F.Z. Xu, S.G. Wu, W.Y. Yu
Published: March 11, 2013
Genet. Mol. Res. 12 (1) : 693-701
DOI: https://doi.org/10.4238/2013.March.11.17
Cite this Article:
F.Z. Xu, S.G. Wu, W.Y. Yu (2013). Intracellular localization and association of MHC class I with porcine invariant chain. Genet. Mol. Res. 12(1): 693-701. https://doi.org/10.4238/2013.March.11.17
About the Authors
F.Z. Xu, S.G. Wu, W.Y. Yu
Corresponding Author
F.Z. Xu
Email: xufazhi1103@sina.com
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-DCLIP-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.
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-DCLIP-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.
Key words: Pig invariant chain, Intracellular localization, MHC I molecule.