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Peptide Loading of MHC
Chapter 93
Peptide Loading of MHC Melissa J. Call
ABSTRACT The proteins of the major histocompatibility complex (MHC) are central to the ability of an organism to protect itself from foreign invasion, be it viral, bacterial, or parasitic. This “protection,” unfortunately for many, also mediates transplant rejection and, when mistakenly directed, results in autoimmune pathology. The two classes of MHC molecules source peptides in different ways and use different strategies to promote peptide loading. This chapter focuses on how immunogenic peptides are generated and the molecules that mediate peptide loading onto MHC proteins.
INTRODUCTION There are two main classes of MHC molecules that display peptide epitopes. Their roles in immunity are distinct and perhaps the simplest way to illustrate their function is to consider some of the roles each plays during the course of a viral infection. A viral infection begins when infectious viral particles penetrate protective barriers such as the skin or mucosal membranes. Once in contact with a cell that supports viral entry, invasion followed by viral replication within the cytoplasm can begin. Any given cell has many countermeasures available to respond to infection, but MHC-mediated responses are particularly important in efficient pathogen clearance and the generation of lasting immunity. In almost all cells of the body, MHCclass I molecules are constitutively expressed and loaded with peptides derived from proteins being synthesized and degraded within the cell, and thus, it is extremely challenging for a virus to replicate using host machinery without supplying viral peptides to the MHC-class I loading pathway. This provides an elegant system for cells to display a “snapshot” of the proteome at the cell surface for perusal by circulating lymphocytes. The role of CD8+ T-cells is to lyse cells that show signs of infection through the display of foreign peptides on surface MHC-class I molecules. Although this drastic response is extremely effective in limiting viral replication, it also has the potential to create catastrophic tissue damage if inappropriately deployed, and therefore, CD8+ T-cell responses are highly regulated. Before a CD8+ T-cell can kill an infected cell it Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00093-2 Copyright © 2013 Elsevier Inc. All rights reserved.
must first become activated in the lymph node by dendritic cells (DCs). Conventional migratory DCs are found with high frequency in peripheral tissues near protective barriers to sense when these are disrupted and to direct T-cell responses accordingly.33 These sentinels straddle the innate and adaptive immune responses. DCs in the periphery are highly phagocytic and responsive to innate “danger signals”22 that warn of infection. Dying cells and virions are rich sources of antigen for DCs, and once activated by the danger signals, DCs undergo changes to initiate an immune response. They alter their chemokine and cytokine expression profiles, leading to the recruitment of other immune cells to the site of infection. They migrate to the draining lymph node and can pass antigen on to other specialized DC subsets that reside there or interact directly with a large population of naïve T-cells. Costimulatory molecules that are required for the productive stimulation of T-cells are upregulated. Antigen processing within the lysosomal pathway is also upregulated, as is MHC-class II, which is loaded with peptides from endocytic compartments before export to the cell surface. In a specialized mechanism of “crosspresentation” that occurs efficiently in certain DC subsets, the peptides from the phagocytic pathway are also loaded onto MHC-class I molecules. The end result of these processes yields a potent antigen-presenting cell that can present peptides to both CD4+ and CD8+ T-cells. Unlike CD8+ T-cells, CD4+ T-cells do not generally lyse the infected cells through direct cytotoxicity, but rather exert their important effects by directing the quality of the adaptive immune response. Through recognition of peptides displayed on MHC-class II molecules, CD4+ T-cells provide positive feedback in the form of cytokines to the DC, enhancing its ability to provoke long-lived and sustainable activation from naïve CD8+ T-cells, which then traffic to the site of infection to lyse infected cells. Activated CD4+ T-cells also promote antibody producing B-cell activation21 through recognition of MHC-class II-peptide complexes generated from processed antigen acquired through surface immunoglobulin receptors. Subsequent antibody class-switching and affinity maturation is critical to the ability of the host to generate sterilizing immunity. 687
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Although this simplistic overview of some MHCdirected processes only touches on a few of the events required for viral clearance, it highlights the features of the MHC system that share critical information among the many cell types that must cooperate to generate a successful immune response. In this context, MHC-class I is available to all cell types to display fragments of proteins currently being synthesized within the cell, while MHCclass II is reserved for the “professional” antigen-presenting cells and endows them with the special ability to display foreign epitopes without being directly infected. DCs are the most efficient antigen-presenting cells and have the ability to transfer the peptides derived from extracellular antigens into the MHC-class I pathway, representing an important link in the crosstalk between MHC-class I and MHC-class II mediated responses. In the following sections, I will go into more detail on how the peptides that are presented by MHC-class I and MHC-class II are generated and the sources from which they are derived.
