Redox-regulated Export of the Major Histocompatibility Complex Class I-Peptide Complexes from the Endoplasmic Reticulum

In contrast to the fairly well-characterized mechanism of assembly of MHC class I-peptide complexes, the disassembly mechanism by which peptide-loaded MHC class I molecules are released from the peptide-loading complex and exit the endoplasmic reticulum (ER) is poorly understood. Optimal peptide binding by MHC class I molecules is assumed to be sufficient for triggering exit of peptide-filled MHC class I molecules from the ER. We now show that protein disulfide isomerase (PDI) controls MHC class I disassembly by regulating dissociation of the tapasin-ERp57 disulfide conjugate. PDI acts as a peptide-dependent molecular switch; in the peptide-bound state, it binds to tapasin and ERp57 and induces dissociation of the tapasin-ERp57 conjugate. In the peptide-free state, PDI is incompetent to bind to tapasin or ERp57 and fails to dissociate the tapasin-ERp57 conjugates, resulting in ER retention of MHC class I molecules. Thus, our results indicate that even after optimal peptide loading, MHC class I disassembly does not occur by default but, rather, is a regulated process involving PDI-mediated interactions within the peptide-loading complex.     

Distinct functions for the glycans of tapasin and heavy chains in the assembly of MHC class I molecules

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 β(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-β(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.     

Localization of phospholipase Cbeta isozymes in the mouse cerebellum

Recent studies demonstrate that processing of N-linked glycans plays an important role in the quality control of major histocompatibility complex (MHC) class I transport from the endoplasmic reticulum (ER) to the Golgi complex and beyond. Here, we investigated the importance of oligosaccharide chain length on the association of MHC class I proteins with molecular chaperones and their intracellular transport from the ER to the Golgi. These data show that calnexin interaction with class I proteins having truncated N-glycans was reduced compared to normal class I molecules, whereas the assembly of class I with calreticulin and TAP was unperturbed by N-glycan chain length. Additionally, these results demonstrate that class I proteins containing truncated N-glycans showed decreased detachment from calreticulin and TAP relative to class I proteins bearing typical oligosaccharides. Taken together, these studies show that N-glycan chain length is an important determinant for the quality control of newly synthesized MHC class I proteins in the ER.     

ER-60, a chaperone with thiol-dependent reductase activity involved in MHC class I assembly.

The assembly of newly synthesized MHC class I molecules within the endoplasmic reticulum and their association with the transporter associated with antigen processing (TAP) is a process involving the chaperones calnexin and calreticulin. Using peptide mapping by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify a new component, we now introduce a third molecular chaperone, the thiol-dependent reductase ER-60 (ERp57/GRP58/ERp61/HIP-70/Q2), into this process. ER-60 is found in MHC class I heavy chain complexes with calnexin that are generated early during the MHC class I assembly pathway. The thiol reductase activity of ER-60 raises the possibility that ER-60 is involved in the disulfide bond formation within heavy chains. In addition, ER-60 is part of the late assembly complexes consisting of MHC class I, tapasin, TAP, calreticulin and calnexin. In a beta2-microglobulin (beta2m)-negative mouse cell line, S3, ER-60-calnexin-heavy chain complexes are shown to bind to TAP, suggesting that beta2m is not required for the association of MHC class I heavy chains with TAP.     

The quality control of MHC class I peptide loading

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 multistep assembly of MHC class I heavy chain with beta(2)-microglobulin 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.     

Modes of Calreticulin Recruitment to the Major Histocompatibility Complex Class I Assembly Pathway

Major histocompatibility complex (MHC) class I molecules are ligands for T-cell receptors of CD8⁺ T cells and inhibitory receptors of natural killer cells. Assembly of the heavy chain, light chain, and peptide components of MHC class I molecules occurs in the endoplasmic reticulum (ER). Specific assembly factors and generic ER chaperones, collectively called the MHC class I peptide loading complex (PLC), are required for MHC class I assembly. Calreticulin has an important role within the PLC and induces MHC class I cell surface expression, but the interactions and mechanisms involved are incompletely understood. We show that interactions with the thiol oxidoreductase ERp57 and substrate glycans are important for the recruitment of calreticulin into the PLC and for its functional activities in MHC class I assembly. The glycan and ERp57 binding sites of calreticulin contribute directly or indirectly to complexes between calreticulin and the MHC class I assembly factor tapasin and are important for maintaining steady-state levels of both tapasin and MHC class I heavy chains. A number of destabilizing conditions and mutations induce generic polypeptide binding sites on calreticulin and contribute to calreticulin-mediated suppression of misfolded protein aggregation in vitro. We show that generic polypeptide binding sites per se are insufficient for stable recruitment of calreticulin to PLC substrates in cells. However, such binding sites could contribute to substrate stabilization in a step that follows the glycan and ERp57-dependent recruitment of calreticulin to the PLC.     

