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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

Native PAGE of E. granulosus AgB and recombinant oligomers.(A) AgB oligomers from bovine and human samples. Lanes 1–2, AgB purified from bovine cyst 1 and 2, respectively; lane 3, hydatid fluid from human cyst. (B) Oligomers formed by AgB recombinant subunits at different temperatures. Lanes 1–2, rAgB8/1; Lanes 3–4, rAgB8/2; Lanes 5–6, rAgB8/3, at 23°C (lanes 1, 3 and 5) and 37°C (lanes 2, 4 and 6). Samples were resolved on 4–20% polyacrylamide gels followed by Coomassie blue staining. The migration of molecular mass markers is indicated on the left of each gel. Protein markers were bovine thyroglobulin (669 kDa), Helicobacter pylori urease (550 kDa), bovine gamma-globulin (158 kDa) and bovine albumin (66 kDa).
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pntd-0001551-g002: Native PAGE of E. granulosus AgB and recombinant oligomers.(A) AgB oligomers from bovine and human samples. Lanes 1–2, AgB purified from bovine cyst 1 and 2, respectively; lane 3, hydatid fluid from human cyst. (B) Oligomers formed by AgB recombinant subunits at different temperatures. Lanes 1–2, rAgB8/1; Lanes 3–4, rAgB8/2; Lanes 5–6, rAgB8/3, at 23°C (lanes 1, 3 and 5) and 37°C (lanes 2, 4 and 6). Samples were resolved on 4–20% polyacrylamide gels followed by Coomassie blue staining. The migration of molecular mass markers is indicated on the left of each gel. Protein markers were bovine thyroglobulin (669 kDa), Helicobacter pylori urease (550 kDa), bovine gamma-globulin (158 kDa) and bovine albumin (66 kDa).

Mentions: AgB was found in different oligomeric states, as detected by both native PAGE and light scattering. In native PAGE, AgB showed oligomers of different sizes, which appeared as a broad smear, with a more defined band of ∼550 kDa (Figure 2A). In DLS, AgB also showed different oligomeric states, which are represented by populations with hydrodynamic radii (Rh) of ∼4 nm, ∼100–200 nm and >2 µm. AgB samples heated at 37°C were also analyzed to evaluate the protein oligomeric states at physiological conditions, but no effect of temperature in AgB oligomerization was observed by native PAGE or DLS (data not shown).


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)

Native PAGE of E. granulosus AgB and recombinant oligomers.(A) AgB oligomers from bovine and human samples. Lanes 1–2, AgB purified from bovine cyst 1 and 2, respectively; lane 3, hydatid fluid from human cyst. (B) Oligomers formed by AgB recombinant subunits at different temperatures. Lanes 1–2, rAgB8/1; Lanes 3–4, rAgB8/2; Lanes 5–6, rAgB8/3, at 23°C (lanes 1, 3 and 5) and 37°C (lanes 2, 4 and 6). Samples were resolved on 4–20% polyacrylamide gels followed by Coomassie blue staining. The migration of molecular mass markers is indicated on the left of each gel. Protein markers were bovine thyroglobulin (669 kDa), Helicobacter pylori urease (550 kDa), bovine gamma-globulin (158 kDa) and bovine albumin (66 kDa).
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0001551-g002: Native PAGE of E. granulosus AgB and recombinant oligomers.(A) AgB oligomers from bovine and human samples. Lanes 1–2, AgB purified from bovine cyst 1 and 2, respectively; lane 3, hydatid fluid from human cyst. (B) Oligomers formed by AgB recombinant subunits at different temperatures. Lanes 1–2, rAgB8/1; Lanes 3–4, rAgB8/2; Lanes 5–6, rAgB8/3, at 23°C (lanes 1, 3 and 5) and 37°C (lanes 2, 4 and 6). Samples were resolved on 4–20% polyacrylamide gels followed by Coomassie blue staining. The migration of molecular mass markers is indicated on the left of each gel. Protein markers were bovine thyroglobulin (669 kDa), Helicobacter pylori urease (550 kDa), bovine gamma-globulin (158 kDa) and bovine albumin (66 kDa).
Mentions: AgB was found in different oligomeric states, as detected by both native PAGE and light scattering. In native PAGE, AgB showed oligomers of different sizes, which appeared as a broad smear, with a more defined band of ∼550 kDa (Figure 2A). In DLS, AgB also showed different oligomeric states, which are represented by populations with hydrodynamic radii (Rh) of ∼4 nm, ∼100–200 nm and >2 µm. AgB samples heated at 37°C were also analyzed to evaluate the protein oligomeric states at physiological conditions, but no effect of temperature in AgB oligomerization was observed by native PAGE or DLS (data not shown).

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