The peptide-loading complex and ligand selection during the assembly of HLA class I molecules

Molecular Immunology 37 (2000) 483 – 492 www.elsevier.com/locate/molimm

Review

The peptide-loading complex and ligand selection during the assembly of HLA class I molecules Anthony W. Purcell * The Department of Microbiology and Immunology, Uni6ersity of Melbourne, Park6ille 3052, Victoria, Australia

Abstract The identification and characterisation of the class I peptide loading complex has resulted in an appreciation of the co-ordinated and multifaceted nature of HLA class I assembly in the lumen of the endoplasmic reticulum. This loading complex consists of the assembling class I heterodimer in association with a number of molecular chaperones. These chaperones can be classified as generic to the folding of most glycoproteins in the endoplasmic reticulum or specific to the class I loading pathway. The functions of the various components of the loading complex in class I molecule assembly are reviewed. A critical component of the class I loading complex is the specialised chaperone tapasin. The role of tapasin in the stabilisation and retention of empty or suboptimally loaded class I molecules and the facilitation of the loading of these molecules with more appropriate ligands is discussed. As such, it is proposed that tapasin is a major determinant of peptide repertoire selection for class I-restricted presentation in normal antigen presenting cells. The potential implications in vaccine design and autoimmunity are discussed. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Chaperone; HLA class I assembly; Ligand selection; Tapasin; Binding motifs

Contents 1. Class I assembly and peptide loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2. Factors that influence class I ligand selection . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3. Class I polymorphism and interaction with the class I assembly complex functional relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. TAP-independent loading pathways of class I . . . . . . . . . . . . . 3.2. Tapasin-independent loading of class I HLA molecules . . . . . . . . 4. Potential editorial roles of components of the peptide-loading complex .

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reveals structural and . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. Defining the sites of interaction between class I molecules and the loading complex . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbre6iations: APC, antigen presenting cell; b2m, b2-microglobulin; CTL, cytotoxic T lymphocyte; ER, endoplasmic reticulum; hc, heavy chain; HLA, human leukocyte antigen; LCL, lymphoblastoid cell line; MHC, major histocompatibility complex; RP-HPL, reversed phase-high performance liquid chromatography; SRP, signal recognition pore; TAP, transporter associated with antigen presentation. * Tel.: +61-3-93449911; fax: +61-3-93471540. E-mail address: [email protected] (A.W. Purcell). 0161-5890/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0161-5890(00)00075-4

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1. Class I assembly and peptide loading The pathway by which class I molecules acquire antigenic peptide ligands does not rely on the relatively inefficient process of simple mass transfer. Following translocation of peptides from the cytoplasm to the lumen of the endoplasmic reticulum (ER), a highly complex process involving the interplay of multiple ER-resident chaperones (Cresswell et al., 1999) facilitates class I loading. Initially, nascent HLA class I heavy chain (hc) is targeted to the ER and is stabilised 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 stabilisation of the newly synthesised, translocated hc polypeptide. As such, complexes of b2-microglobulin (b2m)-free hc can be co-precipitated with both the Grp78 and calnexin chaperones (Degen et al., 1992; Jackson et al., 1994; Nossner and Parham, 1995; Ortmann et al., 1994). Once b2m associates with the class I hc, calnexin is exchanged for another ER-resident chaperone, calreticulin. 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 (Margolese et al., 1993; Vassilakos et al., 1996; Harris et al., 1998). The association of the class I heterodimer with calreticulin is also associated with the recruitment of tapasin and ERp57 into the loading complex (see Fig. 1). The precise contribution of each of these chaperones

