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Transmembrane protein 63A is a partner protein of Haemonchus contortus galectin in the regulation of goat peripheral blood mononuclear cells.

Yuan C, Zhang H, Wang W, Li Y, Yan R, Xu L, Song X, Li X - Parasit Vectors (2015)

Bottom Line: We found that TMEM63A, a functionally unknown protein, from goat PBMCs could bind to Hco-gal-m and -f.In addition, the production of IL-10, IFN-γ and TGF-β induced by rHco-gal-m were also altered.Our results show that TMEM63A is a binding partner of Hco-gal-m/f, and involved in the immune responses of host PBMCs induced by Hco-gal-m for the first time.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China. adorehere@sina.com.

ABSTRACT

Background: Hco-gal-m and -f were two isoforms of galectin cloned from male and female Haemonchus contortus, respectively, and it was demonstrated that recombinant Hco-gal-m and -f could act as immune suppressors. However, little is known about the receptors or binding partners of these galectins in the host. The research of the molecular mechanisms that govern the interactions between these galectins and host molecules will fill a gap in our understanding how parasite galectins interact with host cells.

Methods: A yeast two-hybrid system was used to identify the binding partners of Hco-gal-m and -f in this research. The interaction between rHco-gal-m and candidate binding protein was validated by co-immunoprecipitation. The localization of transmembrane protein 63A (TMEM63A) in peripheral blood mononuclear cells (PBMCs) was detected by immunofluorescence. The distribution of TMEM63A in T cells, B cells and monocytes in PBMCs was detected by flow cytometry. The immunomodulatory effects of Hco-gal-m and TMEM63A on cell proliferation, migration, apoptosis, nitric oxide production and cytokine secretion were observed by co-incubation of rHco-gal-m and TMEM63A-siRNA with goat PBMCs and monocytes.

Results: We found that TMEM63A, a functionally unknown protein, from goat PBMCs could bind to Hco-gal-m and -f. Immunofluorescence showed that TMEM63A was localized to the cell membrane. Flow cytometric analysis revealed that TMEM63A was expressed in the majority of goat PBMCs. After using RNA interference to knockdown expression of TMEM63A, the PBMC proliferation and migration were significantly increased, while the influence of rHco-gal-m on monocyte phagocytosis, PBMC nitric oxide production and migration were potently blocked. In addition, the production of IL-10, IFN-γ and TGF-β induced by rHco-gal-m were also altered.

Conclusions: Our results show that TMEM63A is a binding partner of Hco-gal-m/f, and involved in the immune responses of host PBMCs induced by Hco-gal-m for the first time.

No MeSH data available.


Related in: MedlinePlus

Relative levels of cytokine mRNA transcripts in goat PBMCs pretreated with negative orTMEM63AsiRNA. Goat PBMCs pretreated with TMEM63A siRNA were stimulated with PBS or rHco-gal-m. After the stimulation, changes in cytokine transcript levels in cells were detected by real-time PCR. Each stimuli is shown in one of the two graphs. (A) IL-10; (B) IFN-γ; (C) TGF-β1. Data are presented as the means ± SD; n = 3. Significant differences between the two stimulation conditions for each RNAi group are indicated by an asterisk (*, p < 0.001). A number sign (#) designates that the mean of the TMEM63A knockdown group differs significantly from the mean of the negative siRNA group (p < 0.001) for a stimulation condition. Representative data from one independent experiment with technical triplicates are shown that is representative of three independent experiments.
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Fig8: Relative levels of cytokine mRNA transcripts in goat PBMCs pretreated with negative orTMEM63AsiRNA. Goat PBMCs pretreated with TMEM63A siRNA were stimulated with PBS or rHco-gal-m. After the stimulation, changes in cytokine transcript levels in cells were detected by real-time PCR. Each stimuli is shown in one of the two graphs. (A) IL-10; (B) IFN-γ; (C) TGF-β1. Data are presented as the means ± SD; n = 3. Significant differences between the two stimulation conditions for each RNAi group are indicated by an asterisk (*, p < 0.001). A number sign (#) designates that the mean of the TMEM63A knockdown group differs significantly from the mean of the negative siRNA group (p < 0.001) for a stimulation condition. Representative data from one independent experiment with technical triplicates are shown that is representative of three independent experiments.

Mentions: Goat PBMCs pretreated with ns or TMEM63A siRNA were stimulated by PBS (vehicle) or rHco-gal-m. Then, expression of cytokine mRNA transcripts in PBMCs were detected by real-time PCR. From the result of real-time PCR, IL-10, IFN-γ and TGF-β1 were regulated at a transcriptional level (Figure 8).Figure 8


Transmembrane protein 63A is a partner protein of Haemonchus contortus galectin in the regulation of goat peripheral blood mononuclear cells.

