Molecular machinations of the MHC-I peptide loading complex

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Molecular machinations of the MHC-I peptide loading complex Anthony W Purcell1 and Tim Elliott2 The acquisition of an optimal peptide ligand by MHC class I molecules is crucial for the generation of immunity to viruses and tumors. This process is orchestrated by a molecular machine known as the peptide loading complex (PLC) that consists of specialized and general ER-resident molecules. These proteins collaborate to ensure the loading of an optimal peptide ligand into the antigen binding cleft of class I molecules. The surprising diversity of peptides bound to MHC class I molecules and recapitulation of class I assembly in vitro have provided new insights into the molecular machinations of peptide loading. Coupled with the extraordinary polymorphism of class I molecules and their differential dependence on various components of the PLC for cell surface expression, a picture of peptide loading at the molecular level has recently emerged and will be discussed herein. Addresses 1 Department of Biochemistry and Molecular Biology, Bio21 Institute for Molecular Science and Biotechnology, University of Melbourne, Parkville, Victoria 3010, Australia 2 CRUK Clinical Centre, Cancer Sciences Division, School of Medicine, University of Southampton, MP824, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK Corresponding author: Elliott, Tim ([email protected])

Current Opinion in Immunology 2008, 20:75–81 This review comes from a themed issue on Antigen Processing and Recognition Edited by Emil Unanue and James McCluskey

0952-7915/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2007.12.005

Introduction The recognition of class I MHC molecules by CD8+ T cells is the pivotal event in the detection of infected or malignant cells. CD8+ T cells keep a constant vigil for signs of infection and cancer by surveying the array of peptides presented in complex with class I MHC molecules on the surface of all nucleated cells. The presence of viral or tumor-specific peptide antigens in these complexes acts as a signal for the destruction of the abnormal or infected cells allowing the prevention of tumors and eradication of infected cells. Following translocation of peptides from the cytoplasm to the lumen of the endoplasmic reticulum (ER), a highly orchestrated process involving the interplay of www.sciencedirect.com

multiple ER-resident chaperones and accessory molecules facilitates HLA class I loading (Figure 1). Initially, nascent HLA class I heavy chain (hc) is targeted to the ER and is stabilized by interacting with the chaperones Grp78 and calnexin. These chaperones are involved in the insertion of the class I polypeptide into the lumenal compartment of the ER and stabilization of the translocated hc. Once b2-microglobulin (b2m) associates with the class I hc, rebinding of hc to calnexin is disfavored in preference to binding of the ortholog chaperone, calreticulin [1]. This is the result of conformational changes associated with heterodimer formation and de-glucosylation of the mono-glucosylated N-linked glycan attached to Asparagine 86 of the HLA class I hc [2,3]. The association of the class I heterodimer with calreticulin is also associated with the recruitment of tapasin and ERp57 into the loading complex (see Figure 1). ERp57 is a thiol oxidoreductase [4] involved in assuring correct disulfide bonding of the nascent class I hc [5]. It is well understood that ERp57 and calreticulin collaborate to ensure correct folding and maturation of a number of ER-resident glycoproteins [6]. The prolonged association of ERp57 with both the nascent class I hc and the loading complex is consistent with the notion that this chaperone may also be involved in facilitating conformational breathing of the class I binding cleft to assist in peptide binding. Tapasin is a 48kDa glycoprotein that was first noted to bridge peptide receptive class I heterodimers to the transporter associated with antigen processing (TAP) heterodimer. Colocalization of these complexes to the TAP facilitates the loading of peptides into the antigen binding cleft of the class I molecules. In addition to a bridging function, tapasin is thought to stabilize the peptide receptive state of the class I complex independently of TAPassociation [7], enhance expression of TAP [7,8] and increase peptide binding to the TAP heterodimer [9]. It also prevents premature release of class I molecules from the ER of mammalian cells [10]. Of the accessory molecules and chaperones involved in class I assembly, only TAP and tapasin are uniquely involved in the class I assembly pathway. Recent studies have shown the importance of covalent linkages between tapasin and ERp57. Unlike many other associated chaperones, tapasin forms a relatively stable disulfide linkage with ERp57 trapping a large proportion of cellular ERp57 in the PLC, and there is now evidence that in vivo, the functional unit responsible for peptide editing is the covalent ERp57–tapasin heterodimer [11,12]. Current Opinion in Immunology 2008, 20:75–81

