Essential glycan-dependent interactions optimize MHC class I peptide loading Pamela A. Wearscha, David R. Peapera, and Peter Cresswella,b,1 aDepartment of Immunobiology and bDepartment of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520-8011 Contributed by Peter Cresswell, February 15, 2011 (sent for review January 5, 2011) In this study we sought to better understand the role of the (CNX). Both chaperones have a globular lectin-binding domain glycoprotein quality control machinery in the assembly of MHC and an extended arm known as the P-domain that binds specifi- class I molecules with high-affinity peptides. The lectin-like chap- cally to ERp57, a member of the protein disulfide isomerase (PDI) erone calreticulin (CRT) and the thiol oxidoreductase ERp57 partic- family. ERp57 has a four-domain architecture of abb′a′ in which fi ipate in the nal step of this process as part of the peptide-loading the first and last contain a CXXC active site. CRT and CNX work complex (PLC). We provide evidence for an MHC class I/CRT in- in concert with ERp57 to promote proper folding and disulfide termediate before PLC engagement and examine the nature of that bond formation of newly synthesized glycoproteins. Upon release chaperone interaction in detail. To investigate the mechanism of of the glycoprotein from CRT or CNX, its glycan is deglucosylated peptide loading and roles of individual components, we reconsti- tuted a PLC subcomplex, excluding the Transporter Associated with by GlsII and is no longer a substrate for the chaperones. If the Antigen Processing, from purified, recombinant proteins. ERp57 glycoprotein has not yet acquired its native structure, the enzyme disulfide linked to the class I-specific chaperone tapasin and CRT UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1), a fold- were the minimal PLC components required for MHC class I as- ing sensor, reglucosylates it to reinitiate an interaction with CRT/ sociation and peptide loading. Mutations disrupting the interaction CNX/ERp57. However, a properly folded glycoprotein is not a of CRT with ERp57 or the class I glycan completely eliminated PLC substrate for UGT1 and can be exported to the Golgi. activity in vitro. By using the purified system, we also provide Important roles for CRT and ERp57 in MHC class I assembly direct evidence for a role for UDP-glucose:glycoprotein glucosyl- and PLC function have been established from studies of KO transferase 1 in MHC class I assembly. The recombinant Drosoph- mice and deficient cell lines (2, 3). In the absence of either ila enzyme reglucosylated MHC class I molecules associated with component, the cell surface expression and stability of MHC suboptimal ligands and allowed PLC reengagement and high- class I molecules are reduced. The cause of the defect has been fi af nity peptide exchange. Collectively, the data indicate that elucidated for ERp57-deficient cells. Tapasin forms a stable CRT in the PLC enhances weak tapasin/class I interactions in a man- disulfide-linked heterodimer with ERp57, which is required for ner that is glycan-dependent and regulated by UDP-glucose:glyco- the structural stability and optimal function of the PLC (4, 7, 8). protein glucosyltransferase 1. The disulfide linkage is between Cys95 of tapasin and Cys57 a protein folding | peptide editing of the ERp57 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 he assembly of MHC class I molecules is a critical step in the and the b′ domain of ERp57, but the nature and importance of Tgeneration of immune responses against viruses and tumors, and also a highly specialized example of glycoprotein folding in these interactions within the PLC are controversial, particularly – the endoplasmic reticulum (ER). MHC class I molecules display in regard to glycan-independent substrate binding (11 15). Fi- peptides representative of the cellular protein content to CD8+ nally, the potential roles of GlsII and UGT1 in regulating the T cells, and the stable association of the class I heavy chain (HC), CRT/class I interaction in the PLC have yet to be addressed. By using a variety of biochemical approaches, including re- β2-microglobulin (β2m), and a high-affinity 8- to 10-aa foreign peptide is essential for T-cell activation. As a result, a specialized constitution of a PLC subcomplex entirely from purified com- adaptation of the glycoprotein folding machinery has evolved to ponents, we have investigated the role of ER quality control ensure the loading of MHC class I molecules with optimal peptide components in MHC class I peptide loading. We demonstrate ligands. Following HC assembly with β2m, the empty heterodimer the CRT is recruited to and released from the PLC along with rapidly and stably associates with the peptide-loading complex class I molecules. By examination