Calreticulin binds to the alpha1 domain of MHC class I independently of tapasin

Prior to binding to antigenic peptide, the major histocompatibility complex (MHC) heavy chain associates with an assembly complex of proteins that includes calreticulin, tapasin, and the transporter associated with antigen processing (TAP). Our data show that calreticulin can bind weakly to Ld without tapasin's assistance, and that deglycosylation of the alpha1 domain results in a primary loss of binding to calreticulin rather than tapasin. We have also shown that high amounts of wild-type tapasin are still unable to associate with MHC class I in the absence of the MHC class I/calreticulin interaction, confirming the central role of calreticulin in the formation of the MHC class I assembly complex.     

The optimization of peptide cargo bound to MHC class I molecules by the peptide-loading complex

Summary: Major histocompatibility complex (MHC) class I complexes present peptides from both self and foreign intracellular proteins on the surface of most nucleated cells. The assembled heterotrimeric complexes consist of a polymorphic glycosylated heavy chain, non‐polymorphic β₂ microglobulin, and a peptide of typically nine amino acids in length. Assembly of the class I complexes occurs in the endoplasmic reticulum and is assisted by a number of chaperone molecules. A multimolecular unit termed the peptide‐loading complex (PLC) is integral to this process. The PLC contains a peptide transporter (transporter associated with antigen processing), a thiooxido‐reductase (ERp57), a glycoprotein chaperone (calreticulin), and tapasin, a class I‐specific chaperone. We suggest that class I assembly involves a process of optimization where the peptide cargo of the complex is edited by the PLC. Furthermore, this selective peptide loading is biased toward peptides that have a longer off‐rate from the assembled complex. We suggest that tapasin is the key chaperone that directs this action of the PLC with secondary contributions from calreticulin and possibly ERp57. We provide a framework model for how this may operate at the molecular level and draw parallels with the proposed mechanism of action of human leukocyte antigen‐DM for MHC class II complex optimization.     

HLA-DM, HLA-DO and tapasin: functional similarities and differences.

In both the MHC class II and class I pathways of antigen presentation, accessory molecules influence formation of MHC-peptide complexes. In the MHC class II pathway, DM functions in the loading and editing of peptides; recent work demonstrated that it is acting not only in late endosomal compartments but also in recycling compartments and on the surface of B cells and immature dendritic cells. DM activity is modulated by another accessory molecule, DO, but this modulation is mainly operative in B cells, where it may lead to preferential activation of B cells producing high-affinity antibodies. In the MHC class I pathway of antigen presentation, recent in vivo experiments with knockout mice confirmed the role of tapasin in antigen presentation and indicate that it acts as a peptide editor and as a chaperone for TAP and the MHC class I heavy chain. In the class I loading complex, calreticulin and the thiol-dependent oxidoreductase ER60/ERp57 appear to support the function of tapasin in an as-yet-unknown fashion. The picture emerges that DM and tapasin have analogous functions in shaping the peptide repertoire presented by the respective MHC class II and class I molecules.     

The N-terminal region of tapasin is required to stabilize the MHC class I loading complex.

Tapasin mediates the binding of MHC class I molecules to the transporter associated with antigen processing (TAP). Deletion mutants of tapasin were used to examine the effect of tapasin on interactions within the MHC class I complex. Binding to TAP is mediated by the C-terminal region of tapasin. Michaelis-Menten analysis of peptide transport shows that this interaction is sufficient to increase TAP levels without significantly affecting the intrinsic translocation rate. Weak interactions exist between MHC class I molecules and TAP in the absence of tapasin, and between free heavy chains and TAP-tapasin complexes in the absence of beta2-microglobulin. The N-terminal 50 residues of tapasin constitute the key element which converts the sum of these weak interactions into a stable complex.     

