The Roles Of TMEM63A In The Functional Regulation Of Goat Peripheral Blood Mononuclear Cells By Haemonchus Contortus Galectin

Haemonchus contortus is an important blood-feeding gastrointestinal nematode parasite of small ruminants and abomasums(fourth or true stomach).Infection can lead to anaemia,loss of condition and death of the host,especially lambs,results the host in a low level immunity.Hco-gal-m(Acc.No.AY253330)and Hco-gal-f(Acc.No.AY253331)are two isoforms of galectins which were cloned from male(m)and female(f)Haemonchus contortus,respectively.Recombinant Hco-gal-m/f play as immune suppressors by inducing cell apoptosis in a dose-dependent manner.They also decrease the capacity of cell migration,ROS output and cytokine production by modulating the free radical producing pathway,T cell development pathway and NF-?B pathway.The previous studies showed a significantly reduction in faecal egg output,worm burdens,increased IgG levels,and partial protection against Haemonchus contortus infection.When the goat was immunized with rHco-gal-m/f.Transmembrane protein 63 A was IV type transmembrane protein which can be acted as pathway and participated in transmembrane transport,signal transduction,cell migration and so on.Further studies showed that TMEM63A expressed in the majority of goat PBMCs.However,TMEM63A was validated only in few kinds of mammals with unknown immunity function though they were in a high homology by now.Transmembrane protein 63A(TMEM63A)was proved to be a novel receptor of Haemonchus contortus galectin(Hco-gal-m/f)in goat peripheral blood mononuclear cell(PBMCs).And,there is currently no hint at the immunological function of mammalian TMEM63A.Our previous studies showed that the roles of TMEM63A in the regulation of Hco-gal-m/f on PBMCs were involved in the regulation of cytokine expression.The present study is therefore designed to access the the roles of TMEM63A in the regulation of Hco-gal-m/f on PBMCs by measuring the proliferation capability,phagocytosis capability,apoptosis capability,migration capability and nitric oxide production of goat PBMCs(monocytes).1.TMEM63A-?:cloning and expressionAs TMEM63A is a multiple transmembrane protein.It cannot be expressed as a whole.We selected the longest intramembrane fragment TMEM63A-I.TMEM63A-I is a gene fragment of 648 bp.We get it by RT-PCR,with a pair of primers.TMEM63A-I was cloned into pMD18-T vector.After sequenced and analyzed,We found that the gene was the same with the fragment in GenBank.Then the prokaryotic expression plasmid pET-28a-TMEM63A-? was constructed.After analyzed,the pET-28a-TMEM63A-I was transformed into Escherichia coli BL21.BL21-pET-28a-TMEM63A-? can be induced by isopropyl-B-D-thiogalactopyranoside(IPTG).The molecular weight of TMEM63A-I is about 25KD.SDS-PAGE analysis showed that the recombinant protein was expressed in Inclusion Bodies.The recombinant protein was purified with purification kit and refolded utilizing urea.2.Preparation of polyclonal antibodyThe tag protein of pET-28a(+)was cut by enteropeptidase.Mixed Freund's adjuvant with the recombinant protein without tag.And injected the emulsion into rat.After three times of subcutaneous injections,we get the anti-63-IP polyclonal antibody from the immune rat.The specificity of anti-63-IP polyclonal antibody was checked out by Western blot.Result of Western blot showed that the anti-63-IP polyclonal antibody could recognize by TMEM63A specificitly.The enzyme-linked immunosorbent assay(ELISA)was used to detecte the the anti-63-IP polyclonal antibody,which laid the foundation for the further study of the TMEM63 A function.3.The roles of TMEM63A in the regulation of Hco-gal-m/f on PBMCsThe roles of TMEM63A in the regulation of Hco-gal-m/f on PBMCs was tested by antibody blocking technique and RNA interference technique.Result with antibody blocking technique showed as follows.When it was only treated with 40?g/mL Hco-gal-m/f,the multiplication capability,nitric oxide output capability,migaration capability of PBMCs(monocytes)were inhibited significantly while the phagocytosis capability was promoted significantly,compared with the blank group.When it was treated with 2?/mL anti-63 A IgG?40?g/mL Hco-gal-m/f,the multiplication capability,nitric oxide output capability,migaration capability of PBMCs(monocytes)were promoted while the phagocytosis capability was inhibited compared with the group that monocytes were only treated by 40?g/mL Hco-gal-m/f or the blank group.When it was treated with 2?/mL Neg A IgG,40?g/mL Hco-gal-m/f,the multiplication capability,phagocytosis capability,nitric oxide output capability,migaration capability of PBMCs(monocytes)were not changed significantly compared with the group that PBMCs(monocytes)were only treated by 40?g/mL Hco-gal-m/f.Result with RNA interference technique showed as follows.When it was only treated with 40?g/mL Hco-gal-m/f,the multiplication capability,nitric oxide output capability,migaration capability of PBMCs(monocytes)were inhibited significantly while the phagocytosis capability was promoted compared with the blank group.When it was treated with TMEM63A-siRNA-1,40?g/mL Hco-gal-m/f,the phagocytosis capability of PBMCs was not changed significantly compared with the group that PBMCs(monocytes)were treated by TMEM63A-siRNA-1.When it was treated with TMEM63A-siRNA-1,40?g/mL Hco-gal-m/f,the multiplication capability,nitric oxide output capability,migaration capability of PBMCs(monocytes)were not inhibited significantly compared with the group that PBMCs(monocytes)were only treated by the blank group.When it was treated with non-specific siRNA,40?g/mL Hco-gal-m/f,the multiplication capability,nitric oxide output capability,migaration capability of PBMCs(monocytes)were inhibited significantly compared with the group that PBMCs were treated by non-specific siRNA.Results with antibody blocking technique or RNA interference technique are in the same tendency.In summary,the roles of TMEM63A in the regulation of Hco-gal-m/f on PBMCs are important.