Assembly and Antigen-Presenting Function of MHC Class I Molecules in Cells Lacking the ER Chaperone Calreticulin

Abstract

MHC class I molecules expressed in a calreticulin-deficient cell line (K42) assembled with beta 2-microglobulin (beta2-m) normally, but their subsequent loading with optimal peptides was defective. Suboptimally loaded class I molecules were released into the secretory pathway. This occurred despite the ability of newly synthesized class I to interact with the transporter associated with antigen processing (TAP) loading complex. The efficiency of peptide loading was reduced by 50%-80%, and impaired T cell recognition was observed for three out of four antigens tested. The peptide-loading function was specific to calreticulin, since the defect in K42 could be rectified by transfection with calreticulin but not a soluble form of calnexin, which shares its lectin-like activity.

MHC Class I Quality Control

“…Ten years ago the hypothesis was that the abundant ERp57 found in the PLC was a consequence of its interaction with calreticulin or possibly calnexin (Oliver, Roderick et al 1999). However, coimmunoprecipitation from K42 cells (a calreticulin deficient embryonic mouse fibroblast cell line) showed complexes of calnexin together with ERp57, tapasin and HC suggesting an alternative PLC constellation lacking calreticulin (Gao, Adhikari et al 2002). Coimmunoprecipitation in lysates of K42 cells showed that ERp57 was associated with TAP, but that no calnexin could be detected in these precipitates, suggesting that neither calnexin nor calreticulin are essential for recruitment of ERp57 to the PLC (Suh, Mitchell et al 1996;Gao, Adhikari et al 2002).…”

“…However, coimmunoprecipitation from K42 cells (a calreticulin deficient embryonic mouse fibroblast cell line) showed complexes of calnexin together with ERp57, tapasin and HC suggesting an alternative PLC constellation lacking calreticulin (Gao, Adhikari et al 2002). Coimmunoprecipitation in lysates of K42 cells showed that ERp57 was associated with TAP, but that no calnexin could be detected in these precipitates, suggesting that neither calnexin nor calreticulin are essential for recruitment of ERp57 to the PLC (Suh, Mitchell et al 1996;Gao, Adhikari et al 2002). More recently it has been shown that ERp57 is directly disulfide conjugated to tapasin in the PLC (Peaper, Wearsch et al 2005).…”

“…In the absence of calreticulin the MHC-I molecules are not retained in the ER, and the transport of immature MHC-I to the cell surface is accelerated. In K42 cells, many MHC-I molecules would be loaded with suboptimal peptides, due to lack of calreticulin in the MHC-I quality control, and the pMHC-I complexes would fall apart either before or on the cell surface (Gao, Adhikari et al 2002). It was shown that the peptide-receptive MHC-I molecules were not found in the cis-Golgi or ER as they would be in wild type cells; instead they were found in endosomes and lysosomes.…”

“…The TAPtapasin complex interacts with MHC-I, calreticulin and ERp57 to form a fully functional PLC capable of loading peptides into the peptide-receptive MHC-I binding groove (Garbi, Tan et al 2000;Gao, Adhikari et al 2002;Williams, Peh et al 2002;Garbi, Tanaka et al 2006), see figure 2. The precise binding site to TAP has not yet been mapped, but it has been suggested that the first N-terminal transmembrane helix of TAP binds to the transmembrane domain of tapasin (Koch, Guntrum et al 2006), supported by the fact that soluble human tapasin variants are defective in TAP association, also resulting in MHC-I that does not bind to TAP (Sadasivan, Lehner et al 1996).…”

Generation of a functional, soluble tapasin protein from an alternatively spliced mRNA

“…[4][5][6][7][8][9][10] The first five of these associate to form the peptide-loading complex (PLC), which is believed to promote the assembly of peptide-receptive complexes with peptide within the ER. Unlike calreticulin, calnexin and ERp57, which have general chaperone functions, tapasin has a specialised role in MHC class I assembly.…”