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Echinococcus granulosus antigen B structure: subunit composition and oligomeric states.

Monteiro KM, Cardoso MB, Follmer C, da Silveira NP, Vargas DM, Kitajima EW, Zaha A, Ferreira HB - PLoS Negl Trop Dis (2012)

Bottom Line: AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample.We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties.Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia Molecular de Cestódeos and Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.

ABSTRACT

Background: Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states.

Methodology/principal findings: The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability.

Conclusions/significance: For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.

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Related in: MedlinePlus

Pressure-induced dissociation of AgB recombinant oligomers and its effects on protein secondary structure.High hydrostatic pressure-treatment (A) and circular dichroism spectra (B) of the recombinant AgB oligomers. Protein samples (0.2 mg/ml in 25 mM tris buffer pH 7.5) were submitted to compression (→) and decompression (←) at 25°C, and light scattering was monitored at the steady state [LS was recorded and divided by the initial value (LS/LS0)]. Circular dichroism spectra were recorded before (open symbols) and immediately after (solid symbols) pressure treatment.
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pntd-0001551-g006: Pressure-induced dissociation of AgB recombinant oligomers and its effects on protein secondary structure.High hydrostatic pressure-treatment (A) and circular dichroism spectra (B) of the recombinant AgB oligomers. Protein samples (0.2 mg/ml in 25 mM tris buffer pH 7.5) were submitted to compression (→) and decompression (←) at 25°C, and light scattering was monitored at the steady state [LS was recorded and divided by the initial value (LS/LS0)]. Circular dichroism spectra were recorded before (open symbols) and immediately after (solid symbols) pressure treatment.

Mentions: To compare the structural stabilities of AgB recombinant oligomers, proteins were subjected to treatment with high hydrostatic pressures, during which their oligomeric states were monitored by LS changes (Figure 6A). The LS signal of rAgB8/1 oligomers decreased by ∼60% after compression, which indicates its partial dissociation into smaller species. The rAgB8/2 and rAgB8/3 oligomers dissociated more poorly (LS decreased only ∼30%), even at the highest pressure attained in our experimental setup. The CD spectra recorded before and immediately after the compression of the samples (Figure 6B) indicated that the secondary structure for rAgB8/2 and rAgB8/3 was less perturbed by the pressure treatment than for rAgB8/1. Thus, pressure-induced dissociation experiments showed marked differences in the stabilities of oligomers formed by AgB subunits, with those formed by rAgB8/2 and rAgB8/3 subunits presenting greater structural stability. However, despite of the differences in the stability of recombinant AgB oligomers, the dissociation appears to be irreversible for all of them, since the LS values were maintained after pressure removal (Figure 6A).


Echinococcus granulosus antigen B structure: subunit composition and oligomeric states.

Monteiro KM, Cardoso MB, Follmer C, da Silveira NP, Vargas DM, Kitajima EW, Zaha A, Ferreira HB - PLoS Negl Trop Dis (2012)

Pressure-induced dissociation of AgB recombinant oligomers and its effects on protein secondary structure.High hydrostatic pressure-treatment (A) and circular dichroism spectra (B) of the recombinant AgB oligomers. Protein samples (0.2 mg/ml in 25 mM tris buffer pH 7.5) were submitted to compression (→) and decompression (←) at 25°C, and light scattering was monitored at the steady state [LS was recorded and divided by the initial value (LS/LS0)]. Circular dichroism spectra were recorded before (open symbols) and immediately after (solid symbols) pressure treatment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3295803&req=5

pntd-0001551-g006: Pressure-induced dissociation of AgB recombinant oligomers and its effects on protein secondary structure.High hydrostatic pressure-treatment (A) and circular dichroism spectra (B) of the recombinant AgB oligomers. Protein samples (0.2 mg/ml in 25 mM tris buffer pH 7.5) were submitted to compression (→) and decompression (←) at 25°C, and light scattering was monitored at the steady state [LS was recorded and divided by the initial value (LS/LS0)]. Circular dichroism spectra were recorded before (open symbols) and immediately after (solid symbols) pressure treatment.
Mentions: To compare the structural stabilities of AgB recombinant oligomers, proteins were subjected to treatment with high hydrostatic pressures, during which their oligomeric states were monitored by LS changes (Figure 6A). The LS signal of rAgB8/1 oligomers decreased by ∼60% after compression, which indicates its partial dissociation into smaller species. The rAgB8/2 and rAgB8/3 oligomers dissociated more poorly (LS decreased only ∼30%), even at the highest pressure attained in our experimental setup. The CD spectra recorded before and immediately after the compression of the samples (Figure 6B) indicated that the secondary structure for rAgB8/2 and rAgB8/3 was less perturbed by the pressure treatment than for rAgB8/1. Thus, pressure-induced dissociation experiments showed marked differences in the stabilities of oligomers formed by AgB subunits, with those formed by rAgB8/2 and rAgB8/3 subunits presenting greater structural stability. However, despite of the differences in the stability of recombinant AgB oligomers, the dissociation appears to be irreversible for all of them, since the LS values were maintained after pressure removal (Figure 6A).

Bottom Line: AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample.We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties.Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia Molecular de Cestódeos and Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.

ABSTRACT

Background: Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states.

Methodology/principal findings: The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability.

Conclusions/significance: For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.

Show MeSH
Related in: MedlinePlus