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.     

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.     

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.     

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.     

Aggregate formation by ERp57-deficient MHC class I peptide-loading complexes

The endoplasmic reticulum (ER)-resident proteins TAP, tapasin and ERp57 are the core components of the major histocompatibility complex (MHC) class I peptide-loading complex and play an important role in peptide loading by MHC class I-beta(2)microglobulin dimers. ERp57 and tapasin form a stable disulfide-linked heterodimer within the peptide-loading complex. We demonstrate that ERp57-deficient loading complexes, obtained by expression in a tapasin-negative cell line of a tapasin mutant (C95A) that is not able to form a disulfide bond with ERp57, are prone to aggregation. We studied the assembly, stability and aggregation of the core loading complex using cell lines stably expressing fluorescently tagged tapasin (wild type or C95A mutant) and TAP1. Part of the loading complexes containing the tagged C95A tapasin and TAP1 were sequestered in the ER, without change of their ER transmembrane topology, and were surrounded by a mesh of filaments at the cytosolic side, resulting in formation of protein aggregates with characteristic morphology. Protein aggregates were associated with changes in ER protein turnover but did not affect the cell viability and did not induce the unfolded protein response. Fluorescence resonance energy transfer analysis of the aggregate-free ER fraction revealed that lack of ERp57 did not affect the stoichiometry or stability of tapasin-TAP1 interactions in the assembled 'soluble' core loading complexes. We conclude that the presence of ERp57 is important for the stability of core loading complexes, and that in its absence, the core loading complexes may form stable aggregates within the ER.     

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.     

Tapasin and ERp57 form a stable disulfide-linked dimer within the MHC class I peptide-loading complex

We previously showed that the major histocompatibility complex (MHC) class I chaperone tapasin can be detected as a mixed disulfide with the thiol-oxidoreductase ERp57. Here we show that tapasin is a unique and preferred substrate, a substantial majority of which is disulfide-linked to ERp57 within the cell. Tapasin upregulation by interferon-gamma induces sequestration of the vast majority of ERp57 into the MHC class I peptide-loading complex. The rate of tapasin-ERp57 conjugate formation is unaffected by the absence of beta2-microglubulin (beta2m), and is independent of calnexin or calreticulin interactions with monoglucosylated N-linked glycans. The heterodimer forms spontaneously in vitro upon mixing recombinant ERp57 and tapasin. Noncovalent interactions between the native proteins inhibit the reductase activity of the thioredoxin CXXC motif within the N-terminal a domain of ERp57 to maintain its interaction with tapasin. Disruption of these interactions by denaturation allows reduction to proceed. Thus, tapasin association specifically inhibits the escape pathway required for disulfide-bond isomerization within conventional protein substrates, suggesting a specific structural role for ERp57 within the MHC class I peptide-loading complex.     

The role of ERp57 in disulfide bond formation during the assembly of major histocompatibility complex class I in a synchronized semipermeabilized cell translation system

We have established a semipermeabilized cell system that reproduces the folding and assembly of a major histocompatibility complex (MHC) class I complex as it would occur in the intact cell. The translation of the MHC class I heavy chain (HLA-B27) in this system was synchronized allowing the folding and assembly of polypeptide chains synthesized within a short time frame to be analyzed. This has enabled us to dissect the time course of interaction of both disulfide and nondisulfide-bonded heavy chain with various molecular chaperones during its assembly in a functionally intact endoplasmic reticulum. The results demonstrate that unassembled, nondisulfide-bonded forms of heavy chain interact initially with calnexin. A later and more prolonged interaction of calreticulin, specifically with assembled, disulfide-bonded heavy chain, highlights distinct differences in the roles of these two proteins in the assembly of MHC class I molecules. We also demonstrate that the thiol-dependent reductase ERp57 initially interacts with nondisulfide-bonded heavy chain, but this rapidly becomes disulfide-bonded and indicates that heavy chain folding occurs during its interaction with ERp57. In addition, we also confirm a direct interaction between MHC class I heavy chain and tapasin, emphasizing the role that this protein plays in the later stages of MHC class I assembly.     

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.     

Competition for access to the rat major histocompatibility complex class I peptide-loading complex reveals optimization of peptide cargo in the absence of transporter associated with antigen processing (TAP) association

Major histocompatibility complex (MHC) class I molecules load peptides in the endoplasmic reticulum in a process during which the peptide cargo is normally optimized in favor of stable MHC-peptide interactions. A dynamic multimolecular assembly termed the peptide-loading complex (PLC) participates in this process and is composed of MHC class I molecules, calreticulin, ERp57, and tapasin bound to the transporter associated with antigen processing (TAP) peptide transporter. We have exploited the observation that the rat MHC class I allele RT1-Aa, when expressed in the rat C58 thymoma cell line, effectively competes and prevents the endogenous RT1-Au molecule from associating with TAP. However, stable RT1-Au molecules are assembled efficiently in competition with RT1-Aa, demonstrating that cargo optimization can occur in the absence of TAP association. Defined mutants of RT1-Aa, which do not allow formation of the PLC, fail to become thermostable in C58 cells. Wild-type RT1-Aa, which does allow PLC formation, also fails to become thermostable in this cell line, which carries the rat TAPB transporter that supplies peptides incompatible for RT1-Aa binding. Full optimization of RT1-Aa requires the presence of the TAP2A allele, which is capable of supplying suitable peptides. Thus, formation of the PLC alone is not sufficient for optimization of the MHC class I peptide cargo.     