to the stabilisation and folding of the class I heterodimer remains to be fully elucidated. ERp57 is a thiol oxidoreductase (Hughes and Cresswell, 1998; Lindquist et al., 1998) involved in assuring correct disulfide bonding of the nascent class I hc (Farmery et al., 2000). The prolonged association of ERp57 with both the nascent class I hc and the loading complex (Farmery et al., 2000) 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 (Cresswell et al., 1999). Tapasin is a 48-kDa glycoprotein that was first noted to bridge peptide receptive class I heterodimers to the transporter associated with antigen processing (TAP) heterodimer (Sadasivan et al., 1996; Grandea et al., 1997; Pamer and Cresswell, 1998). Co-localisation 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 stabilise the peptide receptive state of the class I complex independently of TAP association (Lehner et al., 1998), enhance expression of TAP (Lehner et al., 1998; Bangia et al., 1999) and increase peptide binding to the TAP heterodimer (Li et al., 2000). It also retains empty major histocompatibility complex (MHC) class I molecules in the ER of insect cells (Schoenhals et al., 1999) and prevents premature release of class I molecules from the ER of mammalian cells (Barnden et al., 2000). Of the accessory molecules and chaperones involved in class I assembly, only TAP and tapasin are uniquely involved in the class I assembly pathway.

Fig. 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 SRP and initially stabilised 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 recognised by the lectin-like chaperone calnexin. Upon further folding, disulphide 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. Tapasin co-localises 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 scrutinised by CTL.

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Studies in mutant antigen presenting cells (APC) have revealed the crucial role both these molecules play in peptide loading and class I maturation. Variation in the dependence of different HLA alleles on these chaperones for efficient class I assembly and peptide loading has been used to study the influence of these molecules on the composition of the peptide repertoire.

2. Factors that influence class I ligand selection HLA molecules are membrane-bound chaperones of antigenic polypeptides involved in the presentation of antigen to T cells at the immunological synapse (Grakoui et al., 1999). The ligand specificity of the different HLA class I alleles is the result of the interaction of the antigenic oligopeptide (usually eight to 11 amino acids in length) with the binding cleft of the class I molecule (Rammensee, 1995). X-Ray crystallographic studies have revealed the remarkable complementarity at the interface of the floor and ridges of the class I binding cleft and amino acid side chains of the peptide ligand (Maenaka and Jones, 1999). In combination with biochemical analysis of peptides eluted from mature class I molecules (Rammensee et al., 1995), an appreciation of allelic polymorphism and its influence on ligand specificity has arisen. Listings of binding motifs for the common HLA class I allotypes (Rammensee et al., 1995) are now conveniently web based (see, for example, MHCPEP Žhttp://wehih.wehi.edu.au/mhcpep/; SYFPEITHI Žhttp://134.2.96.221/). These binding motifs describe the amino acids located at critical positions along the sequence of the antigenic peptide that are responsible for making highly conserved and energetically important contacts with depressions or pockets in the binding cleft of the class I molecules. Rather than providing a panacea for vaccine design, the use of these binding motifs to predict candidate T-cell determinants from the sequence of pathogenic microorganisms has been somewhat disappointing. This approach is successful in de novo prediction of T-cell epitopes in between 50 and 70% of cases but there are numerous examples of atypical ligands possessing non-motif based sequences, post-translationally modified ligands or of the failure of antigen processing to liberate the candidate oligopeptides (Eisenlohr et al., 1992; Chen et al., 1994b; Haurum et al., 1994; Kohler et al., 1995; Martin et al., 1995; Moulon et al., 1995; Chen et al., 1996; Skipper et al., 1996; Purcell et al., 1998; Andersen et al., 1999). A further consideration in vaccine design that relates more to generation of effector responses, rather than ligand selection per se, is the observation that many cytotoxic T lymphocyte (CTL) responses are focussed on one or two immunodominant peptides during infection (Yewdell and Bennink, 1999). This focused CTL response has led to an appreciation of the complex

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relationships that exist between antigen presentation and the generation of CTL responses (Chen et al., 1994a; Niedermann et al., 1995, 1996; Blum et al., 1997; Deng et al., 1997; Pamer et al., 1997; Brooks et al., 1998; Gallimore et al., 1998; Yewdell and Bennink, 1999; Chen et al., 2000). Moreover, the participation of so few epitopes from the viral genome hamper predictive studies since markers of immunogenicity must take into account not just peptide binding, but the abundance, time of expression, correct processing and transport of the epitope as well as the available CTL repertoire.

