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 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.

Fig. 1.

HC/β₂m heterodimers associate with CRT independently of the PLC. (A) .220.B8 cells were labeled for 30 min and lysed with or without 0.3 mM DSP. Immunoprecipitations were performed with tapasin (R.gp48C), CRT, or β₂m Abs, followed by reimmunoprecipitation with 3B10.7 (HC) or β₂m Abs. (B) Radiolabeled .220.B8.Tpsn cells (60-min pulse) were lysed and subjected to four immunodepletion steps with PaSta1-coupled beads, followed by immunoprecipitation with tapasin (R.gp48C), CRT, or ERp57 Abs. The associated HCs were reimmunoprecipitated with 3B10.7 and analyzed by SDS/PAGE. (C) Cell extracts were prepared from .220.B8 cells in lysis buffer containing the indicated concentrations of recombinant CRT. Samples were then incubated with or without 0.35 μM C60A conjugate for 15 min at RT followed by immunoprecipitation with PaSta1-coupled beads. The bound proteins were eluted and analyzed by immunoblotting with CRT and 3B10.7 Abs.

Fig. 2.

Analysis of the CRT/HC interaction in the PLC. (A) TAP immunoprecipitations were performed from the indicated number of .220.B8.Tpsn cells and analyzed by SDS/PAGE along with recombinant class I HC and CRT standards. Quantitative immunoblotting was performed with 3B10.7 or CRT Abs, and the standard curves are shown in Fig. S2. Two sample sets were processed and averaged per experiment, but only one is shown. (B) Extracts from radiolabeled .220.B8.Tpsn cells (60-min pulse) were subjected to four CRT immunodepletion steps. Immunoprecipitations were then performed with control, PaSta1, or CRT Abs and analyzed by SDS/PAGE. (C) Schematic of the JBM assay. (D) .220.B8.Tpsn cells were pulse-labeled for 30 min and chased for as long as 90 min. Primary immunoprecipitations were performed with 148.3 Ab-coupled beads and secondary immunoprecipitations were performed with 3B10.7 or R.gp48C. Digests were then performed with JBM or EndoH and samples were analyzed by SDS/PAGE. Note that, after JBM digestion, the migration of glucosylated species is more similar to that of the undigested band (red asterisk), whereas the migration of deglucosylated species is more similar to that of the EndoH-treated band (blue asterisk). (E) .220.B8.tpsn cells were labeled for 60 min and sequential immunoprecipitation/pull-down assays were performed with 3B10.7 Ab, GST, or CRT-GST immobilized on beads followed by 3B.10.7 (Upper). The total amount of HC recovered was calculated from two sequential 3B10.7 immunoprecipitations or from three CRT-GST pull-down steps (Lower).

Fig. 3.

In vitro reconstitution of a soluble subcomplex of the PLC. (A) Schematic of the soluble PLC subcomplex. (B) Recombinant proteins (0.5 μM each) were incubated at RT for 1 h and immunoprecipitations were performed with PaSta1-coupled beads. The tapasin-associated CRT and HC were detected by immunoblotting. (C and D) The indicated recombinant proteins—CRT or Y92A, a CRT glycan-binding mutant; disulfide-linked conjugates of sTpn with C60A, an ERp57 mutant that traps tapasin, with 3X, a redox-inactive ERp57 mutant that traps tapasin, or with ΔPDB, an ERp57 mutant that traps tapasin but does not interact with CRT—were incubated at a final concentration of 0.4 μM each for 15 min at RT with [¹²⁵I]-NP. Peptide loading was measured by w6/32 immunoprecipitation and γ-counting.

Fig. 4.

Reglucosylation of MHC class I molecules by UGT promotes reengagement with the PLC. (A) Control or β2m-specific siRNA oligos were introduced into .220.B8.Tpsn cells and samples were harvested at 0, 12, and 24 h after nucleofection. PLC components were detected by immunoprecipitation with 148.3 Ab and quantitative immunoblotting (Left). Amounts of PLC-associated CRT, tapasin, and class I HC were calculated and normalized to TAP (Right). (B) .220.B8.Tpsn cells were labeled with [³⁵S]-methionine for 30 min and chased for 0 to 120 min with or without 2.5 mM CST. Immunoprecipitations were performed with control or 148.3 Abs followed by elution and reimmunoprecipitation with 3B10.7 mAb (Left). Right: PhosphorImager quantification. (C) .220.B8.Tpsn cells were labeled for 60 min and then subjected to immunoprecipitations and enzymatic digestions as shown in the flowchart (Upper). (D) Purified recombinant MHC class I complexes depleted of those with monoglucosylated glycans were incubated with or without UGT and UDP-glucose. Subsequently, [¹²⁵I]-NP loading was measured in the absence or presence of the remaining PLC components (0.4 μM C60A conjugate and CRT).