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. Author manuscript; available in PMC: 2009 Jun 1.
Published in final edited form as: Curr Opin Cell Biol. 2008 Oct 29;20(6):624–631. doi: 10.1016/j.ceb.2008.09.005

The quality control of MHC class I peptide loading

Pamela A Wearsch 1, Peter Cresswell 1
PMCID: PMC2650229  NIHMSID: NIHMS83672  PMID: 18926908

Abstract

The assembly of Major Histocompatibility Complex (MHC) class I molecules is one of the more widely studied examples of protein folding in the endoplasmic reticulum (ER). It is also one of the most unusual cases of glycoprotein quality control involving the thiol oxidoreductase ERp57 and the lectin-like chaperones calnexin and calreticulin. The multi-step assembly of MHC class I heavy chain with β2microglobulin and peptide is facilitated by these ER-resident proteins and further tailored by the involvement of a peptide transporter, aminopeptidases, and the chaperone-like molecule tapasin. Here we summarize recent progress in understanding the roles of these general and class I-specific ER proteins in facilitating the optimal assembly of MHC class I molecules with high affinity peptides for antigen presentation.

Introduction

The MHC class I heavy chain (HC) was one of the first chaperone substrates to be identified in the early 1990’s and since then its assembly with β2m and peptide has been a popular model system for studying endoplasmic reticulum (ER) folding pathways. Loading MHC class I molecules with peptide ligands, however, poses an unusual challenge for the ER quality control machinery. MHC class I molecules display a ‘peptide fingerprint’ of intracellular protein content to CD8+ T cells for immune surveillance of viruses and tumors. This requires diversity of the peptide repertoire as well as stable association so that the class I-peptide complexes survive trafficking and prolonged expression at the cell surface where the encounter with T cells occurs. A specialized pathway for MHC class I assembly has evolved that overcomes these apparently contradictory principles of degeneracy and high affinity.

Peptide loading of MHC class I molecules is the final event in a multi-step assembly pathway that is an adaptation of a quality control cycle that regulates the folding of conventional glycoproteins [1]. This system involves the lectin-like chaperones calnexin (CNX) and calreticulin (CRT), which transiently bind to newly synthesized proteins bearing monoglucosylated N-linked glycans and promote their folding (Figure 1a) [2]. These chaperones also recruit the thiol oxidoreductase ERp57, which isomerizes disulfide bonds to facilitate acquisition of the correct conformation by the glycoprotein. Deglucosylation of the substrate by glucosidase II is coordinated with its release from CNX/CRT, and its folding status is assessed by the enzyme UDP-glucose Glycoprotein Transferase (UGT). This enzyme re-glucosylates the glycan only if the glycoprotein remains incorrectly folded, allowing its re-entry into the quality control cycle.

Figure 1. ER quality control and MHC class I assembly.

Figure 1

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(a) The ER glycoprotein quality control cycle. The composition of the N-linked glycan transferred to newly synthesized proteins is Glc3Man9GlcNAc2. The first two terminal glucose residues are rapidly trimmed by the sequential action of Glucosidases I (Gls1) and II (GlsII) allowing the glycoprotein to enter the CNX/CRT cycle. The lectin-like chaperones specifically bind monoglucosylated glycans and promote protein folding in conjunction with ERp57. Following the release of the substrate from CNX, CRT and ERp57, the glycan is completely deglucosylated by Glucosidase II. If the protein has acquired its native state, it is removed from the cycle and exported to the Golgi. If not, the unfolded protein is recognized and reglucosylated by UGT, allowing it to re-engage with the chaperones. The cycle may continue until the protein has acquired its native structure. (b) MHC class I assembly in the ER. The folding of the MHC class I HC and formation of disulfide bonds in the α2 and α3 domains is assisted by CNX and ERp57. The HC then assembles with β2m to generate an empty heterodimer that is highly unstable for most MHC class I alleles. The HC/β2m dimer is stabilized by association with tapasin, ERp57, CRT, and TAP in the PLC, where it awaits peptide binding. TAP transports peptides generated from cytosolic proteins by the proteasome into the ER lumen where they are further trimmed by ERAAP/ERAP1 (mice and humans) and ERAP2 (humans only). Once high affinity peptides of the appropriate length are generated, they are loaded onto empty MHC class I molecule by the PLC. Peptide binding induces dissociation of MHC class I from the PLC and it subsequently traffics to the cell surface.

