The role of tapasin in MHC class I antigen assembly

The discovery of tapasin has shed new light on the mechanisms of major histocompatibility complex (MHC) class I assembly in the endoplasmic reticulum (ER). Tapasin appears to play an important role in the stable assembly of class I molecules with peptide, however, the precise function of tapasin remains elusive. The pursuit of tapasin function is complicated by the observation that tapasin is not required for successful antigen presentation by all class I molecules. In addition, current data suggest that the putative role of tapasin as a bridging molecule between transporter associated with antigen presentation (TAP) and class I is only of minor importance in tapasin action, and tapasin' s major role appears to be as an active cofactor in the assembly of class I. Furthermore, it is clear that class I molecules can follow multiple pathways for successful assembly in the ER. These pathways may or may not include the interaction of class I molecules with the accessory proteins tapasin, calreticulin, ERp57, or TAP. I would like to suggest that the particular pathway utilized by a given class I molecule depends more upon the availability of appropriate peptides rather than on an intrinsic property of the class I molecule, and that tapasin may serve a peptide editing function.     

The role of tapasin in MHC class I antigen assembly

The discovery of tapasin has shed new light on the mechanisms of major histocompatibility complex (MHC) class I assembly in the endoplasmic reticulum (ER). Tapasin appears to play an important role in the stable assembly of class I molecules with peptide, however, the precise function of tapasin remains elusive. The pursuit of tapasin function is complicated by the observation that tapasin is not required for successful antigen presentation by all class I molecules. In addition, current data suggest that the putative role of tapasin as a bridging molecule between transporter associated with antigen presentation (TAP) and class I is only of minor importance in tapasin action, and tapasin’s major role appears to be as an active cofactor in the assembly of class I. Furthermore, it is clear that class I molecules can follow multiple pathways for successful assembly in the ER. These pathways may or may not include the interaction of class I molecules with the accessory proteins tapasin, calreticulin, ERp57, or TAP. I would like to suggest that the particular pathway utilized by a given class I molecule depends more upon the availability of appropriate peptides rather than on an intrinsic property of the class I molecule, and that tapasin may serve a peptide editing function.     

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.     

Tapasin edits peptides on MHC class I molecules by accelerating peptide exchange

The endoplasmic reticulum (ER) protein tapasin is essential for the loading of high‐affinity peptides onto MHC class I molecules. It mediates peptide editing, i.e. the binding of peptides of successively higher affinity until class I molecules pass ER quality control and exit to the cell surface. The molecular mechanism of action of tapasin is unknown. We describe here the reconstitution of tapasin‐mediated peptide editing on class I molecules in the lumen of microsomal membranes. We find that in a competitive situation between high‐ and low‐affinity peptides, tapasin mediates the binding of the high‐affinity peptide to class I by accelerating the dissociation of the peptide from an unstable intermediate of the binding reaction.     

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.     

What is the role of alternate splicing in antigen presentation by major histocompatibility complex class I molecules?

The expression of major histocompatibility complex (MHC) class I molecules on the cell surface is critical for recognition by cytotoxic T lymphocytes (CTL). This recognition event leads to destruction of cells displaying MHC class I—viral peptide complexes or cells displaying MHC class I—mutant peptide complexes. Before they can be transported to the cell surface, MHC class I molecules must associate with their peptide ligand in the endoplasmic reticulum (ER) of the cell. Within the ER, numerous proteins assist in the appropriate assembly and folding of MHC class I molecules. These include the heterodimeric transporter associated with antigen processing (TAP1 and TAP2), the heterodimeric chaperone-oxidoreductase complex of tapasin and ERp57 and the general ER chaperones calreticulin and calnexin. Each of these accessory proteins has a well-defined role in antigen presentation by MHC class I molecules. However, alternate splice forms of MHC class I heavy chains, TAP and tapasin, have been reported suggesting additional complexity to the picture of antigen presentation. Here, we review the importance of these different accessory proteins and the progress in our understanding of alternate splicing in antigen presentation.     

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.     

Accessory proteins and the assembly of human class I MHC molecules: a molecular and structural perspective

The cell-surface presentation of antigenic peptides by class I major histocompatibility complex (MHC) molecules to CD8+ T-cell receptors is part of an immune surveillance mechanism aimed at detecting foreign antigens. This process is initiated in the endoplasmic reticulum (ER) with the folding and assembly of class I MHC molecules which are then transported to the cell surface via the secretory pathway. In recent years, several accessory proteins have been identified as key components of the class I maturation process in the ER. These proteins include the lectin chaperones calnexin (CNX) and calreticulin (CRT), the thiol-dependent oxidoreductase ERp57, the transporter associated with antigen processing (TAP), and the protein tapasin. This review presents the most recent advances made in characterizing the biochemical and structural properties of these proteins, and discusses how this knowledge advances our current understanding of the molecular events underlying the folding and assembly of human class I MHC molecules in the ER.     

Accessory molecules in the assembly of major histocompatibility complex class I/peptide complexes: how essential are they for CD8+ T‐cell immune responses?

Summary: Assembly of major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum is a highly coordinated process that results in abundant class I/peptide complexes at the cell surface for recognition by CD8⁺ T cells and natural killer cells. During the assembly process, a number of chaperones and accessory molecules, such as transporter associated with antigen processing, tapasin, ER60, and calreticulin, assist newly synthesized class I molecules to facilitate loading of antigenic peptides and to optimize the repertoire of surface class I/peptide complexes. This review focuses on the relative importance of these accessory molecules for CD8⁺ T‐cell responses in vivo and discusses reasons that may help explain why some CD8⁺ T‐cell responses develop normally in mice deficient in components of class I assembly, despite impaired antigen presentation.     