3. Class I polymorphism and interaction with the class I assembly complex reveals structural and functional relationships The 721 B-lymphoblastiod cell line (LCL) and its derivatives were derived by X-irradiation and selected based on reduced expression of HLA molecules (DeMars et al., 1984). The use of these mutant APCs to study antigen processing and presentation has greatly progressed the field. For class I molecules, the ability to continue stable surface expression in cells deficient in various components of the class I loading and assembly pathway 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.

3.1. 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 (Salter et al., 1985). 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 (Cresswell et al., 1999). 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 and 50% (Salter et al., 1985) 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 Fig. 1) that enter the ER via a distinct translocon, the signal recognition pore (SRP) (Henderson et al., 1992; Wei, 1992). Thus, allelic variation in the ligand specificity of class I molecules can result in acquisition of peptide ligands via alternative pathways, such as the SRP-mediated translocation of HLA A2-restricted ligands. This pathway can also be utilised by other HLA molecules by introducing signal sequences in minigene constructs encoding allele-spe-

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Fig. 2. Reduced recovery of peptide ligands associated with HLA B27 molecules expressed on the surface of tapasin-deficient APCs. Matrix assisted laser desorption/ionisation-time of flight mass spectrometry was performed using a Bruker Reflex mass spectrometer (Bruker-Franzen Analytik, GMBH, Bremen, Germany) as described elsewhere (Lopaticki et al., 1998). Aliquots of fractions (1 – 2 ml) were mixed with an equal volume of a-cyano-4-hydroxycinnamic acid (10 mg/ml in acetonitrile-ethanol 1:1 v/v), spotted onto a target and dried for analysis. For comparison of HPLC fractions derived from tapasin-deficient (spectra of negative polarity) and wild-type (spectra of positive polarity) APCs, identical laser irradiance and repetitions were used to ionise each sample. Care was taken to ensure uniform matrix/sample crystals were deposited onto the target, ensuring minimal variation in sample ionisation between different regions on a given target position and between replicate samples. Post source decay experiments were performed to sequence individual peptide species (Purcell and Gorman, 2000) using 14 stepwise decrements in the reflectron potential and increasing the laser irradiance to optimise the production of fragment ions. Assembly of the individual spectra for each reflectron voltage on to a continuous mass scale was performed using FAST software routines within the BRUKER XTOF software package. Identification of fragmented ion species was determined by manually assigning C- and N-terminal ion series and comparing parent m/z and fragmentation data to databases entries using mass spectrometry-FIT routines available through the protein prospector program Žhttp://prospector.ucsf.edu.

cific ligands (Khanna et al., 1994; Fu et al., 1998). Thus, ER-targeted ligands can result in stable expression of introduced class I gene products and presentation of peptide determinants on the surface of TAP-deficient APCs.

3.2. Tapasin-independent loading of class I HLA molecules Loading complexes fail to assemble in tapasin-deficient cells, reflecting the critical nature of this specialised chaperone in class I antigen presentation (Cresswell et al., 1999). The stable surface expression of some naturally occurring class I alleles in tapasin-deficient cells has raised the possibility that some alleles, like HLA A2 in TAP-deficient cells, have evolved additional or alternative peptide acquisition pathways that operate independently of loading complex formation (Peh et al., 1998; Purcell et al., 2000b). HLA B27, in particular, has been shown to exhibit a ‘tapasin-independent’ phenotype relative to other HLA A and B alleles (Sadasivan et al., 1996; Grandea et al., 1997; Ortmann et al., 1997; Lewis et al., 1998; Peh et al., 1998). Despite displaying discrete differences in the

kinetics of antigen presentation (Peh et al., 1998) and surface stability, HLA B27 is expressed at similar steady-state levels on the surface of wild-type and tapasin-deficient APCs (Peh et al., 1998; Purcell et al., 2000b). The reduced surface stability of these complexes was also revealed by biochemical analysis of the peptides associated with these molecules (Purcell et al., 2000a), which demonstrated an overall reduction in peptide recovery from purified B27 complexes isolated from the surface of the tapasin-deficient cells. Fig. 2 demonstrates the reduction in specific peptide ligands contained in matched reversed phase-high performance liquid chromatography (RP-HPLC) fractions derived from B27 expressed on tapasin-deficient (spectra of negative polarity) and wild-type APCs (spectra of positive polarity). This fraction was typical of the 45 fractions examined (Purcell et al., 2000a). The dominant peptide observed in the mass spectrum (1146.6 Da) of this fraction was subsequently sequenced using a sensitive and novel mass spectrometric technique (Purcell and Gorman, 2000; Purcell et al., 2000a) and determined to be derived from the 40S ribosomal protein S6 (amino acids 139–147). This peptide (SRIRKLFNL) was recovered 2.7-fold less efficiently from the tapasin-

