Molecular cloning and site-directed mutagenesis of leucine-based sorting motifs of the porcine invariant chain

Abstract

ABSTRACT. Invariant chain (Ii) is a transmembrane protein that associates with MHC class II molecules in the endoplasmic reticulum. The cytoplasmic tail of Ii contains two leucine residues able to direct Ii to the endocytic pathway. We obtained the pig Ii gene by RT-PCR. Mutated Ii was prepared via site directed mutagenesis by the PCR Megaprimer method to study the effect of the two leucines on the localization of pig Ii. These mutated fragments were ligated to the vector pmCherry-C1. The recombinant plasmids were transiently transfected into COS-7 cells with Lipofectamine TM 2000. Fluorescence of fusion proteins (mCherry-Ii) was observed with a fluorescent microscope. Amino acid sequence alignment showed that pig Ii has domains similar to those seen in other mammalian Ii, including the cytoplasmic, transmembrane, class II-associated Ii-derived peptide, and trimerization domains. Based on observations with the fluorescent microscope, we found that two leucine-based motifs are required for pig Ii intracellular localization, and that both motifs independently mediate this function in Ii.

The intracellular localization and association of porcine Ia-associated invariant chain with the MHC class I-related porcine neonatal Fc receptor for IgG

“…5), indicating that the Ii chain may retain the FcRn H chain in the ER until its assembly into a complex with β2m (Ye et al, 2008a(Ye et al, , 2008b. This is consistent with studies finding that the Ii chain retains incompletely folded CD1d H chain in the ER (Jayawardena- Wolf et al, 2001), but inconsistent with studies showing that the Ii chain combines with fully folded MHC class I in the ER because Ii cannot associate with a single MHC I molecule, or the α or β2m chain (Powis, 2006;Vigna et al, 1996;Xu et al, 2013aXu et al, , 2013b. FcRn binds IgG in the slightly acidic pH of the gut lumen and releases IgG into the bloodstream of newborn animals at the neutral pH of the interstitium, pH 7.4 (Ghosh et al, 1995;Ye et al, 2008aYe et al, , 2008b.…”

“…However, additional interactions of Ii with other molecules were found in recent studies. Xu et al indicated that Ii and the integrated MHC I molecule can form complexes that colocalize in the endomembranes of cells (Xu et al, 2013a(Xu et al, , 2013b. Ii acts as a receptor for macrophage migration inhibitory factor (MIF), alone or with CD44 (Leng et al, 2003;Naujokas et al, 1993;Shi et al, 2006).…”

“…The DNA fragment was digested and ligated into the plasmids pEGFP-N1 and pcDNA3-FLAG to generate the plasmids pEGFP-N1-FcRn-M and pcDNA3-FLAG-FcRn-M. The construction of expression plasmid pCDNA3-ΔCLIP-Ii for a pig Ii mutant protein lacking the CLIP region (ΔCLIP-Ii) has been described previously (Xu et al, 2013a(Xu et al, , 2013b. All constructs were sequenced to verify the accuracy of the amplification.…”

“…The cDNA fragment encoding pig Ii made in our laboratory was subcloned into the peCherry-C1, pCDNA3, and pEGFP-C1 vectors (Xu et al, 2013a(Xu et al, , 2013b. The cDNA fragments encoding pig β2m and FcRn were subcloned into pEGFP-N1 expression plasmids and pcDNA3-FLAG, respectively.…”

Molecular characterization and tissue-specific expression of invariant chain isoform in Muscovy Duck (Cairina moschata)

“…Ii guides antigen presentation through its N-terminal sorting signal (Landsverk et al, 2009;Xu et al, 2013). Thus, Ii has been used as immune carrier (Chen and Yu, 2010;Wälchli et al, 2014) in the construction of genetic vaccines, in which the class II-associated Ii-derived peptide domain was replaced with sequences encoding for specific antigen epitopes.…”