The convergent roles of tapasin and HLA-DM in antigen presentation

Cytotoxic and helper T cells respond to peptides derived from endogenous and exogenous sources that bind to major histocompatibility complex (MHC) class I and class II molecules and are presented on antigen-presenting cells. MHC class I and class II structures and maturation pathways have evolved to optimize antigen presentation to their respective T cells. The accessory proteins tapasin and HLA-DM (DM) crucially influence the selection of peptides that bind to the MHC molecules. We discuss here the dynamic interactions of tapasin and DM with their corresponding MHC molecules that indicate striking parallels. Utilization of a common mode of peptide selection by two different, but related, biological systems argue for its mechanistic validity.     

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.     

Impaired assembly yet normal trafficking of MHC class I molecules in Tapasin mutant mice

Loading of peptides onto major histocompatibility complex class I molecules involves a multifactorial complex that includes tapasin (TPN), a membrane protein that tethers empty class I glycoproteins to the transporter associated with antigen processing. To evaluate the in vivo role of TPN, we have generated Tpn mutant mice. In these animals, most class I molecules exit the endoplasmic reticulum (ER) in the absence of stably bound peptides. Consequently, mutant animals have defects in class I cell surface expression, antigen presentation, CD8+ T cell development, and immune responses. These findings reveal a critical role of TPN for ER retention of empty class I molecules. Tpn mutant animals should prove useful for studies on alternative antigen-processing pathways that involve post-ER peptide loading.     

Human cytomegalovirus inhibits tapasin-dependent peptide loading and optimization of the MHC class I peptide cargo for immune evasion

The immune evasion protein US3 of human cytomegalovirus binds to and arrests MHC class I molecules in the endoplasmic reticulum (ER). However, substantial amounts of class I molecules still escape US3-mediated ER retention, suggesting that not all class I alleles are affected equally by US3. Here, we identify tapasin inhibition as the mechanism of MHC retention by US3. US3 directly binds tapasin and inhibits tapasin-dependent peptide loading, thereby preventing the optimization of the peptide repertoire presented by class I molecules. Due to the allelic specificity of tapasin toward class I molecules, US3 affects only class I alleles that are dependent on tapasin for peptide loading and surface expression. Accordingly, tapasin-independent class I alleles selectively escape to the cell surface.     

Increased efficiency of folding and peptide loading of mutant MHC class I molecules

Class I molecules, encoded by diverse alleles at several loci of the major histocompatibility complex (MHC) are assembled in the endoplasmic reticulum (ER) from heavy chain, beta2 microglobulin and peptide in association with accessory proteins of the peptide loading complex. We show here, that mutations in the alpha2 domain (Q115A; D122A) of the human class I allele HLA-A2 cause a lack of apparent association with the loading complex and a faster assembly. Despite the drastically reduced association with the TAP loading complex, i. e. less than 20 % of HLA-A2 expressed in the cells can be co-precipitated with either TAP, calreticulin or tapasin, the mutant proteins are expressed on the cell surface in a stable conformation, and bind a complex set of peptides almost identical to that of wild-type HLA-A2. Furthermore, the mutant class I molecules are more rapidly exported from the ER than wild-type HLA-A2 and undergo faster maturation. The mutation Q115A does not destroy a binding site for the loading complex as this HLA-A2 mutant associates with the loading complex when peptide supply is limited. The association of class I molecules with the TAP-associated loading complex appears to be a reflection of how quickly the stable conformation is gained.     

MHC class I assembly: out and about

The assembly of major histocompatibility complex (MHC) class I molecules with peptides is orchestrated by several assembly factors including the transporter associated with antigen processing (TAP) and tapasin, the endoplasmic reticulum (ER) oxido-reductases ERp57 and protein disulfide isomerase (PDI), the lectin chaperones calnexin and calreticulin, and the ER aminopeptidase (ERAAP). Typically, MHC class I molecules present endogenous antigens to cytotoxic T lymphocytes (CTLs). However, the initiation of CD8(+) T-cell responses against many pathogens and tumors also requires the presentation of exogenous antigens by MHC class I molecules. We discuss recent developments relating to interactions and mechanisms of function of the various assembly factors and pathways by which exogenous antigens access MHC class I molecules.     

Assembly of MHC class I molecules within the endoplasmic reticulum

MHC class I molecules bind cytosolically derived peptides within the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T cells. A major focus of our laboratory has been to understand the functions of the diverse proteins involved in the intracellular assembly of MHC class I molecules. These include the molecular chaperones calnexin and calreticulin, which enhance the proper folding and subunit assembly of class I molecules and also retain assembly intermediates within the ER; ERp57, a thiol oxidoreductase that promotes heavy chain disulfide formation and proper assembly of the peptide loading complex; tapasin, which recruits class I molecules to the TAP peptide transporter and enhances the loading of high affinity peptide ligands; and Bap31, which is involved in clustering assembled class I molecules at ER exit sites for export along the secretory pathway. This review describes our contributions to elucidating the functions of these proteins; the combined effort of many dedicated students and postdoctoral fellows.     

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.     

Antigen degradation or presentation by MHC class I molecules via classical and non-classical pathways

Major histocompatibility complex (MHC) class I molecules usually present endogenous peptides at the cell surface. This is the result of a cascade of events involving various dedicated proteins like the peptide transporter associated with antigen processing (TAP) and the ER chaperone tapasin. However, alternative ways for class I peptide loading exist which may be highly relevant in a process called cross-priming. Both pathways are described here in detail. One major difference between these pathways is that the proteases involved in the generation of peptides are different. How proteases and peptidases influence peptide generation and degradation will be discussed. These processes determine the amount of peptides available for TAP translocation and class I binding and ultimately the immune response.