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deficient peptide eluate. The second abundant species that was isolated, was not sequenced, and was recovered 1.7-fold less efficiently from the tapasin-deficient peptide eluate relative to the tapasin positive eluate. This decrease in the yield of specific peptides reflects the quantitative role tapasin plays in facilitating the loading of high-affinity peptide ligands into the binding cleft of class I molecules. Why is a reduction in peptide recovery observed despite equivalent levels of class I expressed on the surface of tapasin-positive and tapasin-negative cell lines? Immunoaffinity chromatography yielded similar amounts of protein consistent with surface expression levels. However, the B27 molecules expressed on the surface of 721.220 cells are less stable by several criteria (Peh et al., 1998; Purcell et al., 2000b), which suggests that molecules expressed on the tapasin-deficient cell line are occupied by a proportion of suboptimal ligands. These ligands are able to partially stabilise B27 molecules on the surface of these cells and contribute to the similar levels of steady-state surface expression on tapasin-deficient cells; however, these ligands are lost during the isolation of the B27 complexes (Purcell et al., 2000a). Importantly, some species contained within different RP-HPLC fractions were recovered up to fivefold more abundantly in the tapasin-deficient eluate, suggesting that this was not an absolute reduction in the recovery of peptides from the tapasin-deficient APC (Purcell et al., 2000a). The identification of species that are removed from the repertoire in cells with functional tapasin reflect the qualitative influence of this chaperone on the peptide repertoire and suggest that some degree of editing may occur during peptide loading. It should be noted, however, that TAP-translocated peptides remain the primary source of the peptides selected by HLA B*2705 under these conditions. TAP-Dependent peptides are also captured and presented by HLA A2 on the surface of tapasin-deficient APC, suggesting that, in tapasindeficient cells, A2 can still acquire TAP-translocated peptides as well as signal sequence-derived peptides prior to egress to the cell surface (Lewis et al., 1998).

4. 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 utilise generic components of the quality control network as well as specialised 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, i.e. b2m and peptide ligands are acquired. Several investigators have proposed that ligand exchange or ligand

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editing occurs during class I assembly (Androlewicz, 1999; Cresswell et al., 1999; Lauvau et al., 1999; Suh et al., 1999; Purcell et al., 2000a). The isolation and characterisation of tapasin-independent ligands of HLA B27 and their removal from the peptide repertoire in tapasin-positive cells is the strongest evidence to date that ligand editing may be mediated via loading complex formation (Purcell et al., 2000a). The extent to which these differences in peptide repertoire reflect generic quality control mechanisms or more specialised editing mechanisms is unclear. It can be envisaged that, under normal circumstances, peptide receptive MHC class I heterodimers acquire antigenic peptide primarily when bridged to the TAP (Cresswell et al., 1999; van Endert, 1999). However, it is possible that a proportion of some class I molecules, like HLA A2 or HLA B27, might initially acquire peptides via alternative TAP or loading complex-independent pathways and then subsequently be localised to the TAP via tapasin binding. 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 less abundant or shorter half-life peptides as well as more optimal peptides. Furthermore, recent studies indicate that class I molecules can associate stably with the TAP for periods of up to several minutes (Marguet et al., 1999) and are associated with tapasin prior to dissociating from the TAP complex (Li et al., 1999). 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 stabilisation 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 recently to be intimately involved in the degradation of abnormally folded or accumulated class I hc (Wilson et al., 2000). 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 (Harris et al., 1998), yet is involved in refolding of most glyco-

