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Participation of ezrin in bacterial uptake by trophoblast giant cells.

Watanabe K, Tachibana M, Kim S, Watarai M - Reprod. Biol. Endocrinol. (2009)

Bottom Line: Here we identified ezrin, a member of ezrin-radixin-moesin (ERM) protein family, as a molecule associated with Hsc70.The expression level of ezrin was higher in TG cells than in trophoblast stem (TS) cells, and ezrin knockdown TG cells showed a reduction in bacterial uptake ability.Ezrin associates with Hsc70 that locates on the membrane of TG cells and participates in the bacterial uptake by TG cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Public Health, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. kentaw@yamaguchi-u.ac.jp

ABSTRACT

Background: Trophoblast giant (TG) cells are involved in systematic removal of bacterial pathogens from the maternal-fetal interface of the placenta. In particular, TG cells have the ability to take up extracellular antigens by active phagocytosis induced by interferon-gamma (IFN-gamma). We previously reported that heat shock cognate protein 70 (Hsc70) present on the surface of TG cells mediated the uptake of Brucella abortus. However, the mechanism of bacterial uptake by TG cells is not completely understood. Here we identified ezrin, a member of ezrin-radixin-moesin (ERM) protein family, as a molecule associated with Hsc70.

Methods: Mouse TG cells were employed in all experiments, and B. abortus was used as the bacterial antigen. Confirmation of the binding capacity of ERM protein was assessed by pull-down assay and ELISA using recombinant Hsc70 and ERM proteins. Ezrin was depleted using siRNA and the depletion examined by immunoblotting or immunofluorescence staining.

Results: The expression level of ezrin was higher in TG cells than in trophoblast stem (TS) cells, and ezrin knockdown TG cells showed a reduction in bacterial uptake ability. Although tyrosine phosphorylation of ezrin was not related to bacterial uptake activity, localization of Hsc70 on the membrane was affected by the depletion of ezrin in TG cells.

Conclusion: Ezrin associates with Hsc70 that locates on the membrane of TG cells and participates in the bacterial uptake by TG cells.

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Expression of ERM proteins in TG cells, and the binding capacity between ezrin and Hsc70. (A) Expression of ERM proteins in TG, TS, and J774 macrophage cells. Immunoblot analyses were performed with anti-ezrin, anti-radixin, and anti-moesin antibodies. (B) Affinity of ERM proteins for Hsc70 shown by pull-down assay. Recombinant Hsc70 and each ERM protein were mixed, and these samples were immunoprecipitated with anti-Hsc70 antibodies or beads only. Detection of proteins was performed by immunoblotting. (C) The binding capacity of ERM proteins to Hsc70 were measured by ELISA. Immunoplates were coated with each ERM protein or BSA (control), and then Hsc70 was added. Data are the averages of triplicate samples from three identical experiments. Error bars represent standard deviation. Statistically significant differences between control and ERM proteins are indicated by asterisks (*, P < 0.01). (D) ELISA was used to determine the binding domain of Hsc70 for ezrin. Immunoplates were coated with full-length Hsc70 (Full Hsc70), ATP-binding domain (ATPase), and peptide-binding domain (PBD) of Hsc70, following which ezrin was added. Data are the averages of triplicate samples from three identical experiments. Error bars represent standard deviation. Statistically significant differences between control and each domain proteins are indicated by asterisks (*, P < 0.01).
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Figure 2: Expression of ERM proteins in TG cells, and the binding capacity between ezrin and Hsc70. (A) Expression of ERM proteins in TG, TS, and J774 macrophage cells. Immunoblot analyses were performed with anti-ezrin, anti-radixin, and anti-moesin antibodies. (B) Affinity of ERM proteins for Hsc70 shown by pull-down assay. Recombinant Hsc70 and each ERM protein were mixed, and these samples were immunoprecipitated with anti-Hsc70 antibodies or beads only. Detection of proteins was performed by immunoblotting. (C) The binding capacity of ERM proteins to Hsc70 were measured by ELISA. Immunoplates were coated with each ERM protein or BSA (control), and then Hsc70 was added. Data are the averages of triplicate samples from three identical experiments. Error bars represent standard deviation. Statistically significant differences between control and ERM proteins are indicated by asterisks (*, P < 0.01). (D) ELISA was used to determine the binding domain of Hsc70 for ezrin. Immunoplates were coated with full-length Hsc70 (Full Hsc70), ATP-binding domain (ATPase), and peptide-binding domain (PBD) of Hsc70, following which ezrin was added. Data are the averages of triplicate samples from three identical experiments. Error bars represent standard deviation. Statistically significant differences between control and each domain proteins are indicated by asterisks (*, P < 0.01).

