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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

Knockdown of the TMEM63A gene and rHco-gal-m-treatment affected the PBMC nitrite oxide production. PBMC can be share as blank group (group 1), ns siRNA group (group 2), ns siRNA/g group (group 3), 63A siRNA group (group 4) and 63A siRNA/g group (group 5), which were incubated with equal volume reduced serum medium (group 1), ns siRNA (group 2 and 3) and TMEM63A-siRNA-1 (group 4 and 5) for 60 h. RHco-gal-m in all RNA interference groups (group 3 and 5) was added 12 h before the end of RNA interference period. The nitrite concentration in the PBMCs was measured by using the Griessassay and used as an indicator of nitric oxide production by the PBMCs. Results presented here were collected from one independent experiment and were representative of three independent experiments. Data were represented as mean ± SD, n = 4; an asterisk and a capped line designates two groups differ significantly (p < 0.001).
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Fig6: Knockdown of the TMEM63A gene and rHco-gal-m-treatment affected the PBMC nitrite oxide production. PBMC can be share as blank group (group 1), ns siRNA group (group 2), ns siRNA/g group (group 3), 63A siRNA group (group 4) and 63A siRNA/g group (group 5), which were incubated with equal volume reduced serum medium (group 1), ns siRNA (group 2 and 3) and TMEM63A-siRNA-1 (group 4 and 5) for 60 h. RHco-gal-m in all RNA interference groups (group 3 and 5) was added 12 h before the end of RNA interference period. The nitrite concentration in the PBMCs was measured by using the Griessassay and used as an indicator of nitric oxide production by the PBMCs. Results presented here were collected from one independent experiment and were representative of three independent experiments. Data were represented as mean ± SD, n = 4; an asterisk and a capped line designates two groups differ significantly (p < 0.001).

Mentions: We measured nitric oxide production by TMEM63A gene knockdown PBMC treated with rHco-gal-m. Treatment with rHco-gal-m significantly suppressed nitric oxide production in the ns siRNA/g group and 63A siRNA/g group compared with ns siRNA group and 63A siRNA group (Figure 6). In the rHco-gal-m-treatment cells, the nitric oxide production in the 63A siRNA/g group was significantly increased compared to the cells in the ns siRNA/g group (Figure 6). After the TMEM63A siRNA-treatments, there was no influence on the PBMC nitric oxide production in the 63A siRNA group compared with the ns siRNA group (Figure 6).Figure 6


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)

Knockdown of the TMEM63A gene and rHco-gal-m-treatment affected the PBMC nitrite oxide production. PBMC can be share as blank group (group 1), ns siRNA group (group 2), ns siRNA/g group (group 3), 63A siRNA group (group 4) and 63A siRNA/g group (group 5), which were incubated with equal volume reduced serum medium (group 1), ns siRNA (group 2 and 3) and TMEM63A-siRNA-1 (group 4 and 5) for 60 h. RHco-gal-m in all RNA interference groups (group 3 and 5) was added 12 h before the end of RNA interference period. The nitrite concentration in the PBMCs was measured by using the Griessassay and used as an indicator of nitric oxide production by the PBMCs. Results presented here were collected from one independent experiment and were representative of three independent experiments. Data were represented as mean ± SD, n = 4; an asterisk and a capped line designates two groups differ significantly (p < 0.001).
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Related In: Results  -  Collection

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Fig6: Knockdown of the TMEM63A gene and rHco-gal-m-treatment affected the PBMC nitrite oxide production. PBMC can be share as blank group (group 1), ns siRNA group (group 2), ns siRNA/g group (group 3), 63A siRNA group (group 4) and 63A siRNA/g group (group 5), which were incubated with equal volume reduced serum medium (group 1), ns siRNA (group 2 and 3) and TMEM63A-siRNA-1 (group 4 and 5) for 60 h. RHco-gal-m in all RNA interference groups (group 3 and 5) was added 12 h before the end of RNA interference period. The nitrite concentration in the PBMCs was measured by using the Griessassay and used as an indicator of nitric oxide production by the PBMCs. Results presented here were collected from one independent experiment and were representative of three independent experiments. Data were represented as mean ± SD, n = 4; an asterisk and a capped line designates two groups differ significantly (p < 0.001).
Mentions: We measured nitric oxide production by TMEM63A gene knockdown PBMC treated with rHco-gal-m. Treatment with rHco-gal-m significantly suppressed nitric oxide production in the ns siRNA/g group and 63A siRNA/g group compared with ns siRNA group and 63A siRNA group (Figure 6). In the rHco-gal-m-treatment cells, the nitric oxide production in the 63A siRNA/g group was significantly increased compared to the cells in the ns siRNA/g group (Figure 6). After the TMEM63A siRNA-treatments, there was no influence on the PBMC nitric oxide production in the 63A siRNA group compared with the ns siRNA group (Figure 6).Figure 6

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