THE MHC LOCUS The MHC locus, located on chromosome 6 in humans and chromosome 17 in mice, is a clustering of around 150 genes that are largely involved in immune regulation and encode many different proteins including both MHCclass I and MHC-class II molecules. In humans, the classical MHC-class I genes are extremely polymorphic and are encoded by HLA-A, HLA-B and HLA-C, the gene products of which dimerize with β2 microglobulin to form functional MHC-class I proteins. More than a thousand polymorphisms have been reported for each gene and many more will probably be found. There are currently 5400 distinct sequences reported for MHC-class I molecules. An individual possesses two alleles at each MHC-class I gene locus, one maternal and one paternal. Thus, an individual expresses up to six different classical MHC-class I gene products at the cell surface. MHC-class II proteins are formed by hetero-dimerization of α- and β-chains. Humans have three classical MHC-class II proteins, HLA-DR, HLA-DP, and HLADQ. The most polymorphic gene encodes the HLA-DR β-chain and the products of this gene combine with an invariant α-chain to make HLA-DR molecules. In HLADP and HLA-DQ both chains are polymorphic. There are currently around 1600 alleles reported for MHC-class II α- and β-chains, although the number of productive combinations that can form functional proteins is more difficult to estimate. Discussion of the nomenclature and diversity of MHC molecules is complex and beyond the scope of this chapter, and the reader is directed to this reference for further information.28
MHC-CLASS I The MHC-Class I Peptide-Binding Site MHC-class I gene products are type I membrane proteins. Each has a short cytoplasmic sequence, transmembrane domain, a membrane-proximal immunoglobulin domain and a membrane-distal peptide-binding groove. This groove is formed by an eight-strand antiparallel β-pleated sheet “floor” supporting two α-helices that flank the bound peptide. The folded protein is joined by β2-microglobulin which assumes a membrane-proximal position next to the class I immunoglobulin domain, underneath the floor of the peptide-binding groove2 (Fig. 1A). The α-helices that line the MHC-class I peptide-binding groove are closely apposed at both ends and MHC-class I molecules are therefore only able to “comfortably” bind peptides of 8–10 amino acids in length. The peptide is maintained in an extended conformation through a series of interactions. The N- and C-termini of the peptide are bound through a conserved network of hydrogen bonds, and key “anchor” residues have their side-chains buried in pockets found along the peptide-binding groove (Fig. 1B). The interactions mediated by the N- and C-termini represent the bulk of the peptide-binding energy4 and this sequenceindependent feature allows MHC-class I to bind extremely diverse peptides (each allele is estimated to have the ability to bind many thousands of different sequences). The series of pockets in the floor of the peptide-binding groove (Fig. 1C) provide distinct chemical environments that favor certain residues over others, and the polymorphisms in MHC-class I are concentrated in these regions to endow each MHC allele with a unique peptide specificity.10 These pockets place restrictions on size, charge, and polarity of anchor residues and can stabilize the peptides through hydrogen bonds, electrostatic interactions, hydrophobic packing, and van der Waals forces. Importantly, in most human MHC-class I alleles, the pocket that houses the peptide C-terminal residue favors hydrophobic residues and allows basic residues, but is incompatible with peptides that have an acidic residue at this position.