A role for calnexin in the assembly of the MHC class I loading complex in the endoplasmic reticulum

Heterodimers of MHC class I glycoprotein and beta(2)-microglobulin (beta(2)m) bind short peptides in the endoplasmic reticulum (ER). Before peptide binding these molecules form part of a multisubunit loading complex that also contains the two subunits of the TAP, the transmembrane glycoprotein tapasin, the soluble chaperone calreticulin, and the thiol oxidoreductase ERp57. We have investigated the assembly of the loading complex and provide evidence that after TAP and tapasin associate with each other, the transmembrane chaperone calnexin and ERp57 bind to the TAP-tapasin complex to generate an intermediate. These interactions are independent of the N:-linked glycan of tapasin, but require its transmembrane and/or cytoplasmic domain. This intermediate complex binds MHC class I-beta(2)m dimers, an event accompanied by the loss of calnexin and the acquisition of calreticulin, generating the MHC class I loading complex. Peptide binding then induces the dissociation of MHC class I-beta(2)m dimers, which can be transported to the cell surface.     

Clustering of peptide-loaded MHC class I molecules for endoplasmic reticulum export imaged by fluorescence resonance energy transfer

Fluorescence resonance energy transfer between cyan fluorescent protein- and yellow fluorescent protein-tagged MHC class I molecules reports on their spatial organization during assembly and export from the endoplasmic reticulum (ER). A fraction of MHC class I molecules is clustered in the ER at steady state. Contrary to expectations from biochemical models, this fraction is not bound to the TAP. Instead, it appears that MHC class I molecules cluster after peptide loading. This clustering points toward a novel step involved in the selective export of peptide-loaded MHC class I molecules from the ER. Consistent with this model, we detected clusters of wild-type HLA-A2 molecules and of mutant A2-T134K molecules that cannot bind TAP, but HLA-A2 did not detectably cluster with A2-T134K at steady state. Lactacystin treatment disrupted the HLA-A2 clusters, but had no effect on the A2-T134K clusters. However, when cells were fed peptides with high affinity for HLA-A2, mixed clusters containing both HLA-A2 and A2-T134K were detected.     

Assembly of HLA DR1 molecules translated in vitro: binding of peptide in the endoplasmic reticulum precludes association with invariant chain.

MHC class II molecules usually bind peptides in the endocytic pathway, but can also present endogenous peptides from newly synthesized proteins in a chloroquine-insensitive manner, suggesting that peptide binding might occur in the endoplasmic reticulum (ER). We used in vitro translation of HLA-DR1 class II molecules in the presence of microsomes to study peptide binding in the ER. Formation of functional class II molecules in vitro depends on formation of disulfide bridges in alpha and beta chains. The class II alpha beta heterodimers made by in vitro translation resemble class II molecules synthesized in cells in (i) their reactivity with conformation-specific antibodies, (ii) their assembly with Ii chain homotrimers, (iii) the generation of SDS-stable dimers upon peptide binding and (iv) their specificity of peptide binding. The assembly of class II molecules occurs via an alpha beta intermediate and can occur post-translationally, but only in intact microsomes. Class II alpha beta heterodimers are able to bind peptides in ER-derived microsomes, a process that precludes subsequent association of class II molecules with Ii chain. This mechanism might explain presentation of endogenous peptides by class II molecules.     

