Class I Molecules and Heavy Chains in the Assembly of MHC Distinct Functions for the Glycans of Tapasin

Complexes of speciﬁc assembly factors and generic endoplasmic reticulum (ER) chaperones, collectively called the MHC class I peptide-loading complex (PLC), function in the folding and assembly of MHC class I molecules. The glycan-binding chaperone calreticulin (CRT) and partner oxidoreductase ERp57 are important in MHC class I assembly, but the sequence of assembly events and speciﬁc interactions involved remain incompletely understood. We show that the recruitments of CRT and ERp57 to the PLC are codependent and also dependent upon the ERp57 binding site and the glycan of the assembly factor tapasin. Furthermore, the ERp57 binding site and the glycan of tapasin enhance b 2 m and MHC class I heavy (H) chain recruitment to the PLC, with the ERp57 binding site having the dominant effect. In contrast, the conserved MHC class I H chain glycan played a minor role in CRT recruitment into the PLC, but impacted the recruitment of H chains into the PLC, and glycan-deﬁcient H chains were impaired for tapasin-independent and tapasin-assisted assembly. The conserved MHC class I glycan and tapasin facilitated an early step in the assembly of H chain– b 2 m heterodimers, for which tapasin–ERp57 or tapasin–CRT complexes were not required. Together, these studies provide insights into how PLCs are constructed, demonstrate two distinct mechanisms by which PLCs can be stabilized, and suggest the presence of intermediate H chain-deﬁcient PLCs. The Journal of Immunology , 2011, 186: 2309–2320. wild-type -CTGCGCGGCTACTAC- CAGCAGAGCGAGGCCGGT-3 9 -ACCGGCCTCGCTCTGCTGG-TAGTAGCCGCGCAG-3 cloned pMSCV-neo ligation-independent cloning plasmid pMSCV-neo LIC enzyme Pme LIC overhangs linearized T4 polymerase exonuclease activity. inserted sequence con- structed annealing two oligos generate a fragment with overhangs complementary to an EcoRI restriction site. The oligo sequences used were 5 9 -AATTAGAGAGTTTAAACTTCCAC-3 9 and 5 9 -AATTGTGGAAGTT-TAAACTCTCT-3 . The lyophilized oligos resuspended in distilled water at a concentration of 20 m M. Prior to annealing, phos-phorylated using T4 polynucleotide kinase (New England Biolabs). The 20- m l 2 m l 10 mM ATP, 12.5 m l 20 mM oligo, 2 m l 3 reaction buffer, 1 m l enzyme, and 2.5 m l distilled incubated for 1 h at 37˚C, and then shifted to 95˚C for an additional 10 min. two oligo reactions were then mixed together at 95˚C and to 37˚C. A 2.5- m l aliquot was then added to a 20- m l ligation reaction composed of 1 m l 45 ng/ m l EcoRI-cut pMSCV-neo, calf intestinal phosphatase, 2 m l 10 3 reaction buffer, and 13.5 l distilled from 37˚C to 16˚C, forms, and intermediate H chain-depleted PLCs are detectable. A , Tapasin’s glycan contributes to the stabilization of CRT, H chain, and b 2 m recruitment to tapasin (Tpn). Such a complex or its subcomplex may facilitate ERp57 recruitment by transient interaction of ERp57 with the tip of CRT’s P domain, thus bringing ERp57 into proximity to tapasin(C95). B , ERp57 becomes conjugated to tapasin(C95). This is shown to displace CRT from tapasin’s glycan, and link CRT to the PLC via P domain-mediated interactions with tapasin(C95)-associated ERp57. In this study, recruitments of CRT and ERp57 to the PLC are shown to be codependent and also de- pendent upon tapasin(C95) and tapasin’s glycan. In IFN- g –treated M553 cells, where there is strong upregulation of MHC class I expression, CRT recruitment to tapasin was largely tapasin(C95) dependent. Tapasin’s glycan and tapasin(C95) both enhanced MHC class I recruitment to the PLC, with dominant stability conferred by tapasin(C95). Thus, the equilibrium is shifted toward B in IFN- g –treated M553 cells, under conditions in which ERp57 is not limiting. In M553 cells not treated with IFN- g , conditions under which MHC class I H chain and b 2 m expression levels are low, CRT recruitment to tapasin was largely tapasin(N233) dependent. Thus, the equilibrium is shifted toward A . MHC class I recruitment was nevertheless largely C95 dependent, suggesting that the A → B transition is favored by the presence of both MHC class I heterodimers and ERp57. Thus, cellular conditions are expected to determine the steady-state prevalence of each complex and their subcomplexes. C and D , In proposed models of the MHC class I assembly pathways based on the data from Figs. 5 and 6, intermediate H chain-depleted complexes are predicted to form. Suboptimally assembled MHC