MHC-Class I Peptide-Loading Complex MHC-class I proteins are directed to the endoplasmic reticulum (ER) on synthesis for folding and secretion to the cell surface. MHC-class I molecules are unable to reach the cell surface until they are loaded with peptide. Immediately after synthesis, MHC-class I heavy chain associates with calnexin, a molecular chaperone that resides in the ER (Fig. 2a). Calnexin is associated with ERp57, a protein thiol oxoreductase that influences disulfide bond formation in the nascent MHC-class I heavy chain. Through interaction with these proteins, the heavy chain is folded
SECTION | IX Handbook of Biologically Active Peptides: Immune/Inflammatory Peptides
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(A)
(B)
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FIGURE 1 (A) Side view of MHC-class I (left) and MHC-class II (right) showing the peptide-binding site at the top and the membrane proximal immunoglobulin domains at the bottom. (B) Looking down on the peptide-binding site, which is formed by α-helices that flank the peptide and supported by an eight-strand β-sheet. Conserved hydrogen bonds that stabilize the peptide backbone are indicated as dashed lines in each structure. Note that in MHC-class II, the α- and β-chains together combine to form the groove, and peptide extends out of the peptide-binding site. The peptide N- and C-termini are indicated with N and C respectively. (C) The same view as (B), but in surface rendering with the peptide removed. The pockets that serve as anchor points for peptide side chains are labeled A–F for MHC-class I and P1–P9 for MHC-class II. PBD files 1S9Y and 1DLH were used to generate this figure. See color plate 23.
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a llowing recruitment of β2-microglobulin, signaling that the MHC-class I molecule is ready for peptide loading (Fig. 2b). In humans, the folded MHC-class I – β2-microglobulin heterodimer then dissociates from calnexin and joins the peptide-loading complex containing calreticulin, transporter associated with antigen processing (TAP), tapasin, and ERp57 (Fig. 2c).19 ERp57 is covalently linked to tapasin through a disulfide bond and this complex bridges
the empty MHC heavy chain to TAP and calreticulin. In addition to its structural role in the peptide-loading complex, biochemical studies suggest that tapasin-ERp57 can modulate the quality of the peptide repertoire by favoring the loading of high-affinity peptides and promoting dissociation of prebound, low affinity peptides.34 TAP is an ATP-dependent transporter that delivers peptides from the cytosol to the ER lumen and comprises two Key
(A) Classical MHC class I Pathway Extracellular
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AA A AA AA A AA AA AA
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FIGURE 2 (A) MHC-class I heavy chains are synthesized in the ER (a) and then partially folded with assistance from calnexin and ERp57 (b). After β2 microglobulin joins the MHC-class I heavy chain the heterodimer is incorporated into the peptide-loading complex and loaded with peptides transported from the cytoplasm to the ER lumen by TAP and trimmed to the correct size by ERAP family proteases (c). Peptide-loaded MHC-class I is then transported via the Golgi to the cell surface (d). Proteasomes cleave proteins in to peptides, which can be selected by TAP for transport into the ER (e). (B) MHC-class II chains and invariant chain are synthesized in the ER (f) and fold together into a nonameric complex with 3 invariant chains, and 3 MHC-class II molecules (g). A region of the invariant chain (CLIP) occupies the peptide-binding groove of MHC-class II. This complex is exported via the Golgi to multivesicular bodies in the endocytic compartment (h). Proteolytic cleavage of invariant chain occurs to release MHC-class II molecules and allow HLA-DM mediated release of CLIP and replacement with peptides from endocytozed proteins (i). MHC-class II molecules are transported to the cell surface for display to CD4+ T-cells (j). See color plate 24.
SECTION | IX Handbook of Biologically Active Peptides: Immune/Inflammatory Peptides
proteins, TAP1 and TAP2.27 Like MHC-class I, TAP exhibits selectivity for peptides that have hydrophobic or basic C-terminal residues and are between 8 and 16 amino acids in length.13 TAP has the ability to transport peptides that are longer than those preferred by MHC-class I, and proteases that trim the N-terminus are present in the ER lumen to ensure peptides of optimal length can be generated. In humans, ERAP1 and ERAP2 fulfill this role. Studies with ERAP1 have determined that this protease exhibits selectivity for peptides between 9 and 16 amino acids in length and displays little activity for peptides longer or shorter than this. A model has been proposed that suggests the peptide C-terminal residue is bound by ERAP1 in a pocket distinct from the active site and that the distance between these two sites determines the selectivity of ERAP1 for peptide length. If accurate, it would suggest that peptide trimming occurs before loading into MHC-class I.20