Mechanisms of Function of Tapasin,a Critical Major Histocompatibility Complex Class I Assembly Factor

For their efficient assembly in the endoplasmic reticulum (ER), major histocompatibility complex (MHC) class I molecules require the specific assembly factors transporter associated with antigen processing (TAP) and tapasin, as well as generic ER folding factors, including the oxidoreductases ERp57 and protein disulfide isomerase (PDI), and the chaperone calreticulin. TAP transports peptides from the cytosol into the ER. Tapasin promotes the assembly of MHC class I molecules with peptides. The formation of disulfide‐linked conjugates of tapasin with ERp57 is suggested to be crucial for tapasin function. Important functional roles are also suggested for the tapasin transmembrane and cytoplasmic domains, sites of tapasin interaction with TAP. We show that interactions of tapasin with both TAP and ERp57 are correlated with strong MHC class I recruitment and assembly enhancement. The presence of the transmembrane/cytosolic regions of tapasin is critical for efficient tapasin–MHC class I binding in interferon‐γ‐treated cells, and contributes to an ERp57‐independent mode of MHC class I assembly enhancement. A second ERp57‐dependent mode of tapasin function correlates with enhanced MHC class I binding to tapasin and calreticulin. We also show that PDI binds to TAP in a tapasin‐independent manner, but forms disulfide‐linked conjugates with soluble tapasin. Thus, full‐length tapasin is important for enhancing recruitment of MHC class I molecules and increasing specificity of tapasin–ERp57 conjugation. Furthermore, tapasin or the TAP/tapasin complex has an intrinsic ability to recruit MHC class I molecules and promote assembly, but also uses generic folding factors to enhance MHC class I recruitment and assembly.     

Peptide-bound major histocompatibility complex class I molecules associate with tapasin before dissociation from transporter associated with antigen processing

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8 T cells. The peptides are generated in the cytosol, then translocated across the membrane of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). TAP is a trimeric complex consisting of TAP1, TAP2, and tapasin (TAP-A) as indicated for human cells by reciprocal coprecipitation with anti-TAP1/2 and anti-tapasin antibodies, respectively. TAP1 and TAP2 are required for the peptide transport. Tapasin is involved in the association of class I with TAP and in the assembly of class I with peptide. The mechanisms of tapasin function are still unknown. Moreover, there has been no evidence for a murine tapasin analogue, which has led to the suggestion that murine MHC class I binds directly to TAP1/2. In this study, we have cloned the mouse analogue of tapasin. The predicted amino acid sequence showed 78% identity to human tapasin with identical consensus sequences of signal peptide, N-linked glycosylation site, transmembrane domain and double lysine motif. However, there was less homology (47%) found at the predicted cytosolic domain, and in addition, mouse tapasin is 14 amino acids longer than the human analogue at the C terminus. This part of the molecule may determine the species specificity for interaction with MHC class I or TAP1/2. Like human tapasin, mouse tapasin binds both to TAP1/2 and MHC class I. In TAP2-mutated RMA-S cells, both TAP1 and MHC class I were coprecipitated by anti-tapasin antiserum indicative of association of tapasin with TAP1 but not TAP2. With crosslinker-modified peptides and purified microsomes, anti-tapasin coprecipitated both peptide-bound MHC class I and TAP1/2. In contrast, anti-calreticulin only coprecipitated peptide-free MHC class I molecules. This difference in association with peptide-loaded class I suggests that tapasin functions later than calreticulin during MHC class I assembly, and controls peptide loading onto MHC class I molecules in the endoplasmic reticulum.     

Apoptosis and altered dendritic cell homeostasis in lupus nephritis are limited by anti-CD154 treatment

Once MHC class I heavy chain binds beta(2)-microglobulin (beta(2)m) within the endoplasmic reticulum, an assembly complex comprising the class I heterodimer, TAP, TAPasin, calreticulin, and possibly Erp57 is formed before the binding of high affinity peptide. TAP-dependent delivery of high affinity peptide to in vitro translated K(b)beta(2)m complexes within microsomes (TAP(+)/TAPasin(+)) was studied to determine at which point peptide binding becomes resistant to thermal denaturation. It was determined that the thermal stability of K(b)-beta(2)m-peptide complexes depends on the timing of peptide binding to K(b)beta(2)m relative to TAP binding high affinity peptide. Premature exposure of the TAP complex to high affinity peptide before its association with class I heavy chain results in K(b)beta(2)m-peptide-TAP complexes that lose peptide upon exposure to elevated temperature after solubilization away from microsome-associated proteins. These findings suggest that the order in which class I heavy chain associates with endoplasmic reticulum-resident chaperones and peptide determines the stability of K(b)beta(2)m-peptide complexes.     