76 Antigen Processing and Recognition

Figure 1

The class I assembly pathway involves a network of ER-resident chaperones: Nascent class I hc is co-translationally inserted into the ER via the signal recognition pore (SRP) and initially stabilized by interacting with Grp78. During insertion the signal sequence peptide is cleaved and may be available for class I binding under special circumstances. In human class I molecules a single amino acid residue (Asn86) bears a N-linked glycan. This carbohydrate is recognized by the lectin-like chaperone calnexin. Upon further folding, disulfide bond formation and association with b2m, the class I heterodimer (hc-b2m) is bound by the chaperone calreticulin. At this stage the oxidoreductase ERp57 and tapasin are recruited to the loading complex. A relatively stable disulfide bond forms between tapasin and ERp57, forming the core functional unit of the PLC. Tapasin co-localizes this peptide receptive loading complex to the TAP heterodimer, where peptides generated in the cytoplasm by the proteasome are translocated into the lumen of the ER. Following peptide loading, the class I molecules dissociate from the TAP and associated chaperones and are transported to the cell surface where they are scrutinized by CTL.

Class I polymorphism and interaction with the class I assembly complex reveals structural and functional relationships

Studies in mutant antigen presenting cells (APC) have revealed the crucial role of components of the PLC in peptide loading and class I maturation. For class I molecules, the ability to continue stable surface expression in cells deficient in various components of the PLC, resides in the intrinsic properties of the class I allele and also in the nature and abundance of appropriate ligands that remain available for loading in these mutant APCs. TAP-independent loading pathways of class I

The mutant cell line T2 was derived from the X-irradiation mutant 721.174, which lacks genes for class II, LMP, and TAP following fusion with the CEM.T1 T-LCL [13]. Despite lacking the TAP gene, loading complexes consisting of class I heterodimer, calreticulin, and tapasin can be detected in lysates of 721.174 and T2 cells [14]. T2 retains the haplotype of 721.174 (i.e. A2+, B5+, Cw1+, DR , DQ , DP ) but no HLA B or HLA C expression can be detected on the cell surface. HLA A2 expression reaches between 20–50% of wild-type levels. This surface expression, in the absence of TAP-mediated translocation of peptide antigen, has been attributed to the binding of signal sequence derived peptides (see Figure 1) that enter the ER via a distinct translocon, the signal recognition pore (SRP) [15,16]. Thus, allelic variation in the ligand specificity of class I molecules can result in acquisition of Current Opinion in Immunology 2008, 20:75–81

peptide ligands via alternative pathways, such as the SRPmediated translocation of HLA A2-restricted ligands. This pathway can also be utilized by other HLA molecules by introducing signal sequences in minigene constructs encoding allele-specific ligands [17–20]. ERtargeted ligands can result in stable expression of introduced class I gene products and presentation of peptide determinants on the surface of TAP-deficient APCs. Tapasin-independent loading of class I HLA molecules

Loading complexes fail to assemble in tapasin-deficient cells, reflecting the crucial nature of this specialized chaperone in class I antigen presentation. However, the stable surface expression of a number of naturally occurring class I alleles in tapasin-deficient cells has raised the possibility that some alleles have evolved additional or alternative peptide acquisition pathways that operate independently of loading complex formation [21,22,23,24]. Low levels of HLA A2 are observed on the surface of tapasin-deficient APC, and although tapasin is required to bridge HLA A2 to the TAP this molecule still acquires TAP-translocated peptides as well as signal sequence-derived peptides before egress to the cell surface [25]. Moreover, HLA B27 has been shown to exhibit a ‘tapasin-independent’ phenotype relative to other HLA A and B alleles. Despite displaying discrete differences in the kinetics of antigen presentation [21] and surface stability, HLA B27 is expressed at similar steady-state levels on wild type and tapasin-deficient www.sciencedirect.com

Molecular machinations of the MHC-I peptide loading complex Purcell and Elliott 77

APCs [21,24]. The reduced surface stability of these complexes was also revealed by biochemical analysis of the class I associated peptides [26], demonstrating an overall reduction in peptide recovery from purified B27 complexes isolated from the surface of tapasin-deficient cells. The identification of peptides that are removed from the repertoire in cells with functional tapasin reflects the qualitative influence of this chaperone on the peptide repertoire and suggest that some degree of editing occurs during peptide loading. A more dramatic demonstration of natural HLA polymorphism impacting on tapasin dependence is observed in the HLA B44 family of alleles, whereby single amino acid polymorphism at position 116 of the HLA hc dictates dependence on tapasin for cell surface expression [22,23]. An investigation into the assembly and peptide loading of these alleles showed that whereas HLAB*4402 was unable to load with stabilizing peptides in the absence of tapasin, HLA-B*4405 was relatively successful and as a consequence was expressed at the cell surface. In the presence of tapasin, HLA-B*4402 loaded rapidly and extensively with high affinity peptides. For HLAB*4405, the ‘tapasin effect’ was far less pronounced, leading to the conclusion that this allele was less dependent upon tapasin for its loading [17], consistent with the observation that it binds less efficiently to the PLC [18]. As a consequence, in the presence of tapasin, HLAB*4402 was more efficiently loaded with peptides than HLA-B*4405 [17]. Similarly, a single amino acid difference at position 114 of hc of mutant HLA B44 molecules [19] and polymorphism at positions 152, 114, and 116 in HLA B27 allotypes [24,27] can control tapasin dependence for cell surface expression. Deficiencies in the more generic components of the PLC have also resulted in decreased cell surface expression of class I molecules. For example, murine H2b molecules expressed on the surface of APC deficient in calreticulin fail to contain optimized peptide ligands [28]. Potential editorial roles of components of the peptide loading complex