The Classical MHC-Class I Pathway The source of peptides displayed by classical MHC-class I molecules and the pathways that generate them have been topics of intense research. Initially, it was assumed that peptides were simply “borrowed” from natural turnover processes, but because proteins often have half-lives of hours to days within a cell, the kinetics of this process did not seem to be congruous with the observation that viral peptides could be displayed by MHC-class I molecules in less than an hour after infection. Although natural turnover processes do account for some of the peptides displayed by MHC-class I, it is now thought that the bulk of peptides are acquired through the cleanup of incomplete synthesis products. Estimates indicate that up to 40% of the products generated by protein synthesis are rapidly degraded because of errors that might include early termination, misfolding, missplicing, and frameshift errors. Whether protein synthesis is simply a messy business or whether these errors are made specifically to generate peptides for the MHC-class I pathway is not known and remains a matter of debate.9 Proteins are targeted to the proteasome after becoming tagged with poly-ubiquitin chains, the reasons for which are diverse and include normal protein turnover, the destruction of improperly synthesized proteins described above, as well as regulatory proteins controlling cell cycle progression and intracellular signaling. The proteasome is a multisubunit complex11 with a cylindrical catalytic core that is gated at each end by regulatory cap particles that are defined by their sedimentation values. The addition of the 19S regulatory caps to the 20S proteasomal core constitutes the 26S proteasome. Poly-ubiquitin chains of the proteins to be degraded are recognized by proteins in the 19S regulatory cap and, in an ATP-dependent process, ubiquitin is removed and the protein unwound to allow entry into the 20S proteasomal core. It is here that cleavage occurs, generating
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peptide fragments between 2 and 25 amino acids in length18 (Fig. 2e). In humans the cylindrical 20S proteasomal core is made up of four heptameric rings. The alpha subunits, α1–-7, flank each end of the cylinder and form narrow openings that are modulated by the 19S regulatory cap. Sandwiched between the α rings are two antiparallel β rings made up of β1–β7 subunits.11 Only three of these contain catalytically active sites, but between them they are able to cut after almost any amino acid: β5 is chymotrypsin-like, cleaving after bulky hydrophobic residues; β2 is trypsin-like and prefers to cleave after basic residues; and β1, which is caspase-like, cleaves after acidic and branched amino acids. The pattern of cleavage for a particular protein is not random, and the same fragments are generated each time a protein is processed because the activity of the catalytic triad in each β subunit is sensitive to the sequence context around a cleavage site. The combination of limited time within the proteasome chamber and preferred sequences determining initial cleavage sites results in consistent, though difficult to predict, peptide products for any given protein. Peptides that are longer than seven amino acids in length are potential ligands for MHC-class I molecules, but most do not survive exposure to cytosolic proteases. A nine amino acid peptide in the cytosol has a half-life of less than 10 s, and therefore only peptides that are rapidly transported into the ER lumen have the opportunity to fold with MHCclass I heavy chains awaiting export to the cell surface.
The Immunoproteasome Almost all cells in the body have the ability to express interferon (IFN) α/β in response to infection. This hallmark of stress, among others, is recognized by natural killer (NK) cells, which in turn secrete IFNγ. IFNγ has potent effects on antigen processing in both the MHC-class I and II pathways and is particularly important in antiviral responses. Under normal cellular conditions, the proteasome is actually quite poor at generating peptides suitable for MHC-class I binding because most products are smaller than the minimal length required for ER import and MHCclass I loading. Additionally, all MHC-class I molecules studied to date prefer hydrophobic or basic anchors at the peptide C-terminus and acidic residues in particular seem to be excluded. This means that peptides generated by β1 cleavage, which occurs after acidic residues, are essentially lost from the MHC-class I repertoire. However, IFNγ exposure causes expression of three new β subunits, β1i, β2i, and β5i, which replace β1, β2, and β5 in the 20S proteasome catalytic core and all but eliminate the caspase-like activity. Thus most peptides processed by this alternative proteasome, termed the “immunoproteasome,” contain suitable C-terminal anchor residues for MHC-class I binding. In addition to replacement of three β subunits, the immunoproteasome also uses an alternative regulatory cap known
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as PA28, REG, or 11S Activator, which is IFNγ-inducible and significantly increases the maximal velocity of peptide hydrolysis compared with the normal 26S proteasome.6 Interestingly, although the expression of the immunoproteasome is IFNγ dependent in most cell types, the alternative subunits are constitutively expressed in some cells of hematopoietic origin, including DCs and lymphoid cells, emphasizing their specialization for antigen processing and presentation. Mice lacking all three alternative catalytic subunits exhibited alterations in the MHC-class I peptide repertoire that were significant enough to cause the rejection of splenocytes transferred from wild type mice despite being matched at all MHC loci.17
A Specialized Thymic Proteasome Within the thymus, the organ where T-cells reactive to the self are deleted to prevent autoimmunity, the cells that mediate this “negative selection” express both the proteasome and the immunoproteasome. Interestingly, there is a third “thymoproteasome” that uses an alternative β5 subunit in a distinct population of thymic stromal cells responsible for “positive selection,” a process that ensures T-cells receive tonic stimulation by MHC for survival in the periphery. The thymoproteasome generates peptides that display significantly reduced affinity for MHC-class I, and it has been noted that mice deficient in this alternative β5 subunit have severe defects in positive selection.26 The mechanism behind this observation is not yet understood, but it has been proposed that unstable MHC-class I peptide complexes may be somehow required for this process or that the thymoproteasome generates unique stimulatory peptides to drive positive selection.