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proteins in the ER. It is unlikely, therefore, that calreticulin exerts a direct influence on the peptide repertoire, other than to ensure the bound peptides elicit proper conformational assembly of the class I molecule. ERp57 has recently been shown to associate with both disulfide bonded and non-disulfide-bonded hc, suggesting that this molecule is directly responsible for class I hc disulfide bond formation (Farmery et al., 2000). 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 are required during peptide loading (Cresswell et al., 1999; Farmery et al., 2000). 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 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 oxidoreductase activity, this may facilitate peptide binding or peptide exchange. Whether ERp57 has any influence on the nature of the peptide ligand remains unclear. Tapasin has a number of distinct functions that predicate a role for this chaperone in peptide editing or exchange. These functions include retention of class I complexes in the ER (Schoenhals et al., 1999; Barnden et al., 2000), co-localisation of class I to the TAP heterodimer (Sadasivan et al., 1996), increasing the level of TAP expression, and possibly affecting peptide translocation from the cytosol into the lumen of the ER (Bangia et al., 1999; Li et al., 2000). In addition, as highlighted from studies with soluble tapasin (Lehner et al., 1998), this chaperone may stabilise class I complexes and influence peptide loading independently of TAP association. Biochemical studies of the ligands associated with HLA B27 in tapasin-deficient cells have revealed both quantitative and qualitative influences of tapasin on the class I peptide repertoire (Purcell et al., 2000a). 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 in 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. This region of the class I molecule forms a loop in the a2 domain (see later and Fig. 3) and, conceivably, by interacting with this region tapasin could influence peptide binding by modulating

cleft conformation (Yu et al., 1999a,b). The defined functions of tapasin (Cresswell et al., 1999), combined with recent studies (Barnden et al., 2000; Purcell et al., 2000a), reflects the integral role tapasin plays in both peptide loading of class I molecules and orchestrating ligand optimisation.

5. Defining the sites of interaction between class I molecules and the loading complex Studies that have examined point mutants of naturally occurring class I molecules have defined some of the structural determinants necessary for interaction of the class I heterodimer with the loading complex. Elliott and colleagues (Lewis et al., 1996; Lewis and Elliott, 1998) demonstrated that a point mutant of HLA A2, in which Threonine at position 134 was substituted with a Lysine (T134K), failed to associate with components of the loading complex. This resulted in the rapid transport of these molecules to the cell surface in association with a high proportion of suboptimal ligands. In contrast, a second mutant of HLA A2, S132C, demonstrated a prolonged interaction with the

Fig. 3. A putative HLA class I binding surface involved in loadingcomplex formation. Three-dimensional model of the putative binding sites for components of the class I loading complex. The class I hc and b2m (darker lines) are shown as Ca backbone traces with space-filling representations highlighting the side chains of amino acid residues mapped in previous studies to be important in loading complex formation. The minimal peptide ligand ARAAAAAAA is shown occupying the antigen binding cleft as a space-filling model (darker shading). The structure is based on the published co-ordinates of HLA B*2705 (Madden et al., 1991) and visualised by the Swiss PDB viewer (Žhttp://www.expasy.ch/spdbv/; Guex and Peitsch, 1997).