of the CRT/HC stoichiometry (PLC), which facilitates the final peptide-binding step (1). The and the HC glycosylation state, we found no evidence for glycan- functions of the MHC class I-specific components of the PLC are independent interactions within the PLC. Consistent with this, in fi β well de ned. Tapasin interacts with both the HC/ 2m dimer as vitro reconstitution of the PLC required the lectin- and ERp57- well as Transporter Associated with Antigen Processing (TAP), binding activities of CRT and could be enhanced by UGT1- thereby retaining the empty complexes in proximity to the peptide mediated glucosylation. Taken together, the data support impor- supply. More importantly, tapasin association stabilizes class I tant roles for CRT and the quality control machinery in regulating fi molecules and promotes loading with high-af nity peptides. class I peptide loading by the PLC. However, the optimal activity of tapasin requires the presence of calreticulin (CRT) and ERp57, two ER proteins involved in – general glycoprotein folding, in the PLC (2 4). Author contributions: P.A.W., D.R.P., and P.C. designed research; P.A.W. and D.R.P. per- The ER glycoprotein quality control machinery is a complex formed research; P.A.W., D.R.P., and P.C. analyzed data; and P.A.W. and P.C. wrote the system that uses the structural state of N-linked glycans to dic- paper. tate the fate of newly synthesized proteins (1, 5, 6). Initially, a The authors declare no conflict of interest. Glc3Man9GlcNAc2 glycan is transferred to polypeptides during Freely available online through the PNAS open access option. translocation and subsequently trimmed by glucosidase I (GlsI) 1To whom correspondence should be addressed. E-mail: [email protected]. and glucosidase II (GlsII) to a monoglucosylated glycan, which is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the substrate for the lectin-like chaperones CRT and calnexin 1073/pnas.1102524108/-/DCSupplemental. 4950–4955 | PNAS | March 22, 2011 | vol. 108 | no. 12 www.pnas.org/cgi/doi/10.1073/pnas.1102524108 Downloaded by guest on September 25, 2021 Results and Discussion to the conjugate with this assay. However, a significant portion of CRT Associates with MHC Class I HC/β2m Heterodimers Before these complexes likely dissociate during the extract preparation Incorporation into the PLC. Work from our laboratory has dem- given the relatively low affinity of CRT for glycosylated class I K ∼ μ onstrated that the tapasin/ERp57 conjugate associates with TAP HC ( d of 1 M) (14) and the rapid rate of dissociation de- ∼ in cells lacking expression of MHC class I HC or β2m (8, 16), termined from kinetic experiments (t1/2 of 10 min at 4 °C; Fig. suggesting that this core serves as a scaffold onto which the S1). Consistent with our projections, the addition of excess remaining PLC components assemble. Furthermore, the GlsII recombinant CRT to the lysis buffer enhanced HC association inhibitor castanospermine (CST) prevents the interaction of MHC with the conjugate (Fig. 1C), presumably because of preservation β class I molecules with the PLC in intact cells (17) and a cell-free of CRT/HC/ 2m complexes in the assembly pathway. In agree- ment, Del Cid et al. (11) have proposed a recruiting role for assay (4). This suggests that CRT escorts HC/β2m dimers to tapasin, but this has not been demonstrated experimentally. Ini- CRT in the PLC based on reduced class I association with the fi tially, we sought to identify PLC-independent complexes of HLA- PLC in CRT-de cient cells. However, that interpretation is B8 with CRT by using tapasin-negative .220.B8 cells. We prepared complicated by the analyses being performed at steady state, i.e., extracts from radiolabeled cells in the absence or presence of the effect of CRT on the class I /tapasin interaction may be a DSP, a chemical cross-linker, and performed sequential immu- result of PLC stabilization. Although the two effects cannot be noprecipitations to detect CRT-associated proteins. As shown in discriminated biochemically, CRT may serve an important role β Fig. 1A, a CRT/HC interaction was observed in untreated extracts for both. The discovery that an HC/ 2m/CRT assembly in- and stabilized by DSP. β m, which is not glycosylated, also coim- termediate exists provides the most direct evidence for CRT- 2 A B munoprecipitated with CRT from cross-linked cell extracts, mediated recruitment (Fig. 1 and ), whereas the coupling of known interactions between PLC components is sufficient to demonstrating the presence of CRT-associated HC/β2m hetero- dimers (Fig. 1A). To determine whether this complex exists in the support CRT-mediated stabilization of existing class I/ tapasin presence of the PLC, we performed tapasin immunodepletions interactions (1). from radiolabeled .220.B8.Tpsn cells.