Crosspresentation MHC-class I is expressed ubiquitously and, for the most part, displays peptides derived from intracellular sources, with the notable exception of certain specialized cell types such as CD8αα+ DCs that have the ability to load proteins derived from extracellular sources onto MHC-class I. This specialized pathway, termed crosspresentation, allows priming of cytotoxic CD8+ T-cells by professional antigenpresenting cells that are not directly infected. Endocytic receptors seem to be critical regulators of crosspresentation, with antigens targeted to DEC205, for instance, being efficiently processed for MHC-class I loading, while those targeted to DCIR2 are not. Although it was initially assumed that these markers simply distinguished cells with different inherent abilities to crosspresent, follow-up experiments determined that introduction of DEC205 into the CD8− DC subset that does not normally cross-present, is sufficient to enable cross-presentation to occur.16 The evidence suggests that crosspresentation is proteasome dependent, and therefore a mechanism by which antigen can escape the
Chapter | 93 Peptide Loading of MHC
phagolysosome to reach the cytoplasm must exist. It is considered unlikely that antigens access the cytoplasm through permeabilized phagosomal membranes and research has instead focused on channels that have the ability to translocate proteins to the cytoplasm. The process of ER associated protein degradation (ERAD), which translocates misfolded proteins from the ER to the cytoplasm for degradation by the proteasome provides an attractive solution to this problem, but requires either the recruitment of ER membrane, or more controlled delivery of ERAD components to the phagosomal pathway. Once antigen has reached the cytoplasm it may intersect with the classical MHC-class I loading pathway, but evidence suggests that peptide loading during crosspresentation may actually occur in the phagocytic compartment itself, with the recruitment of proteasomes and peptide-loading machinery directly to the vesicles.15 Although the mechanics of cross-presentation remain controversial, it is clear that this process is extremely important for CD8+ T-cell function.
Nonclassical MHC-Class I Although the products of the classical MHC-class I alleles HLA-A, -B, and -C have a crucial role in binding peptides for display to cytotoxic T-cells, the nonclassical HLA-E, -F, and -G alleles have more poorly defined roles. These proteins do bind peptide, but they are much less polymorphic than HLA-A, -B, and -C, and this is reflected in their restricted peptide repertoire. The best studied of these alleles is HLA-E, which is recognized by the CD94/NKG2 receptor system and seems to have a major role in regulating NK cell activity.32 HLA-E primarily binds peptides that are derived from the signal sequences of other MHC-class I molecules and is believed to convey information about normal MHC biosynthesis to NK cells. NK cells are reactive to alterations in the MHC-class I pathway that are commonly caused by viral proteins evolved to inhibit MHC-mediated immune responses. Normal operation of the pathway results in peptide-loaded HLA-E binding to inhibitory receptors on NK cells and suppression of cytotoxicity, but loss of this signal in virally infected cells can trigger killing. In a striking evolutionary “tit-for-tat,” human cytomegalovirus encodes a homolog of the MHC-class I leader peptide to maintain HLA-E at the cell surface while other virulence factors downregulate classical MHC-class I expression.31
MHC-CLASS II MHC-Class II Peptide-Binding Site Nascent MHC-class II molecules form from α- and β-chains that are assembled in the ER. Like the MHC-class I heavy chain, the β-chain forms a membrane-proximal immunoglobulin domain, but unlike its class I counterpart,
SECTION | IX Handbook of Biologically Active Peptides: Immune/Inflammatory Peptides
only contributes to half of the peptide-binding domain. The α-chain helps complete the peptide-binding domain and also contributes a membrane-proximal immunoglobulin domain so that, unlike MHC-class I, MHC-class II does not require β2 microglobulin (Fig. 1). Although the overall fold of MHC-class II molecules resembles that of MHC-class I, there are significant differences that endow these closely related structures with very different peptidebinding properties.5 The peptide is accommodated in the groove in an extended conformation similar to that of a type II poly-proline helix and importantly, because the helices in MHC-class II molecules do not close together at each end of the peptide-binding groove as with MHC-class I proteins, MHC-class II molecules can bind much longer peptides. This means that polar contacts cannot always be made to free N- and C-termini of the peptide and interactions are instead dominated by a conserved network of hydrogen bonds to polar backbone atoms along the entire length of the peptide (Fig. 1B). Structural and biochemical studies indicate that the strongest binding energy is contributed by hydrogen bonds concentrated at the N-terminus of the bound peptide. These sequence-independent interactions are further complemented by anchoring pockets that, like MHC-class I, favor peptides with compatible amino acids (Fig. 1C). The register that any given peptide binds to MHC class II is defined by the residue that binds in the P1 pocket. The peptide residues are sequentially numbered in an N- to C-terminal direction. P1, P4, P6, and P9 residues interact with the major pockets in MHC, while P2, P5, and P8 are major TCR contact points. The P1 and P4 pockets of HLA-DR molecules contribute the most to peptide stability. A natural polymorphism at residue 86 of the HLA-DR β-chain influences the size of the P1 pocket. A glycine at this position provides a large pocket that can accommodate bulky hydrophobic residues while a valine at this position presents a shallower pocket that prefers smaller aliphatic residues.