Mentions: The ERM family consists of three closely related proteins; sequencing of cDNAs has revealed that the amino acid sequence identity among the ERM proteins is 70-80% [21]. We therefore investigated the expression of ERM proteins in TG cells, TS cells, and J774 macrophage cells by immunoblotting. All ERM proteins were detectable in J774 cells, but only moesin was not detected in TS and TG cells (Fig. 2A). The expression level of ezrin in TG cells was higher than that in TS cells. Next, to confirm the ability of ERM proteins to bind to Hsc70, we tested them by pull-down assay with each recombinant ERM proteins or Hsc70. Immunoblotting of the precipitated proteins showed the association between ERM proteins and Hsc70, but there was no association when only beads were present (Fig. 2B). The association was also confirmed by ELISA using ERM protein-coated immunoplates. Hsc70 bounded to the ezrin-coated wells most strongly (Fig. 2C). It has been reported that Hsc70 contains three functional domains, i.e., ATP-binding domain (ATPase), the peptide-binding domain (PBD), and the carboxy-terminal domain (CTD) [22]. To identify the binding domain of Hsc70, the full-length, ATPase domain, and peptide-binding domain of Hsc70 were constructed, and the ability of each recombinant Hsc70 to bind to ezrin was confirmed by ELISA. Ezrin reacted with PBD of Hsc70 (Fig. 2D). Furthermore, we examined the localization of ezrin in TG cells. Using Triton X-100, it was observed that ezrin co-localized with the actin cytoskeleton inside permeabilized TG cells, and with Hsc70 on TG cell membranes (Fig. 3).


Participation of ezrin in bacterial uptake by trophoblast giant cells.

Watanabe K, Tachibana M, Kim S, Watarai M - Reprod. Biol. Endocrinol. (2009)

Expression of ERM proteins in TG cells, and the binding capacity between ezrin and Hsc70. (A) Expression of ERM proteins in TG, TS, and J774 macrophage cells. Immunoblot analyses were performed with anti-ezrin, anti-radixin, and anti-moesin antibodies. (B) Affinity of ERM proteins for Hsc70 shown by pull-down assay. Recombinant Hsc70 and each ERM protein were mixed, and these samples were immunoprecipitated with anti-Hsc70 antibodies or beads only. Detection of proteins was performed by immunoblotting. (C) The binding capacity of ERM proteins to Hsc70 were measured by ELISA. Immunoplates were coated with each ERM protein or BSA (control), and then Hsc70 was added. Data are the averages of triplicate samples from three identical experiments. Error bars represent standard deviation. Statistically significant differences between control and ERM proteins are indicated by asterisks (*, P < 0.01). (D) ELISA was used to determine the binding domain of Hsc70 for ezrin. Immunoplates were coated with full-length Hsc70 (Full Hsc70), ATP-binding domain (ATPase), and peptide-binding domain (PBD) of Hsc70, following which ezrin was added. Data are the averages of triplicate samples from three identical experiments. Error bars represent standard deviation. Statistically significant differences between control and each domain proteins are indicated by asterisks (*, P < 0.01).
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Related In: Results  -  Collection