Quality control in the ER is essential for the correct folding and export of proteins destined for the secretory pathway or transport to the cell surface. Class I molecules utilize generic components of the quality control network as well as specialized chaperones unique to class I assembly. Like many other multi-subunit proteins, class I molecules are retained in the ER until assembly into its ternary structure is completed. Several investigators have proposed that ligand exchange or ligand editing occurs during class I assembly [14,26,29–31,32], although this has been refuted in other studies suggesting tapasin acts merely as a facilitator of peptide binding [33]. Recent studies clearly show that the immunological function of tapasin is to edit the cargo of peptides that is bound to class I molecules in favor of those that have longer halfwww.sciencedirect.com

lives when expressed at the cell surface [34,35]. Furthermore, the isolation and characterization of tapasin-independent ligands of HLA B27 and their removal from the peptide repertoire in tapasin-positive cells provide strong evidence that ligand editing is mediated via loading complex formation [26,32]. Mechanism of peptide loading/editing

Under normal circumstances peptide receptive MHC class I heterodimers acquire antigenic peptide primarily when bridged to the TAP [14,36]. Some alleles like HLA B*4405, however, appear not to use the PLC or to use it transiently when acquiring peptide ligands [23]. This allows such alleles to rapidly acquire peptide ligands in the face of viral inhibition or tumor escape mutations. As a result the cohort of class I complexes that egress to the cell surface are relatively unstable owing to the lack of peptide optimization while within the PLC. It is likely that such complexes will contain a high proportion of suboptimal ligands with the relative stability of the complexes dependent on the particular class I alleles and the lumenal availability of high affinity peptide ligands. Physical association with the TAP may allow a degree of peptide exchange with more optimal peptides; however, under some circumstances this increased time may also facilitate the acquisition of low abundance or shorter half-life peptides as well. Furthermore, class I molecules can associate stably with the TAP for periods of up to several minutes [37] and are associated with tapasin before dissociating from the TAP complex [38]. These studies also revealed that once dissociated from the TAP complex, peptide-loaded molecules were not immediately transported to the cell surface, suggesting that a window of opportunity for additional editing of bound peptides may exist after TAP-mediated loading. How might such a peptide editing process proceed and which molecules are important in the process? The co-translational insertion and initial stabilization of class I hc proceeds via generic pathways common to all glycoproteins. The chaperone calnexin associates with class I hc via a lectin-like specificity for monoglucosylated N-linked glycans. Further action of glucosidases removes this terminal glucose moiety and is associated with conformational changes involving the formation of disulfide bonds and heterodimer formation. Calnexin subsequently relinquishes its substrate to calreticulin. It is unlikely that calnexin influences ligand selection by the class I molecules it chaperones, although it has been shown to be intimately involved in the degradation of abnormally folded or accumulated class I hc [39]. Calreticulin co-associates with other components of the loading complex; class I heterodimers, ERp57, and tapasin. Calreticulin has both a lectin-like specificity and a protein determinant [2], yet is involved in refolding of most glycoproteins in the ER. Studies of peptide editing in Current Opinion in Immunology 2008, 20:75–81

78 Antigen Processing and Recognition

Figure 2

Molecular model of PLC contacts on HLA B*4405. Three-dimensional model of the putative binding sites for components of the class I loading complex. The class I hc and b2m are shown as ribbon traces with regions known to interact with tapasin and other PLC components highlighted in red. The peptide ligand EEFGRAFSF is shown occupying the antigen binding cleft as a stick model (green). The structure is based on the published coordinates of HLA B*4405 [46] and visualized by the Swiss PDB viewer (http://www.expasy.ch/spdbv/ [47]). (a) The view of the complex from above is shown. Key F pocket amino acid side chains are shown for residues Tyr 116 and Asp 114 in yellow. (b) Shows a side view of the complex highlighting regions 125–136 of the a2 domain and 219–233 of the a3 domain thought to interact with PLC components. (c and d) Show the electrostatic potential of HLA B*4402 and B*4405 F pockets, highlighting the pronounced electronegativity of the B*4402 pocket. Reproduced with permission from reference [23].