Stoichiometric tapasin interactions in the catalysis of major histocompatibility complex class I molecule assembly

The assembly of major histocompatibility complex (MHC) class I molecules with their peptide ligands in the endoplasmic reticulum (ER) requires the assistance of many proteins that form a multimolecular assemblage termed the 'peptide-loading complex'. Tapasin is the central stabilizer of this complex, which also includes the transporter associated with antigen processing (TAP), MHC class I molecules, the ER chaperone, calreticulin, and the thiol-oxidoreductase ERp57. In the present report, we investigated the requirements of these interactions for tapasin protein stability and MHC class I dissociation from the peptide-loading complex. We established that tapasin is stable in the absence of either TAP or MHC class I interaction. In the absence of TAP, tapasin interaction with MHC class I molecules is long-lived and results in the sequestration of existing tapasin molecules. In contrast, in TAP-sufficient cells, tapasin is re-utilized to interact with and facilitate the assembly of many MHC class I molecules sequentially. Furthermore, chemical cross-linking has been utilized to characterize the interactions within this complex. We demonstrate that tapasin and MHC class I molecules exist in a 1 : 1 complex without evidence of higher-order tapasin multimers. Together these studies shed light on the tapasin protein life cycle and how it functions in MHC class I assembly with peptide for presentation to CD8(+) T cells.     

Mutant MHC class I molecules define interactions between components of the peptide-loading complex

Class I histocompatibility molecules, consisting of a heavy chain, beta2-microglobulin and peptide, are assembled in the endoplasmic reticulum (ER) with the assistance of several molecular chaperones and accessory proteins. Peptide binding occurs when assembling class I molecules associate with a loading complex consisting of the transporter associated with antigen processing (TAP) peptide transporter, tapasin, ERp57 and calreticulin (CRT)/calnexin. To assess the physical organization of this complex, we generated a series of mutants in the murine H-2Dd heavy chain and assessed their association with components of the complex. Seven mutations, clustered between amino acids 122 and 136 in the heavy chain alpha2 domain plus one mutation at position 222 in the alpha3 domain, resulted in loss of interaction with tapasin. Association with TAP was always lost simultaneously, supporting the view that tapasin acts as an obligatory bridge between class I molecules and TAP. Compared with previous studies on the HLA-A2 molecule, some differences in points of tapasin interaction were observed. Failure of the H-2Dd mutants to bind tapasin resulted in low cell-surface expression and altered intracellular transport. Most mutants retained a substantial degree of peptide loading, consistent with the view that although tapasin may promote peptide binding to class I, it is not required. A surprising observation was that all mutants lacking tapasin interaction retained normal association with CRT. This contrasts with previous observations on other class I molecules and, combined with differences in tapasin interaction, suggests that the organization of the ER peptide-loading complex can vary depending on the specific class I molecule examined.     

Assembly of tapasin-associated MHC class I in the absence of the transporter associated with antigen processing (TAP)

The assembly of MHC class I molecules is regulated by a multi-protein complex in the endoplasmic reticules (ER) termed the loading complex. Tapasin is suggested to be one of the molecules forming this complex on the basis of its interaction with both the transporter associated with antigen processing (TAP) and MHC class I molecules. To address whether TAP is indispensable for the processing of the assembly of tapasin-associated MHC class I molecules, we studied the association of MHC class I molecules with tapasin, the assembly of tapasin-associated MHC class I with peptides and the peptide-mediated dissociation of MHC class I from tapasin in TAP-mutant T2 cells. In the absence of TAP, MHC class I heavy chain and beta(2)-microglobulin dimers were found to be properly associated with tapasin. The stable MHC class I dimer was required for its association with tapasin in the ER. In the absence of TAP, tapasin retained MHC class I molecules much longer in the ER than in the presence of TAP. This low off-rate of MHC class I from tapasin was due to the absence of high-affinity peptides in the ER of TAP-mutant cells but not to the absence of TAP per se. The introduction of peptides into permeabilized microsomes of TAP-mutant cells led to effective loading of the peptides onto tapasin-associated MHC class I and to the subsequent dissociation of MHC class I from tapasin. These results demonstrate that regulation of the assembly of tapasin-associated MHC class I is independent of the interaction of tapasin with TAP, but is dependent upon the peptides transported by TAP.     