The ER quality control machinery facilitates two discrete stages of MHC class I assembly (Figure 1b) [1]. Interactions with CNX and ERp57 are thought to mediate the early folding events of the MHC class I heavy chain. After β2m association the class I heterodimer is rapidly recruited into the peptide loading complex (PLC), which consists of TAP (Transporter associated with Antigen Processing), the MHC class I-specific chaperone tapasin, CRT and ERp57. Peptides are generated in the cytosol by the proteasome, shuttled into the ER by TAP, and trimmed by aminopeptidases to 8–10 AA, a length appropriate for MHC class I association. Binding of high affinity peptides induces the release of MHC class I-peptide complexes that traffic to the cell surface. In this review we focus on the final events responsible for optimal loading of MHC class I molecules.

Tapasin and the PLC: ‘Specialized Quality Control’

The involvement of tapasin dramatically changes the dynamics of the typically weak and transient interactions observed between CRT, ERp57 and their glycoprotein substrates [2] as the association of many MHC class I alleles with the PLC is quite stable and prolonged [3,4]. Studies of tapasin-deficient cells have indicated that the CRT/ERp57 system by itself cannot promote optimal MHC class I assembly [5,6] and tapasin function is compromised in the absence of CRT or ERp57 [7,8••]. How the general and MHC class I-specific components of the PLC cooperate has been an intense focus of recent research.

The assembly, stability, and function of the PLC are regulated by numerous interactions between its components (Figure 2a). These are summarized below:

  1. Tapasin acts as a bridge between TAP and the remaining PLC components. This interaction is important for stabilizing TAP [9] and the entire PLC assembly [10], and involves the tapasin transmembrane domain and the N-terminal transmembrane domains of TAP1 and TAP2 [10–12].

  2. ERp57, a member of the protein disulfide isomerase (PDI) family that promotes proper disulfide bond formation in newly synthesized glycoproteins [13], is covalently associated with tapasin. This involves a disulfide bond between Cys95 of tapasin and Cys57 of ERp57, which is an active site cysteine [14]. ERp57 typically forms transient mixed disulfides with its substrates, but a number of observations indicate that its association with tapasin is an exception to the quality control rulebook. First, tapasin is an abundant, ER-retained, stable substrate that sequesters 15–80% of the total ERp57 pool (depending on the cell type) in the PLC [8,15,16]. Second, ERp57 binds native tapasin and the interaction occurs independently of the lectin-like chaperones, suggesting that although tapasin is a glycoprotein the conjugate is not a folding intermediate [15]. Finally, all tapasin in the PLC is stably disulfide-linked to ERp57 and once the association occurs there is no subunit exchange [15,17••].

  3. The tip of the CRT P-domain binds to residues in the b’ domain of ERp57 with a Kd of ~2.6 µM [18–20].

  4. CRT binds to a monoglucoyslated N-linked glycan at Asn86 of the MHC class I HC with a Kd of ~1 µM [21]. The glycan is clearly sufficient to mediate HC binding [21] but the existence of additional polypeptide-based CRT interactions has been suggested. MHC class I assembly is suboptimal in CRT knockout cells [7], but re-introduction of a lectin-deficient CRT mutant restored MHC class I expression and stability at the cell surface [22•].

  5. The interaction between the MHC class I HC and tapasin remains poorly defined, likely due to a very low intrinsic affinity [23••,24••]. Mutants of MHC molecules that fail to interact with the PLC have been generated and they fail to load optimally with peptides [25].

Figure 2. Components and interactions that define the MHC class I PLC.

Figure 2

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(a) A schematic of the PLC showing its components and defined intermolecular interactions. Only one unit of each protein is illustrated for simplicity not to indicate the stoichiometry. Some studies have suggested that the ratio of TAP1/2:tapasin:HC is 1:4:4 [4,54] whereas another has proposed that it is 1:2:1 [34]. (b) Overview of the MHC class I peptide binding groove and residues that are important for assembly or PLC interactions. The MHC class I HC is composed of three domains, the first two of which, α1, α2, comprise the peptide binding domain and include a disulfide bond. The third is an Ig-like domain and its disulfide bond is expected to be stable. Shown is the structure of HLA-B*4402 without the α3 domain or bound peptide. The peptide binding platform consists of two antiparallel α-helices overlaying an eight strand β-sheet structure. In this view, the peptide N-terminus binds at the bottom and the C-terminus binds at the top of the groove. Residues that are considered to be important for MHC class I assembly and/or PLC association are highlighted. Blue: Asn86 is the site of N-linked glycosylation and CRT binding. Orange: the α2–1 segment is encoded by residues 138–149. Red: the mutations in the MHC class I HC that have been shown to affect PLC association via tapasin are between 128–136. Green: a disulfide bond is present in the native structure of the α2 between Cys101 and Cys164.