The first N-terminal transmembrane helix of each subunit of the antigenic peptide transporter TAP is essential for independent tapasin binding

The heterodimeric ABC transporter TAP translocates proteasomal degradation products from the cytosol into the lumen of the endoplasmic reticulum, where these peptides are loaded onto MHC class I molecules by a macromolecular peptide-loading complex (PLC) and subsequently shuttled to the cell surface for inspection by cytotoxic T lymphocytes. Tapasin recruits, as a central adapter protein, other components of the PLC at the unique N-terminal domains of TAP. We found that the N-terminal domains of human TAP1 and TAP2 can independently bind to tapasin, thus providing two separate loading platforms for PLC assembly. Moreover, tapasin binding is dependent on the first N-terminal transmembrane helix of TAP1 and TAP2, demonstrating that these two helices contribute independently to the recruitment of tapasin and associated factors.     

Assembly and Function of the Major Histocompatibility Complex (MHC) I Peptide-loading Complex Are Conserved Across Higher Vertebrates

Antigen presentation to cytotoxic T lymphocytes via major histocompatibility complex class I (MHC I) molecules depends on the heterodimeric transporter associated with antigen processing (TAP). For efficient antigen supply to MHC I molecules in the ER, TAP assembles a macromolecular peptide-loading complex (PLC) by recruiting tapasin. In evolution, TAP appeared together with effector cells of adaptive immunity at the transition from jawless to jawed vertebrates and diversified further within the jawed vertebrates. Here, we compared TAP function and interaction with tapasin of a range of species within two classes of jawed vertebrates. We found that avian and mammalian TAP1 and TAP2 form heterodimeric complexes across taxa. Moreover, the extra N-terminal domain TMD0 of mammalian TAP1 and TAP2 as well as avian TAP2 recruits tapasin. Strikingly, however, only TAP1 and TAP2 from the same taxon can form a functional heterodimeric translocation complex. These data demonstrate that the dimerization interface between TAP1 and TAP2 and the tapasin docking sites for PLC assembly are conserved in evolution, whereas elements of antigen translocation diverged later in evolution and are thus taxon specific.     

Tapasin and other chaperones: models of the MHC class I loading complex

MHC (major histocompatibility complex) class I molecules bind intracellular virus-derived peptides in the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T lymphocytes. Peptide-free class I molecules at the cell surface, however, could lead to aberrant T cell killing. Therefore, cells ensure that class I molecules bind high-affinity ligand peptides in the ER, and restrict the export of empty class I molecules to the Golgi apparatus. For both of these safeguard mechanisms, the MHC class I loading complex (which consists of the peptide transporter TAP, the chaperones tapasin and calreticulin, and the protein disulfide isomerase ERp57) plays a central role. This article reviews the actions of accessory proteins in the biogenesis of class I molecules, specifically the functions of the loading complex in high-affinity peptide binding and localization of class I molecules, and the known connections between these two regulatory mechanisms. It introduces new models for the mode of action of tapasin, the role of the class I loading complex in peptide editing, and the intracellular localization of class I molecules.     

Sequence, linkage to H2-K, and function of mouse tapasin in MHC class I assembly

 Assembly of major histocompatibility complex (MHC) class I molecules in human cells is dependent on the accessory protein tapasin, which mediates their interaction with the transporters associated with antigen processing (TAP) and thereby ensures efficient peptide binding. Analysis of a mouse tapasin complementary DNA defined a conserved polypeptide sharing sequences diagnostic of a transmembrane protein related to the immunoglobulin superfamily, and an endoplasmic reticulum retention motif. The mouse tapasin gene was mapped about 70 kilobases from H2-K at the centromeric end of the mouse MHC. Expression of mouse tapasin in a tapasin-deficient human mutant cell line restored the normal assembly and expression of class I alleles. Thus, tapasin is a structurally and functionally conserved component of the MHC class I antigen processing pathway. Its genetic linkage to the class I and TAP subunit genes in the MHC may be of significance in the coordinate expression and functional coadaptation of the diverse gene products. Received: 1 February 1998 / Revised: 23 March 1998     

The polypeptide binding conformation of calreticulin facilitates its cell-surface expression under conditions of endoplasmic reticulum stress

We define two classes of calreticulin mutants that retain glycan binding activity; those that display enhanced or reduced polypeptide-specific chaperone activity, due to conformational effects. Under normal conditions, neither set of mutants significantly impacts the ability of calreticulin to mediate assembly and trafficking of major histocompatibility complex class I molecules, which are calreticulin substrates. However, in cells treated with thapsigargin, which depletes endoplasmic reticulum calcium, major histocompatibility complex class I trafficking rates are accelerated coincident with calreticulin secretion, and detection of cell-surface calreticulin is dependent on its polypeptide binding conformations. Together, these findings identify a site on calreticulin that is an important determinant of the induction of its polypeptide binding conformation and demonstrate the relevance of the polypeptide binding conformations of calreticulin to endoplasmic reticulum stress-induced interactions.     