Yuan C, Zhang H, Wang W, Li Y, Yan R, Xu L, Song X, Li X - Parasit Vectors (2015)

Relative levels of cytokine mRNA transcripts in goat PBMCs pretreated with negative orTMEM63AsiRNA. Goat PBMCs pretreated with TMEM63A siRNA were stimulated with PBS or rHco-gal-m. After the stimulation, changes in cytokine transcript levels in cells were detected by real-time PCR. Each stimuli is shown in one of the two graphs. (A) IL-10; (B) IFN-γ; (C) TGF-β1. Data are presented as the means ± SD; n = 3. Significant differences between the two stimulation conditions for each RNAi group are indicated by an asterisk (*, p < 0.001). A number sign (#) designates that the mean of the TMEM63A knockdown group differs significantly from the mean of the negative siRNA group (p < 0.001) for a stimulation condition. Representative data from one independent experiment with technical triplicates are shown that is representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4404006&req=5

Fig8: Relative levels of cytokine mRNA transcripts in goat PBMCs pretreated with negative orTMEM63AsiRNA. Goat PBMCs pretreated with TMEM63A siRNA were stimulated with PBS or rHco-gal-m. After the stimulation, changes in cytokine transcript levels in cells were detected by real-time PCR. Each stimuli is shown in one of the two graphs. (A) IL-10; (B) IFN-γ; (C) TGF-β1. Data are presented as the means ± SD; n = 3. Significant differences between the two stimulation conditions for each RNAi group are indicated by an asterisk (*, p < 0.001). A number sign (#) designates that the mean of the TMEM63A knockdown group differs significantly from the mean of the negative siRNA group (p < 0.001) for a stimulation condition. Representative data from one independent experiment with technical triplicates are shown that is representative of three independent experiments.
Mentions: Goat PBMCs pretreated with ns or TMEM63A siRNA were stimulated by PBS (vehicle) or rHco-gal-m. Then, expression of cytokine mRNA transcripts in PBMCs were detected by real-time PCR. From the result of real-time PCR, IL-10, IFN-γ and TGF-β1 were regulated at a transcriptional level (Figure 8).Figure 8

Bottom Line: We found that TMEM63A, a functionally unknown protein, from goat PBMCs could bind to Hco-gal-m and -f.In addition, the production of IL-10, IFN-γ and TGF-β induced by rHco-gal-m were also altered.Our results show that TMEM63A is a binding partner of Hco-gal-m/f, and involved in the immune responses of host PBMCs induced by Hco-gal-m for the first time.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China. adorehere@sina.com.

ABSTRACT

Background: Hco-gal-m and -f were two isoforms of galectin cloned from male and female Haemonchus contortus, respectively, and it was demonstrated that recombinant Hco-gal-m and -f could act as immune suppressors. However, little is known about the receptors or binding partners of these galectins in the host. The research of the molecular mechanisms that govern the interactions between these galectins and host molecules will fill a gap in our understanding how parasite galectins interact with host cells.

Methods: A yeast two-hybrid system was used to identify the binding partners of Hco-gal-m and -f in this research. The interaction between rHco-gal-m and candidate binding protein was validated by co-immunoprecipitation. The localization of transmembrane protein 63A (TMEM63A) in peripheral blood mononuclear cells (PBMCs) was detected by immunofluorescence. The distribution of TMEM63A in T cells, B cells and monocytes in PBMCs was detected by flow cytometry. The immunomodulatory effects of Hco-gal-m and TMEM63A on cell proliferation, migration, apoptosis, nitric oxide production and cytokine secretion were observed by co-incubation of rHco-gal-m and TMEM63A-siRNA with goat PBMCs and monocytes.

Results: We found that TMEM63A, a functionally unknown protein, from goat PBMCs could bind to Hco-gal-m and -f. Immunofluorescence showed that TMEM63A was localized to the cell membrane. Flow cytometric analysis revealed that TMEM63A was expressed in the majority of goat PBMCs. After using RNA interference to knockdown expression of TMEM63A, the PBMC proliferation and migration were significantly increased, while the influence of rHco-gal-m on monocyte phagocytosis, PBMC nitric oxide production and migration were potently blocked. In addition, the production of IL-10, IFN-γ and TGF-β induced by rHco-gal-m were also altered.

Conclusions: Our results show that TMEM63A is a binding partner of Hco-gal-m/f, and involved in the immune responses of host PBMCs induced by Hco-gal-m for the first time.

No MeSH data available.


Related in: MedlinePlus