Thus, three of the known points of contact between the luminal components of the PLC, i.e. CRT-MHC class I glycan, tapasin-MHC class I HC, and CRT-ERp57, are weak and reversible. The fourth contact, the covalent linkage between tapasin and ERp57, plays a major role PLC assembly, as might be expected. Steady-state PLC association of CRT and MHC class I is reduced by 50–90% in ERp57-deficient mouse B cells [8••] and by 75% in human cells expressing a C95A tapasin mutant that is not disulfide-bonded to ERp57 [17••]. Thus, the covalent tapasin/ERp57 association affects the stability of the PLC, the recruitment of MHC class I and CRT, or both. Avian tapasin lacks the cysteine that forms the disulfide bond with ERp57 [26], suggesting that mammalian tapasin has evolved to sequester ERp57 and co-opt the normal ER quality control pathway to enhance PLC function.

ERp57 and redox reactions in MHC class I assembly and peptide loading

To address the role of ERp57 in the PLC, Garbi et al made a conditional knockout of ERp57 in mouse B cells [8••]. Total and cell surface MHC class I (H2-Kb) were reduced by ~50% compared to wild-type B cells. MHC class I molecules were transported more rapidly from the ER and had a decreased half-life at the cell surface, suggesting poor peptide binding. In addition, antigen presentation was reduced when peptide supply was limited. A less dramatic phenotype was observed when siRNA was used to knockdown mouse ERp57 in cells expressing H2-Kb. Although significant delays of HC oxidation and folding were observed, several criteria for PLC composition and MHC class I presentation were normal [27]. This could be because the knockdown of ERp57, although high (>90%), was incomplete. When identical knockdown levels were achieved using shRNA for human ERp57, virtually all of the residual ERp57 was sequestered by tapasin and was sufficient to support normal PLC function [17••].

Considerable attention has been given to the role of redox reactions in the PLC. This followed naturally from the identification of ERp57 in the PLC and speculation that its function was to catalyze the formation and/or rearrangement of disulfide bonds in the MHC class I molecule. ERp57 is composed of 4 thioredoxin-like domains, ordered abb’a’, in which the first and last are catalytically active [13]. Several studies identified minor populations of mixed disulfides between ERp57 and the MHC class I HC [28,29•,30] and, in two of these cases, a disulfide-linked tapasin/ERp57/HC triple conjugate was seen [28,30]. The significance of these trapped intermediates should be evaluated by two criteria: 1) ERp57-linked HCs should be associated with β2m and the PLC, and 2) the disulfide bond should involve cysteines in the MHC class I α2 domain (Figure 2b) and Cys406 in the ERp57 a’ domain, since the a domain active site is involved in tapasin conjugation [14]. These standards were best applied by Bulleid and co-workers [30], who identified a tapasin/ERp57/HC triple conjugate, but the association involved a disulfide linkage between the class I HC and tapasin, not ERp57, and involved cytoplasmic, not luminal, cysteine residues.

It has also been proposed that the redox activity of ERp57 can play a negative role in MHC class I assembly [29•]. Since the tapasin-dependent HLA-B*4402 allele was partially reduced in tapasin-deficient cells or cells transfected with C95A tapasin, it was suggested that ERp57 may reduce the α2 disulfide bond. Thus, an additional function of tapasin could be to inhibit this counter-productive activity of ERp57. A complication, however, is that HLA-B*4402 is highly unstable in the absence of tapasin [31] and is poorly recruited to tapasin in the absence of ERp57 conjugation [17••]. An alternate explanation is that in the absence of tapasin or the conjugate, HLA-B*4402 is unstable and reduced by ERp57 following its dissociation from β2m. The latter interpretation is consistent with studies supporting a role for ERp57 in processing free HCs for ER-associated degradation [30,32]. Future studies with tapasin mutants that form the conjugate, but do not stabilize MHC class I, should resolve this matter.

