Essential glycan-dependent interactions optimize MHC class I peptide loading

Abstract

In this study we sought to better understand the role of the glycoprotein quality control machinery in the assembly of MHC class I molecules with high-affinity peptides. The lectin-like chaperone calreticulin (CRT) and the thiol oxidoreductase ERp57 participate in the final step of this process as part of the peptide-loading complex (PLC). We provide evidence for an MHC class I/CRT intermediate before PLC engagement and examine the nature of that chaperone interaction in detail. To investigate the mechanism of peptide loading and roles of individual components, we reconstituted a PLC subcomplex, excluding the Transporter Associated with Antigen Processing, from purified, recombinant proteins. ERp57 disulfide linked to the class I-specific chaperone tapasin and CRT were the minimal PLC components required for MHC class I association and peptide loading. Mutations disrupting the interaction of CRT with ERp57 or the class I glycan completely eliminated PLC activity in vitro. By using the purified system, we also provide direct evidence for a role for UDP-glucose:glycoprotein glucosyltransferase 1 in MHC class I assembly. The recombinant Drosophila enzyme reglucosylated MHC class I molecules associated with suboptimal ligands and allowed PLC reengagement and highaffinity peptide exchange. Collectively, the data indicate that CRT in the PLC enhances weak tapasin/class I interactions in a manner that is glycan-dependent and regulated by UDP-glucose:glycoprotein glucosyltransferase 1.protein folding | peptide editing T he assembly of MHC class I molecules is a critical step in the generation of immune responses against viruses and tumors, and also a highly specialized example of glycoprotein folding in the endoplasmic reticulum (ER). MHC class I molecules display peptides representative of the cellular protein content to CD8 + T cells, and the stable association of the class I heavy chain (HC), β 2 -microglobulin (β 2 m), and a high-affinity 8-to 10-aa foreign peptide is essential for T-cell activation. As a result, a specialized adaptation of the glycoprotein folding machinery has evolved to ensure the loading of MHC class I molecules with optimal peptide ligands. Following HC assembly with β 2 m, the empty heterodimer rapidly and stably associates with the peptide-loading complex (PLC), which facilitates the final peptide-binding step (1). The functions of the MHC class I-specific components of the PLC are well defined. Tapasin interacts with both the HC/β 2 m dimer as well as Transporter Associated with Antigen Processing (TAP), thereby retaining the empty complexes in proximity to the peptide supply. More importantly, tapasin association stabilizes class I molecules and promotes loading with high-affinity peptides. 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).The ER glycoprotein quality control machinery is a complex system that uses the structural state of N-linked glycans to dictate the fa...

Contributions of the Lectin and Polypeptide Binding Sites of Calreticulin to Its Chaperone Functions in Vitro and in Cells

“…The expression of class I molecules at the cell surface was also impaired (20). Similarly, the Y109A mutant was unable to support peptide loading onto class I molecules when the PLC was assembled from purified components in vitro (21). Lectin-deficient Crt has also been reported to be strongly impaired in its ability to bind to newly synthesized monoglucosylated glycoproteins in chemically cross-linked cell lysates (22).…”

“…The polypeptide binding site does not appear to participate in these processes. Given the important contribution of the polypeptide binding site of Crt in suppressing protein aggregation in vitro, we speculated that its apparent lack of involvement in class I biogenesis might be due to Crt functioning more to stabilize the PLC rather than suppressing the aggregation of newly synthesized class I molecules (20,21). Consequently, we turned to another assay where Crt has been shown to play a clearer role in suppressing client protein aggregation.…”

“…1, 11, and 13-19 and references therein). Further detailed studies using class I histocompatibility molecules have shown that interactions with Crt are dependent on a functional lectin site and the presence of monoglucosylated oligosaccharide on the substrate (20,21). The ability of these chaperones to suppress client protein aggregation appears to be due in part to their sequestration of glycoprotein folding intermediates between the globular lectin domain and the flexible extended arm domain.…”

MHC Class I Quality Control

“…This indicates that the glucose residues of the monoglycosylated MHC-I molecule become accessible to the glucosidase II first after its release from the PLC. After the last glucose residues has been removed, the MHC-I molecule can no longer associate with the PLC as its recruitment is dependent on the interaction with calreticulin which only binds to monoglycosylated proteins (Wearsch, Peaper et al 2011). However, it has been proposed that if a deglycosylated MHC-I molecule is loaded with suboptimal peptide it can be rescued back into the PLC via the soluble enzyme UDP-glucose glycoprotein glucosyltransferase 1 (UGT1) (Zhang, Wearsch et al 2011).…”

MHC Class I Quality Control

“…The mono-glucosylated HC will then bind to calnexin for oxidative folding (Nossner and Parham 1995). Sequentially, the immature HC will create a heterodimer with the stabilising polypeptide 2 m. As this occurs, calnexin is replaced by calreticulin, which then recruits the immature heterodimer into the PLC where it can be loaded with high affinity peptides (Wearsch, Peaper et al 2011).…”