Calreticulin‐dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin‐deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub‐optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER–Golgi intermediate compartment (ERGIC) and the cis‐Golgi, together with sub‐optimally loaded class I molecules. Calreticulin that lacks its C‐terminal KDEL retrieval sequence assembles with the peptide‐loading complex but neither retrieves sub‐optimally loaded class I molecules from the cis‐Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.     

Control of MHC class I traffic from the endoplasmic reticulum by cellular chaperones and viral anti-chaperones

MHC class I molecules assemble with peptides in the endoplasmic reticulum (ER). To ensure that only peptide-loaded MHC molecules leave the ER, empty molecules are retained by ER-resident chaperones, most notably the MHC-specific tapasin. ER exit of class I MHC is also controlled by viruses, but for the opposite purpose of preventing peptide presentation to T cells. Interestingly, some viral proteins are able to retain MHC class I molecules in the ER despite being transported. By contrast, other viral proteins exit the ER only upon binding to class I MHC, thereby rerouting newly synthesized class I molecules to intracellular sites of proteolysis. Thus, immune escape can be achieved by reversing, inhibiting or redirecting the chaperone-assisted MHC class I folding, assembly and intracellular transport.     

TAP association influences the conformation of nascent MHC class I molecules

The influence of TAP-MHC class I interactions on peptide binding to the class I heavy chain is assessed during TAP-dependent assembly using Kb-specific Abs that recognize conformational changes induced by assembly with beta2-microglobulin (beta2m) and by peptide binding. A significant portion (45%) of Kb molecules in TAP+, RMA-derived microsomes are associated with the TAP complex as measured by coimmunoisolation of Kb using anti-TAP1 Abs, while only 20% of the Kb heavy chain molecules are isolated as Kbbeta2m complexes with the alpha-Kb-specific Abs, Y-3 or K-10-56. The amount of Kb isolated with Y-3 and K-10-56 increases in proportion to transport and binding of peptide to the Kb molecules within the RMA microsomes. In contrast, less than 5% of the Kb within TAP2-RMA-S microsomes associated with the remaining TAP1 subunit. However, greater than 60% of Kb heavy chain is isolated as K-10-56- and Y-3-reactive Kbbeta2m complexes. We propose that a TAP-MHC class I interaction serves to stabilize the MHC class I:beta2m complex in an immature conformation (Y-3 and K-10-56 nonreactive) prior to high affinity peptide binding, preventing the export of class I molecules complexed with low affinity peptide ligands from the ER.     

Calreticulin promotes folding of functional human leukocyte antigen class I molecules in vitro

The assembly of MHC class I molecules with beta(2)-microglobulin and peptides is assisted by the housekeeping chaperones calnexin, calreticulin, and Erp57 and the dedicated accessory protein, tapasin. Tapasin and calreticulin are essential for efficient MHC class I assembly, but their precise action during class I assembly remains to be elucidated. Previous in vitro studies have demonstrated that the lectin calreticulin interacts with monoglucosylated MHC class I heavy chains, whatever their state of assembly with light chains and peptide, and inhibits their aggregation above physiological temperature. We used a soluble single chain HLA-A2/beta(2)-microglobulin molecule, A2SC, to study the effect of calreticulin on the peptide binding capacity of HLA class I molecules. Calreticulin inhibited the formation of A2SC aggregates both when co-expressed in insect cells and during incubations at elevated temperature. Calreticulin dramatically enhanced acquisition of peptide binding capacity when added to denatured A2SC molecules during refolding at 4 degrees C. However, it had no effect on the rapid loss of A2SC peptide binding capacity at physiological temperature. We conclude that calreticulin promotes the folding of HLA class I molecules to a state in which, at low temperature, they spontaneously acquire peptide binding capacity. However, it does not induce or maintain a peptide-receptive state of the class I-binding site, which is likely to be promoted by one or several other components of the class I loading complexes. By being amenable to complementation with additional proteins, the described system should be useful for identification of these components.     

