The Identification And Functional Research On The Receptors Of Haemonchus Contortus Galectin In Goat Peripheral Blood Mononuclear Cells

The gastrointestinal parasitic nematode Haemonchus contortus(H.contortus)is a major blood feeding parasite of small ruminants,and causes major economic loss worldwide.It is also the most widely used model parasitic nematode in drug discovery,vaccine development and anthelmintic resistance research.Galectins secreted or excreted by parasitic nematodes and other helminths significantly modulate the host immune response.In our laboratory,two galectins were cloned from male and female H.contortus,respectively,and were named as Hco-gal-m(GenBank No.AY253330)and Hco-gal-f(GenBank No.AY253331).Our previous studies showed that recombinant Hco-gal-m/f(rHco-gal-m/f)provided partial protection against H.contortus infection.We also found that rHco-gal-m/f could bind to the surface of goat peripheral blood mononuclear cell(PBMC),influence several signaling pathways,and inhibite the mRNA transcriptions of cytokines in the goat PBMCs.Cell apoptosis,migration and T cell differentiation were also influenced by rHco-gal-m/f.These findings suggested that Hco-gal-m/f could influence many biological processes,especially those relevant to immune response or evasion.However,little is known about the molecular mechanisms which govern the interactions between the galectin and the host.In this case,the discovery of the membrane receptor of Hco-gal-m/f in goat PBMCs will be a challenge for understanding how H.contortus interacts with its host and parasite-host interaction.1.The construction of a yeast two-hybrid systemIn order to indicate the molecular mechanism of the interaction between Hco-gal-m/f and host cells,we constructed a split-ubiquitin yeast two-hybrid system.Total RNA was extracted from goat PBMC.First-strand cDNA was synthesized from the purified RNA.Double-stranded cDNA was amplified and ligated to adaptor,digested with SfiI enzyme,then cloned into the pPR3-N vector.The recombinant plasmid was electro-transformed into E.coli DH10B to obtain a primary cDNA library.The primary library was amplified and determined the size of cDNA inserts.The recombinant plasmids pDHB1-gal-m and pDMB1-gal-f were constructed as baits,and the expression of Hco-gal-m/fin the yeast was confirmed.The results showed that the total RNA was good quality.The size of the inserts varied from 500 to 2500 bp,with an average value of 1000 bp.The primary cDNA library titer was 1×106 cfu/mL and the amplified library titer was 1×109 cfu/mL.A yeast two-hybrid system has been successfully generated and can be used for future screening of proteins interacting with Hco-gal-m/f.2.The identification and verification of the binding partners of Hco-gal-m/fThe yeast two hybrid system was used to identify the binding partners of Hco-gal-m/f.Eighty-one proteins were identified to have potential interaction with the Hco-Gal-m/f protein in the yeast cells.Three potential Hco-Gal-m/f interacting proteins were identified in the retest,which were TMEM147,SERP1 and TMEM63A(NCBI accession number:JQ923484,KF873762 and KF850508).For validating the combination between rHco-gal-m and candidate protein,the result obtained with yeast two hybrid system was confirmed in rHco-Gal-m stimulated(12 h)goat PBMC by two independent co-immunoprecipitaion(co-IP)approaches.The results of the co-IP studies strongly supported that the binding of Hco-Gal-m with TMEM147,SERP1 and TMEM63A proteins in PBMCs was specific.There results further consolidated that these proteins were binding proteins of Hco-gal-m/f in goat PBMCs.The discovery of the binding partners of Hco-gal-m/f in goat PBMCs will be a challenge for understanding how nematode galectin interacts with its host.3.The location and functional research of the binding partners of Hco-Gal-m/fImmunofluorescence(IF)results showed that TMEM147 located in the cell membrane and within the cell,SERP1 within the cell,and TMEM63A in the cell membrane.Flow cytometric analysis revealed that these proteins were expressed in the majority of goat PBMCs.Co-immunoprecipitation after the RNA interference(RNAi)of binding proteins identified that TMEM147,SERP1 and TMEM63A are binding partners and recptors of rHco-gal-m.Analysis of cytokine transcription and secretion after the RNA interference of binding proteins,indicated that the interaction of rHco-gal-m with TMEM147,SERP1 and TMEM63A was involved in regulating cytokines(TNF-?,IL-10,IFN-?,IL-4,TGF-?1 and IL-17)expression.Collectively,our results provided the first evidence that TMEM147 and TMEM63A might be two receptors of Hco-gal-m/f,and SERP1 was a binding partner of Hco-gal-m/f in goat PBMCs.The combination of these proteins involved in the regulation of immune responses in host cells.