Class IMHC molecules: assembly and antigen presentation

Summary: Several years ago, the only factor known to be necessary for the assembly and surface expression of class I MHC was pjm; even for β₂m, it was unclear at what point in class I maturation its role was played. Recent experiments that employed attachment of an endoplasmic reticulum (ER) retention signal to β₂m have shown that the point of time at which β₂m is required is while the class I heavy chain is in the ER. Later association between β₂m and class I is not vital in order for properly folded class I to be expressed at β₂m cell surface. After crystallization of the first class I MHC molecule, it was reahed that not only is antigen presented by class I, but that antigen is presented in the form of a peptide that stabilizes the class I structure and allows its transit to the cell surface. Class I allelic differences influence interactions with both peptide and β₂m, with likely consequences for the ability of the class I heavy chains to present antigen through alternative pathways. Furthermore, it is now also clear that formation of appropriate disulfide bonds in the class I heavy chain is needed before class I can bind peptide antigen securely, a process that may he assisted by an ER chaperone. Many different proteins that are resident in the ER, such as calnexin, transporter associated with antigen processing (TAP), caheticulin, and tapasin, have been found to be integral to class I assembly. TAP, tapasin, and calreticulin hind preferentially to the open form of class I, which can be distinguished with the use of a monoclonal antibody specific for this form. Calreticulin and calnexin contrast in their interactions with class I, despite other similarities between these two chaperones. Overall, class I MHC assembly is now understood to involve the interplay of multiple intra- and intermolecular events in a defined chronological order which ensure continual reporting of cellular contents to cytotoxic T lymphocytes.     

The nature of the MHC class I peptide loading complex

Summary: Peptide binding to major histocompatibility complex (MHC) dass I molecules occurs in the endoplasmic reticulum (ER). Efficient peptide binding requires a number of components in addition co the MHC class I-β₂ microglobulin dimer (β₂m). These include the two subunits of the transporter associated with antigen presentation (TAP1 and TAP2), which are essential for introducing peptides into the ER from the cytosol, and tapasin, an MHC-encoded membrane protein. Prior to peptide binding, MHC class I-β₂m dimers form part of a large multisubnnit ER complex which includes TAP and tapasin. In addition to these specialized components two soluble 'house-keeping' proteins, the chaperone calreticulin and the thiol oxidoreductase ERp57, are also components of this complex. Our current understanding of the nature and function of the MHC class I peptide loading complex is the topic of this review.     

HLA class I polymorphism has a dual impact on ligand binding and chaperone interaction

This article will describe coordinated analyses of how amino acid substitutions in the HLA class I antigen binding groove modify chaperone interaction and peptide ligand presentation. By parallel testing of ligand presentation and chaperone interaction with a series of natural HLA-B subtypes, this study has discovered that position 116 of the HLA-B15 class I heavy chain is pivotal in both peptide selection and control of interaction between the assembly complex and the class I heavy chain. Correlated with these qualitative differences in peptide selection and chaperone association are quantitative differences in the expression levels of the HLA molecules at the cell surface. These parallel studies, therefore, demonstrate that particular HLA class I polymorphisms can simultaneously influence ligand presentation and interaction with intracellular chaperones.     

Comparative analysis of the impact of a free cysteine in tapasin on the maturation and surface expression of murine MHC class I allotypes

Tapasin is a key molecule in the major histocompatibility complex (MHC) class I peptide‐loading complex, interacting with several other proteins in the complex. An amino acid substitution at a free cysteine position in tapasin has been shown to disrupt the covalent association of tapasin with ERp57. In this study, we mutated the free cysteine in mouse tapasin, and analysed the effects on the cell surface expression of the mouse MHC class I molecules K^d and K^b. The C95S substitution in mouse tapasin increased the proportion of open forms relative to folded forms for both types of MHC class I molecules at the cell surface. Furthermore, the C95S substitution resulted in increased association of tapasin with folded K^d. Overall, our studies with these mouse MHC class I allotypes have revealed that the free cysteine 95 in mouse tapasin influences stable expression at the plasma membrane for both MHC class I allotypes, and have shown that tapasin's interaction with folded K^d is elevated by the C95S substitution in tapasin.     

Disulfide bond isomerization and the assembly of MHC class I-peptide complexes

The presence of a disulfide bond inside the peptide binding groove of MHC class I molecules and of the thiol oxidoreductase ERp57 in the class I loading complex suggests that disulfide bond isomerization may play a role in peptide loading. Here we show that ERp57 and tapasin are disulfide linked inside the loading complex. Mutagenesis of cysteine 95 in tapasin not only abolishes formation of the ERp57-tapasin bond but also prevents complete oxidation of the class I heavy chain in the loading complex. The resulting MHC class I-beta2m heterodimers are poorly loaded with high-affinity peptides in the ER but nevertheless escape to the cell surface where they are unstable. These findings suggest a role for disulfide bond isomerization in tapasin-mediated peptide loading.     

