Limits...
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 proliferation. 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 proliferation was measured by CCK-8 incorporation. Cells proliferation index was calculated considering the OD450 values in blank group as 100%. Results presented here were collected from one independent experiment and were representative of three independent experiments. Data were represented as mean ± SD, n = 6; *p < 0.001 versus the ns siRNA group; an asterisk and a capped line designates two groups differ significantly (p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Knockdown of the TMEM63A gene and rHco-gal-m-treatment affected the PBMC proliferation. 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 proliferation was measured by CCK-8 incorporation. Cells proliferation index was calculated considering the OD450 values in blank group as 100%. Results presented here were collected from one independent experiment and were representative of three independent experiments. Data were represented as mean ± SD, n = 6; *p < 0.001 versus the ns siRNA group; an asterisk and a capped line designates two groups differ significantly (p < 0.001).

Mentions: As demonstrated by incorporation of cell counting kit-8 (CCK-8), no PBMC multiplication was induced by the ns siRNA in ns siRNA group compared with blank group (Figure 4). rHco-gal-m-treatment significantly suppressed the proliferation of PBMC in the ns siRNA/g group and 63A siRNA/g group compared with ns siRNA group and 63A siRNA group (Figure 4). No significant difference was observed between ns siRNA/g group and 63A siRNA/g group (Figure 4). After the TMEM63A siRNA-treatment, the PBMC proliferation significantly increased in the 63A siRNA group compared with the ns siRNA group (Figure 4).Figure 4


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 proliferation. 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 proliferation was measured by CCK-8 incorporation. Cells proliferation index was calculated considering the OD450 values in blank group as 100%. Results presented here were collected from one independent experiment and were representative of three independent experiments. Data were represented as mean ± SD, n = 6; *p < 0.001 versus the ns siRNA group; an asterisk and a capped line designates two groups differ significantly (p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Knockdown of the TMEM63A gene and rHco-gal-m-treatment affected the PBMC proliferation. 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 proliferation was measured by CCK-8 incorporation. Cells proliferation index was calculated considering the OD450 values in blank group as 100%. Results presented here were collected from one independent experiment and were representative of three independent experiments. Data were represented as mean ± SD, n = 6; *p < 0.001 versus the ns siRNA group; an asterisk and a capped line designates two groups differ significantly (p < 0.001).
Mentions: As demonstrated by incorporation of cell counting kit-8 (CCK-8), no PBMC multiplication was induced by the ns siRNA in ns siRNA group compared with blank group (Figure 4). rHco-gal-m-treatment significantly suppressed the proliferation of PBMC in the ns siRNA/g group and 63A siRNA/g group compared with ns siRNA group and 63A siRNA group (Figure 4). No significant difference was observed between ns siRNA/g group and 63A siRNA/g group (Figure 4). After the TMEM63A siRNA-treatment, the PBMC proliferation significantly increased in the 63A siRNA group compared with the ns siRNA group (Figure 4).Figure 4

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