Structure And Function Of Chicken C-type Lectin-like Receptor Blec

Chicken Blec gene is located in the major histocompatibility complex(MHC),and highly conserved among different chicken MHC-B haplotypes.The assumed extracellular domain of Blec containing a C-type lectin-like domain(CTLD)suggests that Blec might function as natural killer cell receptor/ligand protein,belong to the family of C-type lectin-like receptor(CTLR),but the hypothesis has not been proved practicely yet.Neither the tissue distribution of Blec protein in the chicken nor its putative ligand protein has been reported.Here,a conserved extracellular region Ala59-Leu176 of blec gene was RT-PCR amplified from one MHC-B haplotype 21 chicken splenocytes and cloned into a prokaryotic expression vector of pET-30a to obtain a recombinant plasmid of pET-Blec.SDS-PAGE results showed that the foreign protein was expressed in inclusion body.High purity protein products(>99%)were obtained followed by denaturation,dilution renaturation and gel filtration chromatography.In order to analyze the tissue distribution of Blec protein in chicken,one strain of Blec specific monoclonal antibody-secreted hybriodome cell of Blec-3D3 was prepared routinely by immuning cleand level BALB/c mice with the purified Blec protein.The peripheral blood lymphocytes of one male 72week–old specific pathogen free chicken were detected with flow cytometry using Blec-3D3.The results showed that 0.8%of CD3~+and 0.86%of CD4~+cells were markered with blec protein.Strong specific positive signals were detected in lymph organs and mucous immune associated tissues by immunol histochemical assay,including smooth muscle cells of the bursa of Fabricius,bronchial bronchial smooth muscle cells,kidney,small intestine smooth muscle,thymus Hastellella,cecal tonsils and splenic medulla,while not in the heart and liver.The purified protein of Blec was screened by crystal and the X-ray diffraction resolution reached1.1?.Structural analysis revealed that the Blec protein has two typical alpha helices,two antiparallel beta-folded and three pairs of conserved disulfide bonds(Cys62-Cys73,Cys151-Cys164 and Cys90-Cys172).The extracellular domain appeared a typical CTLD sturcture.The similarity of CTLD to human and mice typical C-type lectin subgroup 2(CLEC2)family proteins are 47.41%(hLLT1),42.24%(hAICL),42.24%(mCLEC2b),42.24%(hCD69),41.38%(mCLEC2g)and 40.52%(hKACL),suggesting that Blec may have a function similar to CLEC2 family proteins.Mammalian CLEC2 families are ligands for the natural killer cell surface receptor NKRP1 family.To investigate whether Blec protein interacts with chicken MHC class I molecules(for MHC calss I molecule mediated killer function)or the other genetic linkage CTLR BNK(for immunoglobulin-like molecule mediated killer function),prokaryotic expression of BNK(named BNK21)and MHC class I molecules(MHC-I(B21)were cloned,expressed and purified,respectively.The two proteins were cocultured in vitro and SDS-PAGE results showed that there was no obvious interaction between Blec and BNK21 nor with MHC-I(B21).Next,Blec and BNK21were cloned into a eukayocyte express vector to obtain two recombinant plasmids of pCmv-Myc-Blec and p3xFlag-BNK21 followed by transfecting into human embryonic kedney cell line of HEK293T cells.Co-IP assay results showed that Blec might interact with BNK21.In this study,the three-dimensional spatial structure of the first chicken C-type lectin-like receptor of Blec was proved,and the Blec protein is distributed at mucous immunity assocaited tissue and CD3+CD4+cells,might interact with the other C-type lectin like protein of BNK,these results summarily suggests that the protein might paly a role in chicken immunology.