Tapasin Decreases Immune Responsiveness to a Model Tumor Antigen

The T-cell response against cancer is dependent on the cell surface presentation of tumor-associated or tumor-specific peptides by major histocompatibility complex (MHC) class I molecules. We found that tapasin, a chaperone protein that normally assists in the assembly of MHC class I molecules, is undetectable in an unstimulated pancreatic tumor cell line, Panc02, and only very weakly expressed after -interferon stimulation. Transfection of tapasin into the Panc02 cells did not quantitatively increase MHC class I surface expression or detectably affect MHC class I association with peptide and ₂-microglubulin (₂m). However, we found that transfected tapasin downregulated immune reactivity against a model tumor antigen, MUC1. Although tapasin has been previously shown by others to increase immune recognition of particular antigens, our results suggest that tapasin has a negative impact on the presentation of an immunodominant epitope from a specific model tumor antigen.     

F pocket flexibility influences the tapasin dependence of two differentially disease‐associated MHC Class I proteins

The human MHC class I protein HLA‐B*27:05 is statistically associated with ankylosing spondylitis, unlike HLA‐B*27:09, which differs in a single amino acid in the F pocket of the peptide‐binding groove. To understand how this unique amino acid difference leads to a different behavior of the proteins in the cell, we have investigated the conformational stability of both proteins using a combination of in silico and experimental approaches. Here, we show that the binding site of B*27:05 is conformationally disordered in the absence of peptide due to a charge repulsion at the bottom of the F pocket. In agreement with this, B*27:05 requires the chaperone protein tapasin to a greater extent than the conformationally stable B*27:09 in order to remain structured and to bind peptide. Taken together, our data demonstrate a method to predict tapasin dependence and physiological behavior from the sequence and crystal structure of a particular class I allotype. Also watch the Video Abstract     

The murine gamma-herpesvirus-68 MK3 protein causes TAP degradation independent of MHC class I heavy chain degradation

The murine gamma-herpesvirus-68 MK3 protein has an intricate interaction with the peptide loading complex that involves MK3 stabilization, a rapid degradation of MHC class I heavy chains, and a slower degradation of TAP. Here we have used tapasin chimeras to distinguish functionally the different immune evasion mechanisms of MK3. Tapasin was cloned in two alternatively spliced forms that differed by a single transmembrane valine residue. Each restored antigen presentation and MK3 function in tapasin-deficient cells. The transmembrane/cytoplasmic portion of tapasin, linked to the extracellular domain of CD8, also restored TAP stability and MK3 stability in tapasin-deficient cells. MK3 did not associate with or degrade MHC class I in these cells, which lacked the endoplasmic reticulum domain of tapasin, but degraded TAP at least as efficiently as when full-length tapasin was present. The un-degraded MHC class I consequently showed impaired maturation. The fact that MK3 required intact tapasin to degrade MHC class I but only the transmembrane/cytoplasmic portion of tapasin to degrade TAP indicated that these two immune evasion functions operate independently.     

The cell biology of major histocompatibility complex class I assembly: towards a molecular understanding

Major histocompatibility complex class I (MHC I) proteins protect the host from intracellular pathogens and cellular abnormalities through the binding of peptide fragments derived primarily from intracellular proteins. These peptide-MHC complexes are displayed at the cell surface for inspection by cytotoxic T lymphocytes. Here we reveal how MHC I molecules achieve this feat in the face of numerous levels of quality control. Among these is the chaperone tapasin, which governs peptide selection in the endoplasmic reticulum as part of the peptide-loading complex, and we propose key amino acid interactions central to the peptide selection mechanism. We discuss how the aminopeptidase ERAAP fine-tunes the peptide repertoire available to assembling MHC I molecules, before focusing on the journey of MHC I molecules through the secretory pathway, where calreticulin provides additional regulation of MHC I expression. Lastly we discuss how these processes culminate to influence immune responses.     

Productive association between MHC class I and tapasin requires the tapasin transmembrane/cytosolic region and the tapasin C-terminal Ig-like domain

