12-AS in TBS buffer. (A) Emission spectra of 12AS in TBS or upon adding EgAgB8/2 (0.5 μM). (B) Changes in relative 12-AS fluorescence at 440 nm were used to build the binding isotherm of the complex EgAgB8/2–12AS. (C) Emission spectra of 12AS in TBS or upon adding EgAgB8/3 (0.7 μM). (D) Changes in relative 12-AS fluorescence at 440 nm were used to build the binding isotherm of the complex EgAgB8/2–12AS. For both proteins, 12-AS spectra showed a blue shift in emission spectrum that accompanies a strong increase in fluorescence emission. The data were consistent with one binding site per monomer unit of protein and Kd values of 0.16 ± 0.09 μM for EgAgB8/2 and 0.34 ± 0.02 μM for EgAgB8/3 were obtained using SigmaPlot software. The solid line is the theoretical binding curve for complex formation. One representative experiment of three is shown for both EgAgB8 subunits.
Mentions: We found that 12-AS showed a significant increase in its fluorescence emission, accompanied by a blue shift from 456 nm to 446 nm when lipid-free rEgAgB8/2 or rEgAgB8/3 subunits were added to the probe solution (Fig. 6A and 6C). For both subunits, titration experiments described curves that reached saturation, in accordance with a ligand binding stoichiometry consistent with 1:1 binding per monomer (Fig. 6B and 6D), with a Kd of 0.16 ± 0.09 μM for rEgAgB8/2 (r = 0.9976) and 0.34 ± 0.02 μM for rEgAgB8/3 (r = 0.9927). In contrast, negligible enhancement of fluorescent emission was observed for the fatty acid analogue DAUDA upon adding lipid-free rEgAgB8/2 or rEgAgB8/3 (S1 Fig.). Lipidex-treated recombinant subunits EgAgB8/1 and EgAgB8/2 behaved similarly in previous studies since they bound the fatty acid anthroyloxy derivative, 16-AP, but not DAUDA [35]. The Kd values determined for the binding of these probes by our delipidated subunits and by Lipidex-treated subunits were similar, but the latter exhibited a lower value for binding sites for monomer (n value of approximately 0.3), further emphasising the distinction between methods employed to remove bacterial lipids bound to the recombinant subunits. These findings suggest that the hydrophobic Lipidex resin method is not appropriate to achieve adequate delipidation.

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