Major Histocompatibility Complex Class I-ERp57-Tapasin Interactions within the Peptide-loading Complex

Abstract

The endoplasmic reticulum-located multimolecular peptideloading complex functions to load optimal peptides onto major histocompatibility complex (MHC) class I molecules for presentation to CD8 ؉ T lymphocytes. Two oxidoreductases, ERp57 and protein-disulfide isomerase, are known to be components of the peptide-loading complex. Within the peptide-loading complex ERp57 is normally found disulfide-linked to tapasin, through one of its two thioredoxin-like redox motifs. We describe here a novel trimeric complex that disulfide links together MHC class I heavy chain, ERp57 and tapasin, and that is found in association with the transporter associated with antigen processing peptide transporter. The trimeric complex normally represents a small subset of the total ERp57-tapasin pool but can be significantly increased by altering intracellular oxidizing conditions. Direct mutation of a conserved structural cysteine residue implicates an interaction between ERp57 and the MHC class I peptide-binding groove. Taken together, our studies demonstrate for the first time that ERp57 directly interacts with MHC class I molecules within the peptide-loading complex and suggest that ERp57 and protein-disulfide isomerase act in concert to regulate the redox status of MHC class I during antigen presentation.To bind a high affinity, optimized pool of peptides, MHC 6 class I molecules undergo a series of chaperone-mediated interactions within the endoplasmic reticulum (ER) (1, 2). For most MHC class I molecules, this process occurs in the multicomponent MHC class I peptide-loading complex (PLC), which in addition to ␤ 2 -microglobulin (␤2m)-associated MHC class I molecules includes the chaperone calreticulin, the oxidoreductases ERp57 and protein-disulfide isomerase (PDI), the class I-specific accessory molecule tapasin, and the peptide transporter TAP (3-5). A series of experimental reports have indicated that the PLC acts cooperatively, and that the absence of any of the components results in the disruption of MHC class I assembly, and a reduction in the efficiency of antigen presentation to T cells (6 -9).Both ERp57 and PDI are integral components of the PLC (10 -12). These oxidoreductases are related to thioredoxin (TR) and share two functional CXXC motifs (denoted TR1 and TR2) (13-15). Possession of this motif permits family members to catalyze reduction, oxidation, and isomerization of disulfide bonds within substrate polypeptides (16). ERp57 normally appears to be in a GSH-dependent reduced state within the ER (17, 18), and is thus likely to be involved in reduction or isomerization reactions. However, a striking feature of ERp57 is that, in the presence of tapasin, it preferentially forms an interaction through its TR1 motif with an unpaired cysteine in tapasin at position 95 (19). The normal escape pathway that exists to release ERp57 from substrate polypeptides is inhibited by this interaction (20), leading to the whole cellular pool of tapasin being disulfide-linked to ERp57, and a large pool of cellular ERp57 being likewise oc...

Formation of a Major Histocompatibility Complex Class I Tapasin Disulfide Indicates a Change in Spatial Organization of the Peptide-loading Complex during Assembly

“…1D, lanes 6 and 7). These results confirm previously published results that demonstrated a mixed disulfide between heavy chain, ERp57, and tapasin (20) and show for the first time that a disulfide can form between heavy chain and tapasin. It should be stressed that the formation of disulfide bonds in these experiments could well be a result of the prevailing redox conditions within the in vitro translation system (see "Discussion").…”

“…A ternary complex between ERp57, tapasin, and heavy chain has previously been identified that was suggested to include a mixed disulfide between ERp57 and heavy chain via the ␣2 cysteine residues (20). To determine whether the ternary complex we observed in our experimental system also contains a mixed disulfide between ERp57 and the ␣2 cysteines of heavy chain, we used the approach that maximized the formation of this complex.…”

“…Further support for a role of ERp57 and tapasin in regulation of the redox state of heavy chain within the PepLC was provided when a disulfide-bonded ternary complex between heavy chain, ERp57, and tapasin was identified (20). In particular, it was suggested that a mixed disulfide forms between ERp57 and heavy chain within the PepLC.…”

Redox-regulated Export of the Major Histocompatibility Complex Class I-Peptide Complexes from the Endoplasmic Reticulum

“…After initial interaction with the lectin-like chaperone calnexin, MHC class I heavy chain-␤ 2 -microglobulin (␤ 2 m) heterodimers are incorporated into the peptide-loading complex (PLC), a multimolecular unit of ER proteins that promotes optimal peptide loading into MHC class I molecules (Hammerling et al, 1998;Pamer and Cresswell, 1998;Elliott and Williams, 2005). The PLC contains calreticulin, tapasin, transporters associated with antigen processing (TAP) and two thioldependent oxidoreductases, ERp57 and protein disulfide isomerase (PDI) (Garbi et al, 2005;Park et al, 2006;Santos et al, 2007).…”

“…After initial interaction with the lectin-like chaperone calnexin, MHC class I heavy chain-␤ 2 -microglobulin (␤ 2 m) heterodimers are incorporated into the peptide-loading complex (PLC), a multimolecular unit of ER proteins that promotes optimal peptide loading into MHC class I molecules (Hammerling et al, 1998;Pamer and Cresswell, 1998;Elliott and Williams, 2005). The PLC contains calreticulin, tapasin, transporters associated with antigen processing (TAP) and two thioldependent oxidoreductases, ERp57 and protein disulfide isomerase (PDI) (Garbi et al, 2005;Park et al, 2006;Santos et al, 2007).Several functions have been proposed for tapasin in facilitating optimal peptide loading and optimization of the peptide cargo (Grandea and Van Kaer, 2001;Purcell et al, 2001;Momburg and Tan, 2002). Tapasin bridges heavy chain-␤ 2 m heterodimers to TAP (Sadasivan et al, 1996) and thus provides physical proximity between MHC class I molecules and TAP.…”