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Figure 2: Expression of ERM proteins in TG cells, and the binding capacity between ezrin and Hsc70. (A) Expression of ERM proteins in TG, TS, and J774 macrophage cells. Immunoblot analyses were performed with anti-ezrin, anti-radixin, and anti-moesin antibodies. (B) Affinity of ERM proteins for Hsc70 shown by pull-down assay. Recombinant Hsc70 and each ERM protein were mixed, and these samples were immunoprecipitated with anti-Hsc70 antibodies or beads only. Detection of proteins was performed by immunoblotting. (C) The binding capacity of ERM proteins to Hsc70 were measured by ELISA. Immunoplates were coated with each ERM protein or BSA (control), and then Hsc70 was added. Data are the averages of triplicate samples from three identical experiments. Error bars represent standard deviation. Statistically significant differences between control and ERM proteins are indicated by asterisks (*, P < 0.01). (D) ELISA was used to determine the binding domain of Hsc70 for ezrin. Immunoplates were coated with full-length Hsc70 (Full Hsc70), ATP-binding domain (ATPase), and peptide-binding domain (PBD) of Hsc70, following which ezrin was added. Data are the averages of triplicate samples from three identical experiments. Error bars represent standard deviation. Statistically significant differences between control and each domain proteins are indicated by asterisks (*, P < 0.01).
Mentions: The ERM family consists of three closely related proteins; sequencing of cDNAs has revealed that the amino acid sequence identity among the ERM proteins is 70-80% [21]. We therefore investigated the expression of ERM proteins in TG cells, TS cells, and J774 macrophage cells by immunoblotting. All ERM proteins were detectable in J774 cells, but only moesin was not detected in TS and TG cells (Fig. 2A). The expression level of ezrin in TG cells was higher than that in TS cells. Next, to confirm the ability of ERM proteins to bind to Hsc70, we tested them by pull-down assay with each recombinant ERM proteins or Hsc70. Immunoblotting of the precipitated proteins showed the association between ERM proteins and Hsc70, but there was no association when only beads were present (Fig. 2B). The association was also confirmed by ELISA using ERM protein-coated immunoplates. Hsc70 bounded to the ezrin-coated wells most strongly (Fig. 2C). It has been reported that Hsc70 contains three functional domains, i.e., ATP-binding domain (ATPase), the peptide-binding domain (PBD), and the carboxy-terminal domain (CTD) [22]. To identify the binding domain of Hsc70, the full-length, ATPase domain, and peptide-binding domain of Hsc70 were constructed, and the ability of each recombinant Hsc70 to bind to ezrin was confirmed by ELISA. Ezrin reacted with PBD of Hsc70 (Fig. 2D). Furthermore, we examined the localization of ezrin in TG cells. Using Triton X-100, it was observed that ezrin co-localized with the actin cytoskeleton inside permeabilized TG cells, and with Hsc70 on TG cell membranes (Fig. 3).

Bottom Line: Here we identified ezrin, a member of ezrin-radixin-moesin (ERM) protein family, as a molecule associated with Hsc70.The expression level of ezrin was higher in TG cells than in trophoblast stem (TS) cells, and ezrin knockdown TG cells showed a reduction in bacterial uptake ability.Ezrin associates with Hsc70 that locates on the membrane of TG cells and participates in the bacterial uptake by TG cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Public Health, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. kentaw@yamaguchi-u.ac.jp

ABSTRACT

Background: Trophoblast giant (TG) cells are involved in systematic removal of bacterial pathogens from the maternal-fetal interface of the placenta. In particular, TG cells have the ability to take up extracellular antigens by active phagocytosis induced by interferon-gamma (IFN-gamma). We previously reported that heat shock cognate protein 70 (Hsc70) present on the surface of TG cells mediated the uptake of Brucella abortus. However, the mechanism of bacterial uptake by TG cells is not completely understood. Here we identified ezrin, a member of ezrin-radixin-moesin (ERM) protein family, as a molecule associated with Hsc70.

Methods: Mouse TG cells were employed in all experiments, and B. abortus was used as the bacterial antigen. Confirmation of the binding capacity of ERM protein was assessed by pull-down assay and ELISA using recombinant Hsc70 and ERM proteins. Ezrin was depleted using siRNA and the depletion examined by immunoblotting or immunofluorescence staining.

Results: The expression level of ezrin was higher in TG cells than in trophoblast stem (TS) cells, and ezrin knockdown TG cells showed a reduction in bacterial uptake ability. Although tyrosine phosphorylation of ezrin was not related to bacterial uptake activity, localization of Hsc70 on the membrane was affected by the depletion of ezrin in TG cells.

Conclusion: Ezrin associates with Hsc70 that locates on the membrane of TG cells and participates in the bacterial uptake by TG cells.

Show MeSH
Related in: MedlinePlus