ERp57 Does Not Require Interactions with Calnexin and Calreticulin to Promote Assembly of Class I Histocompatibility Molecules, and It Enhances Peptide Loading Independently of Its Redox Activity

Abstract

ERp57 is a thiol oxidoreductase that catalyzes disulfide formation in heavy chains of class I histocompatibility molecules. It also forms a mixed disulfide with tapasin within the class I peptide loading complex, stabilizing the complex and promoting efficient binding of peptides to class I molecules. Since ERp57 associates with the lectin chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, we examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells. Moreover, ERp57 formed a mixed disulfide with tapasin and promoted efficient peptide loading in the absence of interactions with calnexin and calreticulin. These findings suggest that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin chaperones. We also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide binding site in substrate recognition. Finally, by inactivating the redox-active sites of ERp57, we demonstrate that its enzymatic activity is dispensable in stabilizing the peptide loading complex and in supporting efficient peptide loading. Thus, ERp57 appears to play a structural rather than catalytic role within the peptide loading complex. Major histocompatibility complex (MHC)2 class I molecules present antigenic peptides to cytotoxic T lymphocytes (CTL), which leads to the elimination of virus-infected cells. MHC class I molecules are heterotrimers consisting of a transmembrane heavy chain (H chain), a soluble subunit termed ␤ 2 -microglobulin (␤ 2 m), and a peptide ligand of 8 -10 residues. Assembly of class I molecules begins in the endoplasmic reticulum (ER), where the glycosylated H chain binds to the membrane-bound lectin chaperone calnexin (Cnx) and its associated thiol oxidoreductase, ERp57. At this early stage, the two highly conserved disulfide bonds within the H chain are formed, and the H chain assembles with ␤ 2 m. H chain-␤ 2 m heterodimers then enter a peptide loading complex (PLC), where class I molecules acquire peptides for display to CTL. The PLC consists of calreticulin (Crt), the soluble paralog of Cnx, an associated ERp57 molecule, a peptide transporter termed TAP, and tapasin, which is the nucleus of the PLC, bridging the interaction between class I heterodimers and the TAP peptide transporter. Once peptides are translocated into the ER by TAP, a subset bind to receptive H chain-␤ 2 m heterodimers with high affinity, triggering dissociation of class I molecules from the PLC and their subsequent export from the ER to the cell surface (1, 2).Although the functions of most of the participants in class I biogenesis are well understood, the details of how ERp57 functions in...

Essential glycan-dependent interactions optimize MHC class I peptide loading

“…We also produced a Y92A mutant of human CRT based on the analogous mutations of rabbit and mouse CRT that disrupt glycan binding (11,26). Consistent with the crystal structure of the sTpsn/ERp57 conjugate (10) and the characterization of the 3X mutant in ERp57-deficient cells (7,15), inactivation of ERp57 redox activity did not impair the function of the PLC (Fig. 4D).…”

“…Conflicting data regarding the mechanism of CRT and ERp57 activity in the PLC have been reported in studies in intact cells (11,12,15). We sought to address these discrepancies using an in vitro assay for PLC activity.…”

“…The disulfide linkage is between Cys95 of tapasin and Cys57 of the ERp57 a domain active site (9), and the dimer is further stabilized by noncovalent interactions between tapasin and the a′ domain active site (10). CRT interacts with both the HC glycan and the b′ domain of ERp57, but the nature and importance of these interactions within the PLC are controversial, particularly in regard to glycan-independent substrate binding (11)(12)(13)(14)(15). Finally, the potential roles of GlsII and UGT1 in regulating the CRT/class I interaction in the PLC have yet to be addressed.…”

ERp57/GRP58: A protein with multiple functions

“…1). The tapasin-ERp57 complex already highlights the capability of ERp57 to act in different ways, since, contrary to its participation in glycoprotein folding, calreticulin does not constitute an absolute requirement [32], and the redox activity of ERp57 is not involved in the assembly of the peptide loading complex [32,33] except for the formation of a stable disulfide bond between cys-57 of ERp57 and cys-95 of tapasin. Actually a function of this strong complex seems to be the inhibition of the reduction of the disulfide bonds of the heavy chains, which are required for the proper formation of the MHC.…”

MHC Class I Quality Control

“…It has been suggested that the binding to calnexin and calreticulin is required for the recruitment of ERp57 into the PLC (Oliver, van der Wal et al 1997;Oliver, Roderick et al 1999). However, in more recent studies it has been proposed that ERp57 also on its own can recognize and bind some newly synthesized proteins such as MHC-I HC in the absence of calnexin and calreticulin (Zhang, Kozlov et al 2009). An important function of ERp57 is its thiol-dependent reductase activity that catalyse the formation of protein disulide-bonds during the folding of glycoproteins in the ER (Tector and Salter 1995).…”