A Part Of Functional Research On The Receptors Of Haemonchus Contortus Galectin In Goat Peripheral Blood Mononuclear Cells(TMEM63A And TMEM147)

Haemonchus contortus(H.contortus)is a gastrointestinal nematode of ruminants,could induce anemia,weight loss,even death in the infected animals.Galectins is a family of carbohydrate-binding proteins and implicated in the parasite-host interactions.Hco-gal-m and Hcogal-f are two kinds of galectins cloned from male and female H.contortus respectively in our laboratory.It has been reported that the recombinant Hco-gal-m/f(rHco-gal-m/f)could bind to the cell surface of goat peripheral blood mononuclear cells(PBMC),induced the goat PBMC apoptosis,and implicated in the regulation of cytokines production,cell migration,cell proliferation and differentiation.These results suggested that rHco-gal-m/f was an important immunoregulation factor implicated in H.contortus infection in the host immunity system.TMEM63A and TMEM147 are receptors of H.contortus galectin,and located in most of the goat PBMC.It has been suggested that TMEM63A could be involved in the most regulation of Hco-gal-m/f in the goat PBMC,however,the molecular mechanism of these regulation and the function of TMEM147 are unclear.The functional studies of TMEM147 and TMEM63A not only contribute to the molecular mechanism of H.contortus immune evasion,but also provide new clues and strategies for the control of H.contortus.1 Identification of the binding proteins of TMEM63A in goat peripheral blood mononuclear cellsIn order to identify the binding proteins of TMEM63A in goat peripheral blood mononuclear cells.The co-IP and shotgun LC-MS/MS assays were performed to collect and identify the binding proteins of TMEM63A in goat peripheral blood mononuclear cells.The results showed that 43 proteins were totally identified,including MHC class I antigen(MHC I)in antigen processing and presentation,toll-like receptor 9(TLR-9)in toll-like receptor signaling pathway,Gli3 protein(Gli3)in hedgehog signaling pathway,heat shock 27 kDa protein 1(HSP27)in VEGF signaling pathway,Bcl-2-associated X protein(BAX)in P53 signaling pathway and.Deltex-2 protein(DTX2)in Notch signaling pathway.These proteins may be involved in the regulation of Hco-gal-m/f on the goat peripheral blood mononuclear cells..These findings will provide new clues for the better understanding of the molecular mechanisms of Hco-gal-m/f mediated immunomodulation.2 Effect of TMEM63A to PBMC in associated signaling pathway45 key genes in six signaling pathways were selected based on the KEGG database.The real-time quantitative PCR analysis of associated signaling pathway in goat PBMC were performed before and after the knockdown of TMEM63A.Our results showed that,there was no significant change on the mRNA transcription of Gli3 and DTX2 induced by the knockdown of TMEM63A,while the mRNA transcription of MHC 1,TLR-9,HSP27,BAX and the signaling pathways which included the respective protein were significantly altered.These findings provide new clues for the target sites of TMEM63A in hosts and the better understanding of the molecular mechanisms of Hco-gal-m/f mediated immunomodulation.3 The function of TMEM147 in the regulation of Hco-gal-m/f on goat PBMCOur previous studies suggested that TMEM147 could also bind to the H.contortus galectin in the yeast two-hybrid(YTH)screening.In order to explore the different roles of TMEM147 and TMEM63A in the regulation of Hco-gal-m/f on goat PBMC,the RNA interference was used to observe the function of TMEM147.Firstly,cells were treated with siRNA transfection for 48h.Then,rHco-gal-m was added as stimulation for 12h.After that,the cell proliferation,NO production,cell phagocytosis,cell migration and apoptosis were all detected.Our results showed that the knockdown of TMEM147 could significantly affect the regulation of Hco-gal-m/f on cell proliferation,NO production,phagocytosis and apoptosis.However,the regulation of Hco-gal-m/f on cell migration was not significantly influenced.The functional research of TMEM147 will provide new clues for the better understanding of the molecular mechanisms of Hco-gal-m/f mediated immunomodulation and the immune evasion of H.contortus.