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class I loading complex, matured slowly and acquired high-affinity optimal peptide ligands (Lewis et al., 1996). This region of the a2 domain of HLA A2 was also recently shown (Beissbarth et al., 2000) to influence the maturation kinetics and association with calreticulin, tapasin and TAP for two other A2 mutants (Q115A, D122A). In analogous experiments with mutants of the murine class I molecule H-2Ld, Hansen and colleagues (Yu et al., 1999b) have shown this region in the a2 domain (residues 128 – 136 in particular) is important in peptide loading and formation of the class I loading complex. Likewise, regions of the a3 domain have also been implicated in TAP/tapasin association, including amino acids 219 – 233 (Kulig et al., 1998), 222 (Suh et al., 1999) and 227 (Harris et al., 1998; Yu et al., 1999b). These studies have led to the identification of a surface of the class I molecule that may be involved in associating with components of the class I loading complex (as depicted in Fig. 3). The putative loading complex binding surface of the class I heterodimer is located on the a2-side of the molecule and forms a contiguous face with a3 domain amino acid residues 219 – 233. The binding site also includes the N-glycanated residue (amino acid 86 located on the end of the a1 helix) known to be involved in binding to calnexin and calreticulin (Sadasivan et al., 1996). It should be noted that, although not shown in this model, the N-glycan present on the class I molecule during ER residency (such as the Man9GlcNAc2 intermediate) has been predicted to project a significant distance from its site of N-linked attachment (Asn86) (Rudd et al., 1999). The putative binding surface is opposite to the face of the molecule occupied by b2m. Thus, despite the fact that b2m association is important for efficient loading-complex formation (Cresswell et al., 1999), it is likely that it contributes more to the conformation of the class I hc rather than directly to loading-complex assembly. It should be noted that b2m has been observed bound to TAP in class I hc deficient 721.221 cells (Solheim et al., 1997), suggesting a direct interaction with TAP or tapasin. The surface on the class I molecule that these regions contribute to defines a pronounced groove; it is tempting to speculate that this groove forms a docking structure for one or more components of the loading complex. It is also noteworthy that the conserved disulfide pairing between amino acids Cys101 and Cys164 is also located within this region of the a2 domain. This disulfide bond links the a2 helix to the peptide binding floor and isomerism of this disulfide bond has been implicated during peptide binding (Cresswell et al., 1999). Clearly, this may be a site targeted by ERp57 during refolding of nascent class I hc and potentially during peptide loading. The other conserved disulfide pair (Cys203 – Cys259) is located in the a3 domain in close proximity to the a3 loading complex binding surface.

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The potential for viruses or intracellular pathogens to target TAP or tapasin as a mechanism for evading CTL recognition has been highlighted in several studies. For example, the herpes groups of viruses encode proteins that interfere with TAP-mediated translocation of peptides. The 9kDa protein, ICP47, expressed by herpes simplex virus (York et al., 1994) blocks the cytosolic aspect of the TAP translocon, preventing translocation of peptides from the cytosol to the ER. In contrast, the 21 kDa gpUS6 protein from cytomegalovirus inhibits TAP-mediated translocation of peptides at the level of loading-complex formation (Hengel et al., 1997; Lehner et al., 1997). Similarly, interference of tapasin-mediated loading complex co-localisation to the TAP has been observed for the adenoviral protein E19 (Bennett et al., 1999). HLA alleles that encode TAP or tapasin-independent molecules may have evolved in response to such selective pressures by microorganisms. TAP or tapasin-independent class I molecules might permit the host CTL compartment to continue to respond to pathogens that specifically target these aspects of class I assembly. Sampling alternative peptide loading pathways, although beneficial during infection with such microorganisms, may also result in the upregulated presentation of poorly tolerised self-peptides in an inflammatory context. This may be a novel mechanism of triggering autoimmune responses. This hypothesis might be particularly relevant to the development of inflammatory spondyloarthritis, known to be strongly associated with the ‘tapasin-independent’ HLA B27 alleles (Parham, 1996; Khare et al., 1998; Lopez de Castro, 1998). The identification of alternative pathways by which class I molecules can acquire peptide ligands has broad relevance to antigen presentation. Whether this knowledge can be applied to vaccine design remains to be seen. However, it is possible that, with further definition of class I alleles that intrinsically are capable of sampling different peptide acquisition pathways and the definition of the nature of peptide antigen that is available for loading via these alternative pathways, certain sequence features could be incorporated into vaccines. For example, if TAP or tapasin-independent loading of class I could be achieved at a kinetically favorable rate, then perhaps greater ligand density of such determinants could be achieved on the surface of antigen presenting cells. Thus, peptide-based vaccines could incorporate a degree of TAP/tapasin independence to ensure rapid loading and saturation of the class I pathway with the ligand of choice. This increase in determinant density on the surface of professional APC may also correlate with enhanced immunogenicity (Khanna et al., 1994; Sette et al., 1994; Deng et al., 1997; Yewdell and Bennink, 1999) and improve the outcome of the vaccination.

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