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 li... More
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 high-affinity 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.