Molecular architecture of the TAP-associated MHC class I peptide-loading complex

Tapasin organizes the peptide-loading complex (PLC) by recruiting peptide-receptive MHC class I (MHC-I) and accessory chaperones to the N-terminal regions of the TAP subunits TAP1 and TAP2. Despite numerous studies have shown that the formation of the PLC is essential to facilitate proper MHC-I loading, the molecular architecture of this complex is still highly controversial. We studied the stoichiometry of the PLC by blue native-PAGE in combination with Ab-shift assays and found that TAP/tapasin complexes exist at steady state as a mixture of two distinct oligomers of 350 and 450 kDa. Only the higher m.w. complex contains MHC-I and disulfide-linked tapasin/ER60 conjugates. Moreover, we show for the first time to our knowledge that the fully assembled PLC comprises two tapasin, two ER60, but only one complex of MHC-I and calreticulin. Based hereon we postulate that the TAP subunits alternate in the recruitment and loading of a single MHC-I.     

Major histocompatibility complex class I-ERp57-tapasin interactions within the peptide-loading complex

The endoplasmic reticulum-located multimolecular peptide-loading complex functions to load optimal peptides onto major histocompatibility complex (MHC) class I molecules for presentation to CD8(+) T lymphocytes. Two oxidoreductases, ERp57 and protein-disulfide isomerase, are known to be components of the peptide-loading complex. Within the peptide-loading complex ERp57 is normally found disulfide-linked to tapasin, through one of its two thioredoxin-like redox motifs. We describe here a novel trimeric complex that disulfide links together MHC class I heavy chain, ERp57 and tapasin, and that is found in association with the transporter associated with antigen processing peptide transporter. The trimeric complex normally represents a small subset of the total ERp57-tapasin pool but can be significantly increased by altering intracellular oxidizing conditions. Direct mutation of a conserved structural cysteine residue implicates an interaction between ERp57 and the MHC class I peptide-binding groove. Taken together, our studies demonstrate for the first time that ERp57 directly interacts with MHC class I molecules within the peptide-loading complex and suggest that ERp57 and protein-disulfide isomerase act in concert to regulate the redox status of MHC class I during antigen presentation.     

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.     

ERp57 interacts with conserved cysteine residues in the MHC class I peptide-binding groove

The oxidoreductase ERp57 is a component of the major histocompatibility complex (MHC) class I peptide-loading complex. ERp57 can interact directly with MHC class I molecules, however, little is known about which of the cysteine residues within the MHC class I molecule are relevant to this interaction. MHC class I molecules possess conserved disulfide bonds between cysteines 101-164, and 203-259 in the peptide-binding and alpha3 domain, respectively. By studying a series of mutants of these conserved residues, we demonstrate that ERp57 predominantly associates with cysteine residues in the peptide-binding domain, thus indicating ERp57 has direct access to the peptide-binding groove of MHC class I molecules during assembly.     

Major histocompatibility complex class I molecules expressed with monoglucosylated N-linked glycans bind calreticulin independently of their assembly status

The assembly of major histocompatibility complex (MHC) class I molecules with peptides in the endoplasmic reticulum (ER) is a critical step in the presentation of viral antigens to CD8+ T cells. This process is subject to quality control restrictions that prevent free class I heavy chains (HCs) and peptide-free HC-beta(2)-microglobulin (beta(2)m) dimers from exiting the ER. The lectin-like chaperone calreticulin associates with HC-beta(2)m heterodimers prior to peptide binding, but its precise role in regulating the subsequent events of peptide association and ER to Golgi transport remains undefined. In vitro analysis of the assembly process has been limited by the specificity of calreticulin for monoglucosylated N-linked glycans, which are transient biosynthetic intermediates. To address this problem, we developed a novel expression system using Saccharomyces cerevisiae glycosylation mutants to produce class I HC bearing N-linked oligosaccharides with the specific structure Glc(1)Man(9)GlcNAc(2). The monoglucosylated glycan proved to be both necessary and sufficient for in vitro binding of calreticulin to MHC class I molecules. Calreticulin bound as efficiently to peptide-loaded MHC class I complexes as it did to folding intermediates created in vitro, namely free class I HC and empty HC-beta(2)m heterodimers. Thus, calreticulin is unable to discriminate between native and non-native MHC class I conformations and therefore unlikely to play a role in the recognition and release of peptide-loaded complexes from the ER. Furthermore, the recombinant expression system developed in this study can be used to produce a broad range of calreticulin substrates to elucidate its general mechanism of activity in vitro.