Identification of specific glycoforms of major histocompatibility complex class I heavy chains suggests that class I peptide loading is an adaptation of the quality control pathway involving calreticulin and ERp57

Glycosylation analysis was used to probe the sequence of events accompanying the binding of antigenic peptides to the major histocompatibility complex class I heavy chains. Free heavy chains were isolated from the beta(2)-microglobulin-negative cell line Daudi and from the B-lymphoblastoid cell line Raji. Heavy chains were also isolated from Raji cells in multimolecular complexes (peptide loading complexes) containing the transporter associated with antigen processing, tapasin and ERp57 with and without the lectin-like folding chaperone, calreticulin. Calreticulin is a soluble protein that recognizes primarily the terminal glucose of Glc(1)Man(7-9)GlcNAc(2) glycans. This paper shows that monoglucosylated glycoforms of heavy chain, which exist transiently in the endoplasmic reticulum in the initial stages of the glycosylation processing pathway, are present in the peptide loading complex. The data are consistent with a model in which the release of peptide-loaded major histocompatibility complex class I molecules from calreticulin, induced by deglucosylation of the heavy chain N-linked glycan, signals the dissociation of the complex. This is consistent with the hypothesis that the class I loading process is an adaptation of the quality control mechanism involving calreticulin and ERp57.     

The group II chaperonin TRiC protects proteolytic intermediates from degradation in the MHC class I antigen processing pathway

MHC class I molecules present precisely cleaved peptides of intracellular proteins on the cell surface. For most antigenic precursors, presentation requires transport of peptide fragments into the ER, but the nature of the cytoplasmic peptides and their chaperones is obscure. By tracking proteolytic intermediates in living cells, we show that intracellular proteolysis yields a mixture of antigenic peptides containing only N-terminal flanking residues for ER transport. Some of these peptides were bound to the group II chaperonin TRiC and were protected from degradation. Destabilization of TRiC by RNA interference inhibited the expression of peptide-loaded MHC I molecules on the cell surface. Thus, the TRiC chaperonin serves a function in protecting proteolytic intermediates in the MHC I antigen processing pathway.     

Impaired assembly results in the accumulation of multiple HLA-C heavy chain folding intermediates

Class I MHC H chains assemble with beta2-microglobulin (beta2m) and are loaded with peptide Ags through multiple folding steps. When free of beta2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in this study, detected a preferential association of L31-reactive, beta2m-free H chains with calnexin in beta2m-defective cells, and with calreticulin and TAP in beta2m-expressing cells. In beta2m-defective cells, the accumulation of calnexin-bound H chains stoichiometrically exceeded their overall accumulation, a finding that supports both chaperoning preferences and distinct sorting abilities for different class I folds. No peptide species, in a mass range compatible with that of the classical class I ligands, could be detected by mass spectrometry of acidic eluates from L31-reactive HLA-Cw1 H chains. In vitro assembly experiments in TAP-defective T2 cells, and in cells expressing an intact Ag-processing machinery, demonstrated that L31 H chains are not only free of, but also unreceptive to, peptides. L31 and HC10, which bind nearly adjacent linear epitopes of the alpha1 domain alpha helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the alpha1 domain alpha helix and peptide-anchoring residues of the class I H chain. Thus, unlike certain murine free H chains, L31-reactive H chains are not the immediate precursors of conformed class I molecules. A model inferring their precursor-product relationships with other known class I intermediates is presented.     

Dynamics of free versus complexed β2-microglobulin and the evolution of interfaces in MHC class I molecules

In major histocompatibility complex (MHC) class I molecules, monomorphic β₂-microglobulin (β₂m) is non-covalently bound to a heavy chain (HC) exhibiting a variable degree of polymorphism. β₂M can stabilize a wide variety of complexes ranging from classical peptide binding to nonclassical lipid presenting MHC class I molecules as well as to MHC class I-like molecules that do not bind small ligands. Here we aim to assess the dynamics of individual regions in free as well as complexed β₂m and to understand the evolution of the interfaces between β₂m and different HC. Using human β₂m and the HLA–B*27:09 complex as a model system, a comparison of free and HC-bound β₂m by nuclear magnetic resonance spectroscopy was initially carried out. Although some regions retain their flexibility also after complex formation, these studies reveal that most parts of β₂m gain rigidity upon binding to the HC. Sequence analyses demonstrate that some of the residues exhibiting flexibility participate in evolutionarily conserved β₂m–HC contacts which are detectable in diverse vertebrate species or characterize a particular group of MHC class I complexes such as peptide- or lipid-binding molecules. Therefore, the spectroscopic experiments and the interface analyses demonstrate that β₂m fulfills its role of interacting with diverse MHC class I HC as well as effector cell receptors not only by engaging in conserved intermolecular contacts but also by falling back upon key interface residues that exhibit a high degree of flexibility.