Charactorization Of The Binding Of Properties Of The Chimeric Fusion Protein Ii-NDV-F With MHC Molecules

Major histocompatibility complex(MHC) classâ…¡molecules are polymorphic cell surface glycoproteins that serve the immune system as peptide receptors.The invariant chain(Ii) is a non-polymorphic typeâ…¡transmembrane protein and acts as a chaperone during MHC classâ…¡(MHCâ…¡) biosynthesis and binding foreign peptide antigens.In biosynthesis Ii assists initial folding and oligomerisation of the MHCâ…¡subunits.The final assembled classâ…¡-Ii complex is a nonamer,consisting of a single Ii trimer associated with three classâ…¡heterodimers.The association of Ii with MHC classâ…¡molecules involves the direct occupancy of the peptide-binding groove with a region of Ii known as the CLIP (classâ…¡-associated invariant chain peptide,amino acids 81-104) domain.Occupancy of the classâ…¡peptide binding site with CLIP plays an important role in preventing MHC classâ…¡molecules from the loading of endogenetic antigenic peptides in the endoplasmic reticulum(ER).In the absence of Ii,unassembled MHCⅡαandβchains are largely retained within ER.So far,the studies on the association of MHCâ…¡with Ii have been mainly focusing on mammal,especially human,and have never been shown in poultry.Analysis of the putative amino acid sequence of Ii indicated that the CLIP region(especially aa 91-99: MRMATPLLM) is completely conserved between human,mouse and rat.But this striking conservation does not extend to other species,including poultry.Whether can chicken Ii associate with single MHCⅡαorβchain and what does its CLIP play? Can the Ii replaced CLIP with exogenous antigens peptides associate with MHCâ…¡subunits? For studying these questions,we respectively constructed the eukaryotic expression plasmids pEGFP-C1-Ii,pDsRed2-N1-α,pDsRed2-N1-βthat fused red or enhanced green fluorescent protein.The recombinant plasmids were transiently transfected into COS-7 cells with Lipofectamine 2000.Immunofluorescence microscopy was carried out to detect the expression and cellular localization of Ii and single MHCâ…¡subunit,and immunoprecipitation was used for analyzing the association of chicken Ii orΔCLIP- Ii with the MHCâ…¡subunits.The results indicated that the fusion genes ofα,βor Ii was predominantly expressed and localized in cellular endomembrane system,and changed the cytoplasm localization of the fluorescent protein.Chicken Ii with individual MHCⅡαorβpolypeptides colocalized in cellular endomembrane system,and its CLIP region plays a key role on the assembly of Ii with MHCâ…¡subunits.Furthermore,we also found that the Ii replaced CLIP with Newcastle disease virus F₃₄₃ epitopes(NDV F₃₄₃) can not only efficiently load F₃₄₃ antigenic peptides into the vesicles of endomembrane system but also. restore the association with MHCâ…¡subunits in the transfected cells.Thus,MHCâ…¡polymer assembly is not blocked as long as the basic steric molecular structure of Ii is maintained,and different binding models exist in different species or MHCâ…¡isotypes. Moreover,in the designing of effective DNA vaccines,chicken Ii could act as a potential "carrier" of the antigenic peptides for the delivery of an antigenic peptide to the MHC classâ…¡pathway.Both MHCâ… and MHCâ…¡molecules are synthesized in the ER,yet they were sorted in trans-Golgi.MHCâ… molecules were transported to the cellular surface by vesicles,and MHCâ…¡molecules must be transported to lysosome.The different sorting route of MHCâ… and MHCâ…¡molecules may be related to the guidance of Ii.To investigate the intracellular localization and association of chicken major histocompatibility complex classâ… subunits with invariant chain,we carried out a series of the experiments.At first,the sequences of MHCⅠαandβ_2m subunits were amplified with RT-PCR,and sequentially the prokaryotic expressive vector pET-32a-MHCⅠαand pET-32a-MHCⅠβ_2m containing signal peptides was constructed and expressed by Rosetta bacteria.Secondly,we constructed the eukaryotic expression plasmids of MHCⅠαand MHCⅠβ_2m that fused red or enhanced green fluorescent protein,respectively.The recombinant plasmids of MHCâ… subunits and pEGFP-C1-Ii that can express enhanced green fluorescent protein were transiently transfected into COS-7 cells.The colocalization was found in endocytic compartments when GFP-Ii,MHCⅠα-RFP and MHCⅠβ_2m-RFP were co-expressed in COS-7 cells. Immunoprecipitation of Ii showed that GFP-Ii co-isolated with MHCⅠα-GFP and MHCⅠβ_2m-GFP subunits when COS-7 cells were transiently transfected with pEGFP-C1-Ii, pEGFP-N1-MHCⅠαand pEGFP-N1-MHCⅠβ_2m.Thus,Chicken Ii can not effectively associate with singleαorβ_2m subunits from chicken MHCâ… molecules,but Ii and the integrated MHCâ… molecule can form the complexes that are colocalized in endomembrane system of COS-7 cells.