cells that lack calreticulin have shown that calreticulin exerts no direct influence on the peptide repertoire, other than to ensure the bound peptides elicit proper conformational assembly of the class I molecule [35]. ERp57 associates with both partially and fully oxidized hc, suggesting that this molecule is directly responsible for class I hc disulfide bond formation [5]. The prolonged association of ERp57 with the peptide loading complex, which contains predominantly correctly disulfide bonded class I hc, suggests that additional modification of disulfide bonds is required during peptide loading [5,14]. Two conserved disulfide bond pairings occur in class I molecules. Isomerism of the Cys101-Cys164 disulfide pairing has been postulated to be involved during peptide Current Opinion in Immunology 2008, 20:75–81

loading. This cysteine pairing connects the floor of the peptide binding groove (Cys101) with the a2 helical region (Cys164). The reduction of this disulfide bridge could result in a hinge-like movement within the antigen binding cleft, facilitating the binding or exchange of peptide ligands. If ERp57 regulates the formation of this bond via its oxidoreductase activity; this may facilitate peptide binding or peptide exchange. As discussed earlier, tapasin has a number of distinct functions that predicate a role for this chaperone in peptide editing or exchange. Biochemical studies of the ligands associated with HLA B27 in tapasin-deficient cells have revealed both quantitative and qualitative www.sciencedirect.com

Molecular machinations of the MHC-I peptide loading complex Purcell and Elliott 79

influences of tapasin on the class I peptide repertoire [26,32]. The qualitative influences of tapasin included the removal of some species from the peptide repertoire, which could be defined as a form of ligand editing. Whether this is a direct influence of tapasin on the bound peptides or whether this effect is mediated via the generic quality control mechanisms of the other chaperones associated with the class I loading complex is unclear. The argument for a direct role of tapasin in ligand editing centers around the putative tapasin binding site on class I MHC molecules that includes amino acid residues 128– 136 [34,40,41]. This region of the class I molecule forms a loop in the a2 domain (Figure 2) and conceivably by interacting with this region tapasin could influence peptide binding by modulating cleft conformation [42,43]. It is probably significant that amino acid positions 114 and 116 that control both the ability of some alleles to optimize their peptide cargo spontaneously, and the difference in sensitivity to tapasin between different MHC I alleles lie in the vicinity of the peptide binding groove that accommodates the C-terminal side-chain and carboxy-terminus of bound peptides [22,42,44]. This part of the peptide binding groove immediately opposes the tapasin binding site of the MHC I molecule (Figure 2a and b), and observations have been made that have led to the suggestion that a molecular relay might exist that communicates, via a conformational change, peptide binding on the ‘inside’ of the peptide binding groove to the ‘outside’ of the MHC I molecule that interacts with cofactors such as tapasin. One molecular model of peptide editing is therefore that tapasin stabilizes a more ‘open’ conformation of MHC I, enhancing the off-rate of bound peptides to this conformation. In combination with a peptide-induced conformational change that disrupts the interaction between MHC I and tapasin, this would lead to a two-stage filter for peptides bound to MHC I: the first involving tapasin and the second distal to tapasin during which class I molecules that lose their peptide cargo before their export from the ER are retained or returned to the PLC for further loading. Other cofactors such as calreticulin could be involved in this step [28,45], which could also involve the reduction and reoxidation of the intramolecular disulfide bond in the peptide binding domain of MHC I [12]. How some alleles manage to load peptide in the apparent absence of PLC formation has been explained on the basis of comparative structural analysis of HLA B44 family members, whereby the hydrophobicity of the antigen binding cleft dictates the propensity of the cleft to remain in the open conformation with more hydrophobic clefts requiring more immediate satiation with a peptide ligand [23]. Thus, the nature of the antigen binding cleft appears to ultimately dictate the chaperone dependence of MHC I molecules, and remarkably single amino acid polymorphisms can alter the biophysical nature of the cleft and the kinetic requirements for ligation with antigenic peptides. Through a process of natural selection MHC allomorphs have arisen www.sciencedirect.com

with cleft polymorphism that not only subtly alters peptide ligand repertoire but also dramatically alters the cellular trafficking and chaperone requirements of these molecules, conferring upon the host advantages such as countering viral or tumor immune evasion mechanisms that target PLC components.

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