title: Intracellular localization and association of MHC class I with porcine invariant chain.
abstract: The objective was to investigate the intracellular localization and association of pig major histocompatibility complex (MHC) class I subunits with invariant chain (Ii). Pig MHC class I subunit cDNAs were cloned by RT-PCR and eukaryotic expression plasmids of α and β2m were constructed with fusions to red or enhanced green fluorescent protein (pDsRed2-N1-α, pEGFP-N1-α, pDsRed2-N1-β2m, and pEGFP-N1-β2m). A pig Ii mutant with a deleted CLIP region (DCLIP-Ii) was constructed by overlap extension PCR. Wild-type Ii and mutant Ii were cloned into pEGFP-C1 (pEGFP-C1-Ii, pEGFP-C1-DCLIP-Ii). The recombinant plasmids of MHC I subunits and pEGFP-C1-Ii (pEGFP-C1-ΔCLIP-Ii) were transiently cotransfected into COS-7 cells with Lipofectamine 2000. Immunofluorescence microscopy was performed to detect expression and intracellular localization of Ii and MHC I subunits, and immunoprecipitation was used to analyze their association. Our results indicated that pig Ii associates with integrated MHC I subunits to form oligomers, but cannot associate with single MHC I subunits. Furthermore, deletion of the Ii CLIP sequence blocks association with integrated MHC I subunits. Thus, pig Ii cannot associate with a single MHC I molecule, the α or β2m chain, but Ii and the integrated MHC I molecule can form complexes that colocalize in the endomembranes of COS-7 cells. The Ii of CLIP plays a key role in assembly of Ii and MHC I.
authors.affiliation: Anhui Agricultural University, Hefei, China. xufazhi1103@sina.com
authors.forename: F Z
authors.initials: FZ
authors.lastname: Xu
authors.forename: S G
authors.initials: SG
authors.lastname: Wu
authors.forename: W Y
authors.initials: WY
authors.lastname: Yu
mesh_terms: D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014162:Transfection; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000944:Antigens, Differentiation, B-Lymphocyte; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D008164:Luminescent Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin; C045084:invariant chain