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Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1

View Article: PubMed Central - PubMed

ABSTRACT

The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system.

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Results of the internalization of LTB inhibition. mø264.7 cells were treated with 100 nM ganglioside M1 (GM) for 5, 15, or 30 min, respectively. The LTB was added and incubated for another 5, 15, or 30 min, respectively. After fixing in a solution of 4% formaldehyde and penetrating with 0.5% Triton-X100, the samples were blocked with 10% BSA/PBS and incubated with the rabbit-anti-RAB5A and mouse-anti-His-tag of LTB. Then the samples were incubated with secondary DyLight 488 labeled goat anti-rabbit IgG and Cy3 labeled goat anti-mouse IgG to detect the RAB5A (green) and LTB (red), respectively. Images were taken using a confocal laser scanning microscope (FluoView1000, Olympus) with a 20× objective using the sequential scanning mode (200×) and processed using the FluoView software (Olympus).
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ijms-17-01419-f005: Results of the internalization of LTB inhibition. mø264.7 cells were treated with 100 nM ganglioside M1 (GM) for 5, 15, or 30 min, respectively. The LTB was added and incubated for another 5, 15, or 30 min, respectively. After fixing in a solution of 4% formaldehyde and penetrating with 0.5% Triton-X100, the samples were blocked with 10% BSA/PBS and incubated with the rabbit-anti-RAB5A and mouse-anti-His-tag of LTB. Then the samples were incubated with secondary DyLight 488 labeled goat anti-rabbit IgG and Cy3 labeled goat anti-mouse IgG to detect the RAB5A (green) and LTB (red), respectively. Images were taken using a confocal laser scanning microscope (FluoView1000, Olympus) with a 20× objective using the sequential scanning mode (200×) and processed using the FluoView software (Olympus).

Mentions: To evaluate LTB binding and entry in live mø264.7 cell through surface GM1, several molecular markers were examined by confocal microscopy at different time points. Figure 4 and Figure 5 confirm the results that LTB entered through GM1 binding and was processed via ENS pathway (Figure 4 and Figure 5). The results showed that RAB5A colocalized with LTB at 15 and 30 min (Figure 4). RAB5A was required for the fusion of plasma membranes and early endosomes during exogenous antigen processing. Therefore, it confirmed that LTB was processed through ENS pathway. However, CALR was observed to interact with LTB in ER at 15 min. There was no CALR-LTB colocalization signal detectable at 30 min in ER (Figure 4). Sequently, LAMP-1 was observed to colocalize with LTB in lysosome at 30 min only (Figure 4). The results suggested that LTB was endocytosed in ENS-lysosomal-ER system and that the process might last for 30 min.


Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1
Results of the internalization of LTB inhibition. mø264.7 cells were treated with 100 nM ganglioside M1 (GM) for 5, 15, or 30 min, respectively. The LTB was added and incubated for another 5, 15, or 30 min, respectively. After fixing in a solution of 4% formaldehyde and penetrating with 0.5% Triton-X100, the samples were blocked with 10% BSA/PBS and incubated with the rabbit-anti-RAB5A and mouse-anti-His-tag of LTB. Then the samples were incubated with secondary DyLight 488 labeled goat anti-rabbit IgG and Cy3 labeled goat anti-mouse IgG to detect the RAB5A (green) and LTB (red), respectively. Images were taken using a confocal laser scanning microscope (FluoView1000, Olympus) with a 20× objective using the sequential scanning mode (200×) and processed using the FluoView software (Olympus).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037698&req=5

ijms-17-01419-f005: Results of the internalization of LTB inhibition. mø264.7 cells were treated with 100 nM ganglioside M1 (GM) for 5, 15, or 30 min, respectively. The LTB was added and incubated for another 5, 15, or 30 min, respectively. After fixing in a solution of 4% formaldehyde and penetrating with 0.5% Triton-X100, the samples were blocked with 10% BSA/PBS and incubated with the rabbit-anti-RAB5A and mouse-anti-His-tag of LTB. Then the samples were incubated with secondary DyLight 488 labeled goat anti-rabbit IgG and Cy3 labeled goat anti-mouse IgG to detect the RAB5A (green) and LTB (red), respectively. Images were taken using a confocal laser scanning microscope (FluoView1000, Olympus) with a 20× objective using the sequential scanning mode (200×) and processed using the FluoView software (Olympus).
Mentions: To evaluate LTB binding and entry in live mø264.7 cell through surface GM1, several molecular markers were examined by confocal microscopy at different time points. Figure 4 and Figure 5 confirm the results that LTB entered through GM1 binding and was processed via ENS pathway (Figure 4 and Figure 5). The results showed that RAB5A colocalized with LTB at 15 and 30 min (Figure 4). RAB5A was required for the fusion of plasma membranes and early endosomes during exogenous antigen processing. Therefore, it confirmed that LTB was processed through ENS pathway. However, CALR was observed to interact with LTB in ER at 15 min. There was no CALR-LTB colocalization signal detectable at 30 min in ER (Figure 4). Sequently, LAMP-1 was observed to colocalize with LTB in lysosome at 30 min only (Figure 4). The results suggested that LTB was endocytosed in ENS-lysosomal-ER system and that the process might last for 30 min.

View Article: PubMed Central - PubMed

ABSTRACT

The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system.

No MeSH data available.


Related in: MedlinePlus