Invariant Chain Processing and HLA-DM-Mediated Peptide Exchange Each MHC-class II heterodimer assembles in the ER with the invariant chain, a trimeric chaperone that is exported through the Golgi and trans-Golgi network to intersect with the endosomal pathway in multivesicular antigen-processing compartments (Fig. 2f–h).30 The invariant chain serves a number of purposes. A fragment of the invariant chain is embedded in the peptide-binding site to prevent opportunistic binding of proteins/peptides in the ER29 and to stabilize the MHC-class II peptide-binding site, which is prone to collapse and aggregation if left unoccupied. The invariant chain cytoplasmic domain also contains trafficking signals that target MHC-class II to the appropriate subcellular location for peptide loading to occur.
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As the invariant chain occupies the peptide-binding groove, it must be dislodged from MHC-class II to allow new peptide acquisition, and two important processes ensure that this is achieved. First, proteolytic degradation of invariant chain occurs which releases MHC-class II from all but the invariant-chain-derived fragment protecting the peptide-binding site. This fragment is termed CLIP and typically corresponds to residues approximately 80–105 depending on the fine mapping of protease cleavage sites. Degradation of invariant chain involves a number of proteases that are sensitive to the protease inhibitor leupeptin,3 and although the complete pathway has not been elucidated, two proteases in particular play nonredundant roles in the latter steps of invariant chain processing: these are cathepsin S (in the periphery) and cathepsin L (in the thymus).14 Attempts to identify proteases that have a nonredundant role in early steps of invariant chain processing have failed, and this no doubt reflects the susceptibility of the invariant chain to cleavage by multiple enzymes. HLA-DM ensures that the protective CLIP peptide is then exchanged for exogenously derived peptides from the endocytic pool. HLA-DM is a heterodimeric MHC-class II protein that does not itself bind peptides.23 HLA-DM resides in late endosomal compartments and is responsible for “editing” the peptide repertoire to promote formation of high-affinity complexes with very stably bound peptides. To achieve this, HLA-DM binds to MHC-class II molecules that contain suboptimal P1 anchor residues and stabilizes the empty groove to encourage bound peptides to leave and give new peptides access to sample the site. Peptides that supply optimal P1 anchor residues then cause HLA-DM to dissociate from MHC-class II and are protected from further editing by stable P1 interactions between peptide and MHC that block HLA-DM binding.1 HLA-DM is negatively regulated by HLA-DO in a process that is thought to be mediated by competitive binding to HLA-DM in a way that prohibits interaction with MHC-class II complexes. HLA-DO expression depends on cell type and activation state, and is thought to be an important regulatory pathway in B-cells.8
Properties of CLIP The CLIP peptide binds promiscuously to many MHC-class II alleles, albeit with widely variable affinity. For instance, the half-life of a MHC-class II allele that is associated with susceptibility to rheumatoid arthritis (DRB1*0401) in complex with CLIP is in the order of minutes, while another common allele (DRB1*0101) forms complexes that are stable for days. These differences in stability are unsurprising given that MHC-class II is highly polymorphic and those polymophisms are concentrated within the peptide-binding groove. In this context, perhaps what is more surprising is that there exists a single peptide that seems to interact
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with most of the MHC-class II molecules. Only one crystal structure of an MHC-class II molecule in complex with CLIP has been determined. In this structure, methionine 90 of the invariant chain occupies the P1 pocket of the class II molecule HLA-DR3 (DRB1*0301). Almost all of the MHC-class II alleles analyzed thus far through mutagenesis studies appear to bind CLIP in this register, with the notable exceptions of two HLA-DQ alleles associated with celiac disease in which threonine 94 occupies the P1 pocket.35 The peptide-binding site of MHC-class II molecules is open at each end, giving peptides the ability to shift register in the binding site. This, no doubt, allows different alleles to select different segments of the CLIP sequence according to variable motif preferences.
