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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

Equilibrium partition of 12-AS between EgAgB8 subunits and SUVs.The Kp for 12-AS partitioning was determined by measuring 12-AS fluorescence after addition of SUVs into a solution containing 7.5 μM EgAgB8/2 or EgAgB8/3 and 0.5 μM 12-AS in buffer TBS at 25°C (15:1 mol:mol). (A), (B) Emission spectra changes of 12AS bound to EgAgB8/2 or EgAgB8/3 respectively, upon incremental addition of EPC-SUVs containing NBD-PC. (C), (D) Changes in relative 12-AS fluorescence were used to obtained Kp values, fitting the Equation 2 described in Materials and Methods to the data, employing Solver Add-In from Excel (solid line). Kp values of 0.62 ± 0.09 and 0.88 ± 0.15 were obtained for rEgAgB8/2 and rEgAgB8/3, respectively. One representative experiment of two is shown for both EgAgB8 subunits.
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pntd.0003552.g007: Equilibrium partition of 12-AS between EgAgB8 subunits and SUVs.The Kp for 12-AS partitioning was determined by measuring 12-AS fluorescence after addition of SUVs into a solution containing 7.5 μM EgAgB8/2 or EgAgB8/3 and 0.5 μM 12-AS in buffer TBS at 25°C (15:1 mol:mol). (A), (B) Emission spectra changes of 12AS bound to EgAgB8/2 or EgAgB8/3 respectively, upon incremental addition of EPC-SUVs containing NBD-PC. (C), (D) Changes in relative 12-AS fluorescence were used to obtained Kp values, fitting the Equation 2 described in Materials and Methods to the data, employing Solver Add-In from Excel (solid line). Kp values of 0.62 ± 0.09 and 0.88 ± 0.15 were obtained for rEgAgB8/2 and rEgAgB8/3, respectively. One representative experiment of two is shown for both EgAgB8 subunits.

Mentions: How lipids carried by EgAgB complexes may be distributed within the parasite is unknown, so, we attempted to characterise the capacity of these subunits to transfer lipids to membranes. Since fatty acids, but not cholesterol, were bound by delipidated rEgAgB8 subunits, assays were designed to examine the transfer of 12-AS to phospholipid artificial membranes (SUVs). To establish the conditions for the transfer measurements we used the Kd values obtained for rEgAgB8/2 and rEgAgB8/3 to ensure that less than 5% of 12-AS remained free in solution. We also determined the Kp of 12-AS between rEgAgB8 subunits and vesicles to assure unidirectional transfer of 12-AS from rEgAgB8 to the vesicles. In order to do this, SUVs containing a FRET acceptor of the anthroyloxy group donor (NBD-PC) were added to a solution of 12-AS:EgAgB8/2 or 12-AS:EgAgB8/3 complex. The 12-AS fluorescence decay upon incremental increase in SUV concentration for both 12-AS:EgAgB8 complexes is shown in Fig. 7, indicating the transfer of 12-AS from EgAgB8 subunits to NBD-PC-containing vesicles. Using Eq. 2 (see Materials and Methods) a KP value of 0.62 ± 0.09 was obtained for rEgAgB8/2 and of 0.88 ± 0.15 for rEgAgB8/3. Both indicate that there is a slightly higher preference of 12-AS for the phospholipid membranes. Once these conditions were established, we analysed the transfer rates of 12-AS bound to rEgAgB8/2 or rEgAgB8/3 to the vesicles. Firstly we examined the transfer rates as a function of SUV concentration to determine whether the limiting step for ligand transfer is the effective protein-membrane interaction or the dissociation of the protein-ligand complex, as has been previously established for other LBPs [49–56]. A representative time trace of 12-AS fluorescence change upon SUV addition to EgAgB8/2:12-AS or EgAgB8/3:12-AS complexes is shown in Fig. 8A. We found that the 12-AS transfer rate from rEgAgB8/2 to EPC-SUVs increased significantly from 0.039 ± 0.003 s−1 to 0.084 ± 0.005 s−1 (p < 0.05) when SUV:protein ratio raised from 10:1 to 40:1. In the case of rEgAgB8/3, a trend towards an increase in 12-AS transfer rate (from 0.07 ± 0.02 s−1 to 0.08 ± 0.02 s−1) was observed, but this trend did not reach statistical significance (Fig. 8B). These results using zwitterionic SUVs suggest that the mechanism of 12-AS ligand transfer differed between EgAgB subunits; for rEgAgB8/2 the limiting step for transfer is the direct contact with the vesicle (collisional mechanism), whereas for rEgAgB8/3 the dissociation of 12-AS from the complex seems to be the limiting step (diffusional mechanism).