The current model of antigen assembly with major histocompatibility complex (MHC) class I molecules posits that interactions between the tapasin N-terminal immunoglobulin (Ig)-like domain and the MHC class I peptide-binding groove permit tapasin to regulate antigen selection. Much less is known regarding interactions that might involve the tapasin C-terminal Ig-like domain. Additionally, the tapasin transmembrane/cytoplasmic region enables tapasin to bridge the MHC class I molecule to the transporter associated with antigen processing (TAP). In this investigation, we made use of two tapasin mutants to determine the relative contribution of the tapasin C-terminal Ig-like domain and the tapasin transmembrane/cytoplasmic region to the assembly of MHC class I molecules. Deletion of a loop within the tapasin C-terminal Ig-like domain (Δ334-342) prevented tapasin association with the MHC class I molecule K(d). Although tapasin Δ334-342 did not increase the efficiency of K(d) folding, K(d) surface expression was enhanced on cells expressing this mutant relative to tapasin-deficient cells. In contrast to tapasin Δ334-342, a soluble tapasin mutant lacking the transmembrane/cytoplasmic region retained the ability to bind to K(d) molecules, but did not facilitate K(d) surface expression. Furthermore, when soluble tapasin and tapasin Δ334-342 were co-expressed, soluble tapasin had a dominant negative effect on the folding and surface expression of not only K(d), but also D(b) and K(b). In addition, our molecular modeling of the MHC class I-tapasin interface revealed novel potential interactions involving tapasin residues 334-342. Together, these findings demonstrate that the tapasin C-terminal and transmembrane/cytoplasmic regions are critical to tapasin's capacity to associate effectively with the MHC class I molecule.     

Selective loading of high-affinity peptides onto major histocompatibility complex class I molecules by the tapasin-ERp57 heterodimer

Major histocompatibility complex (MHC) class I glycoproteins bind peptides in the endoplasmic reticulum after incorporation into the peptide-loading complex, whose core is the transporter associated with antigen processing. Other components are the chaperone calreticulin, the thiol oxidoreductase ERp57, and tapasin. Tapasin and ERp57 have been shown to exist in the peptide-loading complex as a disulfide-linked heterodimer. Here, using a cell-free system, we demonstrate that although recombinant tapasin was ineffective in recruiting MHC class I molecules and facilitating peptide binding, recombinant tapasin-ERp57 conjugates accomplished both of those functions and also 'edited' the repertoire of bound peptides to maximize their affinity. Thus, the tapasin-ERp57 conjugate is the functional unit of the peptide-loading complex that generates MHC class I molecules with stably associated peptides.     

An extra molecule in addition to human tapasin is required for surface expression of beta2m linked HLA-B4402 on murine cell

Murine MHC class I can be readily expressed on the surface of human cell lines, but human class I molecules are expressed on mouse cells at a reduced level. Both human beta-2-microglobulin (beta(2)m) and tapasin (Tpn) have been demonstrated to be required for proper human MHC class I surface expression. Here we report that besides beta(2)m and tapasin, an extra unidentified component is also critical for the expression of certain human class I alleles. By covalently linking HLA-B4402 heavy chain to beta(2)m (beta(2)m-B44) a pre-assembled class I molecule has been created, which can be efficiently expressed and travel to the surface in human cells. In spite of being able to express inside cells, the linked beta(2)m-B44 molecule does not express on the surface of a murine fibroblast. Further investigation shows that lack of appearance on the surface is not due to quick degradation of unloaded class I, since provision of HLA-B4402 binding peptide could not rescue impaired surface expression. Co-expression with human tapasin does not rescue the defect excluding tapasin as the critical component for expression and indicating that a novel component of human origin is required for efficient surface expression of beta(2)m-B44 in murine cells. Surprisingly, not only did the beta(2)m-B44 construct fail to express on murine cells but also the surface expression of native murine MHC class I Kb was greatly reduced in transfected cells. It is likely that the expressed linked chain competitively associates with a component of class I processing in murine cells, reducing the exit rate of assembled mouse class I molecules. The results together suggest an unknown mechanism, which leads to the trapping of class I molecules in the ER.     

Identification of four new HLA-Cw alleles in the Sudanese population

The major histocompatibility complex (MHC) is the most polymorphic region of the human genome. Human leukocyte antigen-C (HLA-C) genes are located in the class I region of MHC. Most polymorphisms of HLA class I antigens are present in exons 2 and 3, which encode the alpha1 and alpha2 domains of the HLA-A heavy chain, involved in both peptide binding and HLA-restricted recognition by the T-cell receptor. Four new HLA-Cw alleles were identified in the Sudanese population during HLA class I and class II sequencing-based typing at the HLA-C locus of case-control study of Sudanese HIV patients, in individuals from different ethnic background. Based on the localization of the affected amino acid positions in an outer loop of the alpha-helix forming the side of the peptide-binding groove, we do not expect the replacement mutations to have an effect on peptide binding or T-cell receptor interaction.