The redox activity of ERp57 is not essential for its functions in MHC class I peptide loading

Abstract

ERp57 is an oxidoreductase that, in conjunction with calnexin and calreticulin, assists disulfide bond formation in folding glycoproteins. ERp57 also forms a mixed disulfide with the MHC class I-specific chaperone tapasin, and this dimeric conjugate edits the peptide repertoire bound by MHC class I molecules. In cells unable to form the conjugate, because of tapasin mutation in human studies or ERp57 deletion in mouse studies, peptide loading is impeded. Subtle differences between the mouse and human systems have been observed. Here, we address these differences and expand the analysis to investigate the role of ERp57 redox functions in MHC class I peptide loading. We show in human cells that in the absence of conjugate formation MHC class I recruitment and/or stabilization in the MHC class I peptide-loading complex is impaired, similar to observations in mouse cells. However, we found no role for the enzymatic activities of either the a or a domain redox sites of ERp57 in peptide loading. Our data argue that the function of ERp57 in peptide loading is likely caused by other ERp57 functional domains or a combinatorial feature of the tapasin-ERp57 conjugate.antigen presentation ͉ antigen processing ͉ human ͉ protein folding ͉ quality control S table loading of peptides onto MHC class I/␤ 2 -microglobulin (␤ 2 m) dimers requires coordinated action within the peptide-loading complex (PLC), which consists of TAP1, TAP2, tapasin, ERp57, calreticulin (CRT), MHC class I heavy chain (HC), and ␤ 2 m (1). Tapasin is a critical component of the PLC, but the in vivo role of ERp57 in peptide loading and editing is not entirely resolved. ERp57 is an oxidoreductase that promotes proper disulfide bond formation in folding glycoproteins through its association(s) with calnexin (CNX) and/or CRT (2). Like protein disulfide isomerase, ERp57 is composed of four domains with the a and aЈ domains containing redox active CXXC motifs. During the biosynthetic folding of MHC class I HC, it appears to act in a manner consistent with models of glycoprotein quality control (3). However, within the PLC, Cys-57 of ERp57 forms a disulfide bond with tapasin Cys-95, and tapasin inactivates the substrate dissociation step, or ''escape pathway,'' of ERp57, making this interaction very stable (4).We examined MHC class I assembly in human B lymphoblastoid cells expressing HLA-B*4402 and a tapasin construct in which Cys-95 was mutated to Ala (C95A) to prevent conjugate formation (5). PLC formation was qualitatively normal except for the absence of ERp57, but the stability of peptide-MHC class I complexes assembled in these cells was decreased, consistent with association with a pool of lower-affinity peptides. In mouse B cells lacking ERp57, surface expression of H2-K b molecules was reduced by Ϸ50%, and turnover was faster than in ERp57-expressing cells. H2-K b recruitment into the PLC was affected, and its trafficking through the Golgi was accelerated. Furthermore, presentation of an H2-K b -restricted epitope derived from ovalbumin was reduced in E...

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.…”

Stability of peptide–HLA‐I complexes and tapasin folding facilitation – tools to define immunogenic peptides

“…Ataxin 324-333 (QVFPGLLERV) was modified with substitutions of F to Y, and R to K, resulting in the QVY peptide, QVYPGLLEKV. Ribosomal protein L10a [8][9][10][11][12][13][14][15][16] (TLYEAVREV) was modified with substitution of E to K, resulting in the TLY peptide, TLYEAVRKV. Cadherin 17 precursor 2-10 (ILQAHLHSL) was modified with substitutions of Q to Y, and of S to K, resulting in the ILY peptide, ILYAHLHKL.…”

“…Since its discovery, tapasin has been attributed several functions -to increase the available local peptide concentration by bridging HLA-I molecules to TAP [2][3][4], to stabilize TAP expression and improve its capacity to bind peptide prior to the translocation step [5][6][7][8], to keep HLA-I molecules peptide-receptive in the PLC [9][10][11], to recycle empty or suboptimally loaded HLA-I molecules back to the ER [4,[12][13][14], and, to act as a peptide-editor, allowing exchange of suboptimal peptides for optimal peptides [1,15,16]. However, the exact molecular mechanisms of the tapasin-mediated HLA-I quality control remain unknown.…”

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

“…While this manuscript was in preparation, Cresswell and coworkers (49) reported that when the C60A/C406A/C409A mutant of human ERp57 was expressed in cells stably knocked down for endogenous ERp57, it fully supported PLC assembly and the generation of stable, peptide-loaded human HLA*B4402 molecules. Although consistent with our findings in the mouse system, the interpretation of their results is complicated by the presence of residual conjugate between tapasin and endogenous wild type ERp57 that remained following the ERp57 knockdown.…”