Several independent lines of evidence argue that the oxidoreductase activity of ERp57 is not required for PLC function. First, the redox state of MHC class I HC is not altered in ERp57-deficient B cells [8••]. Second, the recruitment, stability, and redox state for PLC-associated HLA-B*4402 were identical when wild-type and C60A/C406A/C409A mutant ERp57, in which the a domain active site is coupled to tapasin and the a’ domain activity is eliminated, were introduced into human cells with stable knockdown of the endogenous protein [17••]. It therefore seems likely that the inactivation of the ERp57 a domain active site by covalent tapasin association and the lack of a role for the a’ domain active site is a consequence, not the purpose, of sequestration by tapasin.

The available data best supports a structural role of ERp57 in the PLC as indicated in Figure 2a. The stable association of ERp57 with tapasin provides a platform for the simultaneous recruitment of HC/β2m dimers and CRT, customizing the ER quality control machinery for MHC class I assembly with peptides. Although the above studies do not favor a role for ERp57 in the isomerization of the MHC class I α2 disulfide bond, one report has argued that PDI associates with the PLC and may perform such a function [33•]. siRNA knockdown of PDI led to differences in the redox state of MHC class I HC. Although PDI is likely important for some aspect of MHC class I assembly, several laboratories have failed to detect PDI in the PLC [1,29•,34] and this observation remains controversial.

Mechanism of MHC class I peptide loading

The primary role of tapasin is to stabilize empty MHC class I molecules and promote the binding of high affinity peptides, a process known as ‘peptide editing’ [31,35,36]. Experimental approaches involving intact cells, while supporting this idea, are limited in helping us understand the mechanism of PLC activity. Surface expression and thermostability of MHC class I molecules is commonly used as a surrogate for measuring the affinity of bound peptides, but can be complicated by post-ER events such as dissociation of unstable complexes [37] and loading by alternative pathways [10]. Although cell-free assays are better suited to dissect the mechanism of tapasin/PLC activity, none were developed until last year.

The major obstacle was efficiently reconstituting the MHC class I-tapasin association. Attempts to study the interaction of the two proteins in isolation were unsuccessful ([23••] and PW, unpublished data), suggesting that the intrinsic affinity of tapasin for peptide-free class I molecules is weak (Figure 2a). Two strategies have been used to bypass this problem. One used soluble versions of tapasin and MHC class I HC modified at their C-termini with leucine zippers to force the interaction of tapasin and MHC class I HC/β2m dimers [23••]. The association and dissociation rates of peptides were both found to be enhanced by tapasin. Since a peptide lacking a COOH group was less susceptible to tapasin-mediated dissociation than one without an NH2 group, the authors proposed that tapasin works by disrupting hydrogen bonds between the peptide C-terminus and the binding groove. In the second strategy, the luminal sub-complex of the PLC was reconstituted using recombinant tapasin/ERp57 conjugate and cell extracts containing CRT and empty HC/β2m complexes [24••]. The disulfide-linked tapasin/ERp57 heterodimer, but not tapasin alone, supported the assembly and peptide loading function of the luminal sub-complex. Catalysis of peptide loading was demonstrated for several peptides and MHC class I alleles. Furthermore, in competition assays using mixtures of peptides with variable binding affinities, the tapasin/ERp57 conjugate could maximize the affinity of the bound peptides, i.e. could mediate peptide editing. These studies confirmed the proposed peptide loading activities for tapasin, yet underscored the functional importance of its interactions with ERp57 and CRT.

A fundamental and poorly understood question is how MHC class I molecules in the PLC are stabilized in a peptide-receptive state and structurally modified so as to preferentially bind high affinity peptides. Since attempts to crystallize empty HC/β2m heterodimers have been unsuccessful, the best insights have come from molecular dynamics simulations of the peptide binding domain platform in the presence and absence of bound peptide. Conformational fluctuations were primarily observed at the end of the groove where the peptide C-terminus binds, particularly in the α2–1 segment [38] which is proximal to defined HC mutations affecting PLC association [25] (Figure 2b). Interestingly, this region is also highly disordered in the crystal structure of the peptide-free MHC class I-like molecule M10.5 [39]. Furthermore, comparative simulations for two HLA-B alleles differing by a single amino acid residue have suggested why some MHC class I alleles are more dependent on tapasin-mediated assembly than others [40•,41]. The predicted flexibility of the α2–1 region was greater for tapasin-dependent HLA-B*4402 than tapasin-independent HLA-B*4405, which caused disruptions in the peptide contact interface. Thus, the authors proposed that certain alleles are to an extent tapasin-independent because their structure favors a relatively stable, peptide-receptive binding groove, whereas others require tapasin and the PLC to stabilize the α2 domain helix in a conformation favorable for peptide loading.

