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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

TMEM63A localized to the cell surface in PBMCs. Goat PBMC were first fixed and then permeabilized with detergent prior to IF analysis. Then, cells were incubated with rat anti-TMEM63A-NO IgG (TMEM63A) or negative rat IgG (Control). DIO (green), DAPI (blue) and Cy3-conjugated secondary antibodies (red) were used for triple staining. (A and D): cell membrane (green) and nuclei (blue) staining of cells; (B and E): staining of target proteins (red); (C and F): a merged image of the three colors. TMEM63A only localized to the cell membranes. Scale bars represent 5000 nm.
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Fig2: TMEM63A localized to the cell surface in PBMCs. Goat PBMC were first fixed and then permeabilized with detergent prior to IF analysis. Then, cells were incubated with rat anti-TMEM63A-NO IgG (TMEM63A) or negative rat IgG (Control). DIO (green), DAPI (blue) and Cy3-conjugated secondary antibodies (red) were used for triple staining. (A and D): cell membrane (green) and nuclei (blue) staining of cells; (B and E): staining of target proteins (red); (C and F): a merged image of the three colors. TMEM63A only localized to the cell membranes. Scale bars represent 5000 nm.

Mentions: We observed the locations of TMEM63A in intact and permeabilized PBMCs by IF. Cellular membranes were stained with VybrantDiO and then confocal imaging was used to visualize both the membrane and sphere locations [43]. Nuclei were stained with DAPI to observe the nuclear morphology [44]. Confocal microscopy images showed that TMEM63A only localized to the cell surface (Figure 2B and C). In the control group, no red fluorescence was observed (Figure 2E and F).Figure 2


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)

TMEM63A localized to the cell surface in PBMCs. Goat PBMC were first fixed and then permeabilized with detergent prior to IF analysis. Then, cells were incubated with rat anti-TMEM63A-NO IgG (TMEM63A) or negative rat IgG (Control). DIO (green), DAPI (blue) and Cy3-conjugated secondary antibodies (red) were used for triple staining. (A and D): cell membrane (green) and nuclei (blue) staining of cells; (B and E): staining of target proteins (red); (C and F): a merged image of the three colors. TMEM63A only localized to the cell membranes. Scale bars represent 5000 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: TMEM63A localized to the cell surface in PBMCs. Goat PBMC were first fixed and then permeabilized with detergent prior to IF analysis. Then, cells were incubated with rat anti-TMEM63A-NO IgG (TMEM63A) or negative rat IgG (Control). DIO (green), DAPI (blue) and Cy3-conjugated secondary antibodies (red) were used for triple staining. (A and D): cell membrane (green) and nuclei (blue) staining of cells; (B and E): staining of target proteins (red); (C and F): a merged image of the three colors. TMEM63A only localized to the cell membranes. Scale bars represent 5000 nm.
Mentions: We observed the locations of TMEM63A in intact and permeabilized PBMCs by IF. Cellular membranes were stained with VybrantDiO and then confocal imaging was used to visualize both the membrane and sphere locations [43]. Nuclei were stained with DAPI to observe the nuclear morphology [44]. Confocal microscopy images showed that TMEM63A only localized to the cell surface (Figure 2B and C). In the control group, no red fluorescence was observed (Figure 2E and F).Figure 2

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