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)

Equilibrium partition of 12-AS between EgAgB8 subunits and SUVs.The Kp for 12-AS partitioning was determined by measuring 12-AS fluorescence after addition of SUVs into a solution containing 7.5 μM EgAgB8/2 or EgAgB8/3 and 0.5 μM 12-AS in buffer TBS at 25°C (15:1 mol:mol). (A), (B) Emission spectra changes of 12AS bound to EgAgB8/2 or EgAgB8/3 respectively, upon incremental addition of EPC-SUVs containing NBD-PC. (C), (D) Changes in relative 12-AS fluorescence were used to obtained Kp values, fitting the Equation 2 described in Materials and Methods to the data, employing Solver Add-In from Excel (solid line). Kp values of 0.62 ± 0.09 and 0.88 ± 0.15 were obtained for rEgAgB8/2 and rEgAgB8/3, respectively. One representative experiment of two is shown for both EgAgB8 subunits.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003552.g007: Equilibrium partition of 12-AS between EgAgB8 subunits and SUVs.The Kp for 12-AS partitioning was determined by measuring 12-AS fluorescence after addition of SUVs into a solution containing 7.5 μM EgAgB8/2 or EgAgB8/3 and 0.5 μM 12-AS in buffer TBS at 25°C (15:1 mol:mol). (A), (B) Emission spectra changes of 12AS bound to EgAgB8/2 or EgAgB8/3 respectively, upon incremental addition of EPC-SUVs containing NBD-PC. (C), (D) Changes in relative 12-AS fluorescence were used to obtained Kp values, fitting the Equation 2 described in Materials and Methods to the data, employing Solver Add-In from Excel (solid line). Kp values of 0.62 ± 0.09 and 0.88 ± 0.15 were obtained for rEgAgB8/2 and rEgAgB8/3, respectively. One representative experiment of two is shown for both EgAgB8 subunits.
Mentions: How lipids carried by EgAgB complexes may be distributed within the parasite is unknown, so, we attempted to characterise the capacity of these subunits to transfer lipids to membranes. Since fatty acids, but not cholesterol, were bound by delipidated rEgAgB8 subunits, assays were designed to examine the transfer of 12-AS to phospholipid artificial membranes (SUVs). To establish the conditions for the transfer measurements we used the Kd values obtained for rEgAgB8/2 and rEgAgB8/3 to ensure that less than 5% of 12-AS remained free in solution. We also determined the Kp of 12-AS between rEgAgB8 subunits and vesicles to assure unidirectional transfer of 12-AS from rEgAgB8 to the vesicles. In order to do this, SUVs containing a FRET acceptor of the anthroyloxy group donor (NBD-PC) were added to a solution of 12-AS:EgAgB8/2 or 12-AS:EgAgB8/3 complex. The 12-AS fluorescence decay upon incremental increase in SUV concentration for both 12-AS:EgAgB8 complexes is shown in Fig. 7, indicating the transfer of 12-AS from EgAgB8 subunits to NBD-PC-containing vesicles. Using Eq. 2 (see Materials and Methods) a KP value of 0.62 ± 0.09 was obtained for rEgAgB8/2 and of 0.88 ± 0.15 for rEgAgB8/3. Both indicate that there is a slightly higher preference of 12-AS for the phospholipid membranes. Once these conditions were established, we analysed the transfer rates of 12-AS bound to rEgAgB8/2 or rEgAgB8/3 to the vesicles. Firstly we examined the transfer rates as a function of SUV concentration to determine whether the limiting step for ligand transfer is the effective protein-membrane interaction or the dissociation of the protein-ligand complex, as has been previously established for other LBPs [49–56]. A representative time trace of 12-AS fluorescence change upon SUV addition to EgAgB8/2:12-AS or EgAgB8/3:12-AS complexes is shown in Fig. 8A. We found that the 12-AS transfer rate from rEgAgB8/2 to EPC-SUVs increased significantly from 0.039 ± 0.003 s−1 to 0.084 ± 0.005 s−1 (p < 0.05) when SUV:protein ratio raised from 10:1 to 40:1. In the case of rEgAgB8/3, a trend towards an increase in 12-AS transfer rate (from 0.07 ± 0.02 s−1 to 0.08 ± 0.02 s−1) was observed, but this trend did not reach statistical significance (Fig. 8B). These results using zwitterionic SUVs suggest that the mechanism of 12-AS ligand transfer differed between EgAgB subunits; for rEgAgB8/2 the limiting step for transfer is the direct contact with the vesicle (collisional mechanism), whereas for rEgAgB8/3 the dissociation of 12-AS from the complex seems to be the limiting step (diffusional mechanism).

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