Peptide processing in the ER

The assembly of MHC class I molecules is further facilitated by ER aminopeptidases that favor the generation of suitable peptides. Many of the peptides transported by TAP are longer than the 8–10 residues suitable for MHC class I binding. Although the appropriate C-termini of the transported peptides are generated in the cytosol, the N-termini can be extended. In 2002 a soluble interferon-γ–inducible ER aminopeptidase, named ERAAP in mice and ERAP1 in humans, was isolated [42–44]. Recent studies using knockout mice have revealed a role for ERAAP/ERAP1 in the final trimming of peptides to achieve optimal binding to MHC class I molecules. The absence of ERAAP caused reduced MHC class I surface expression yet variable effects on antigen presentation suggesting that it can both create and destroy MHC ligands [45–48]. The importance of ERAAP function is further highlighted by the presence of many unstable and likely N-terminally extended MHC class I-bound peptides in ERAAP-knockout mice [49••].

The mechanism of ERAAP/ERAP1 activity, in particular its length-based cleavage specificity, is currently a matter of debate. First, MHC class I molecules could serve as a template for trimming in the ER, thus limiting the length of peptides to 8–9 AA. Support for this hypothesis derived from analysis of peptide processing in mouse cells with or without expression of ERAAP/ERAP1 and the appropriate MHC class I allele [50•]. No evidence for a physical association between the PLC and ERAAP/ERAP1 has been reported, however. Evidence for an alternative hypothesis originated from in vitro studies with purified enzyme [51]. Using large peptide panels, the authors found that peptides shorter than 8–9 AA in length were poor substrates of ERAP1. This gave rise to the ‘Molecular Ruler’ hypothesis, which suggested that the enzyme itself was responsible for limiting peptide degradation. Similar observations have not been made, however, in studies of ERAAP/ERAP1 processing in cells. The discrepancy may lie in the experimental shortcomings of the two approaches. Analysis of peptide trimming within the ER and in the presence of MHC class I molecules is the most physiologically relevant system, but a larger set of peptides has been analyzed in vitro which perhaps might better allow broad conclusions. Establishing the crystal structure of ERAAP/ERAP1 and defining its active site should settle the argument.

A second, related ER aminopeptidase called L-RAP was identified in human cells [52] and renamed ERAP2 in light of its ability to trim peptides for MHC class I binding [53]. A mouse counterpart has not been identified. Knockdown of ERAP1 or ERAP2 in human cells had minor effects on MHC class I surface expression and variable effects on antigen presentation in a few cases that were more pronounced if both aminopeptidases were depleted [53]. The two enzymes appear to have complementary functions; ERAP1 cleaves substrates with hydrophobic N-terminal residues, whereas ERAP2 prefers those with basic residues [51,53]. ERAP1 may be the dominant proteolytic activity contributing to peptide processing in the ER, but further studies of ERAP2 are warranted.

Concluding remarks

The assembly of MHC class I molecules involves general and specific mechanisms and thus represents a unique case of ER quality control. The early stages of MHC class I HC folding proceed in a conventional manner, but the involvement of tapasin, TAP, and ER aminopeptidases with CRT and ERp57 customizes the quality control machinery to ensure that MHC class I molecules preferentially bind high affinity peptides. Although considerable progress has been made in understanding MHC class I assembly, many unanswered questions remain. In particular, precisely how loading of MHC class I molecules and dissociation from the PLC is initiated is poorly understood. One aspect of the quality control cycle that has been tested only recently is the requirement for UGT in the folding of glycoprotein substrates [55]. It remains an interesting possibility that discrimination between empty and peptide-occupied MHC class I molecules by this enzyme may play a role in peptide loading.

Acknowledgments

We apologize to our colleagues if their papers are not mentioned; citations and discussions were limited due to space constraints. The authors would like to thank Drs. David Peaper and Ralf Leonhardt for helpful discussions. PAW and PC are supported by the Howard Hughes Medical Institute.

Footnotes

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References and recommended reading

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