Generation of Peptides for MHC-Class II Loading Antigen that is taken up by phagocytosis, receptor-mediated endocytosis or pinocytosis must traverse the endocytic pathway before loading onto MHC-class II molecules can be accomplished. The endocytic pathway has distinct compartments. Early endosomes are the principal sorting organelles where decisions are made as to whether molecules are exported back to the cell surface (recycling endosomes) or delivered to late endosomes. Early endosomes are mildly acidic and while MHC peptide complexes exhibit extraordinarily long half-lives, under acidic conditions peptide release is accelerated and therefore recycling endosomes are a possible site of peptide exchange. Late endosomes, however, are the principle location of MHC-class II loading (pH ~5.5) (Fig. 2h). There is some debate as to whether there are distinct late endosomes that are specialized for MHC-class II loading (typically referred to as MIIC) or whether the components required for loading are distributed throughout all late endosomes in antigen-presenting cells.25 The final destination in the endocytic pathway is the lysosome. Lysosomes are highly acidified and even MHC-class II molecules, which are acid resistant, are unable to survive for long in this environment. In immature DCs, MHC-class II is targeted to lysosomes for degradation until maturation signals are encountered. MHC-class II is then directed to the late endosomes for peptide loading and then to the cell surface for peptide display. The proteases with the largest role in generating antigenic peptide fragments for MHC-class II are the cathepsins. Cathepsins are primarily cysteine proteases with pH- and redox-dependent activity and are distributed throughout the endosomal compartment.14 Expressed as proforms, they require proteolytic cleavage themselves to become activated. Although some cathepsins can accomplish their own activation through autocatalytic proteolysis, others require a second protease, which itself might display pH dependence. In this manner, the activity of certain cathepsins can
Chapter | 93 Peptide Loading of MHC
be restricted to different parts of the endocytic pathway, and this is believed to influence the rate of proteolysis to ensure large enough protein fragments reach the compartments where peptide loading takes place. MHC-class II molecules bind peptides in extended conformations, and for this to occur, denaturation of secondary structure must take place. In the endosomal pathway proteins are denatured by a combination of events: acidification interferes with salt bridges and hydrogen bonds; IFNγ-inducible lysosomal thiol reductase (GILT) reduces cysteine-mediated disulfide bonds;12 and endoproteases such as cathepsins B, D, F, G, H, K, S, or asparagine endopeptidase (AEP) cleave exposed flexible areas of proteins to cause catastrophic destabilization.14 It is probable that MHC-class II molecules then bind to large polypeptide fragments, which are trimmed from the C- and N-termini by cathepsins with carboxy and amino proteolytic activity, respectively. This allows MHC-class II, which is assisted by HLA-DM, to bind securely to a peptide epitope and protect it from further proteolysis.7 Model antigens have been used in an attempt to determine the relative contribution of proteases to the generation of epitopes recognized by T-cells. Each cathepsin’s activity destroys some epitopes while creating others, but mice that are deficient in individual cathepsins (with the exception of Cathepsin S and L, which are required for invariant chain processing) show only minor defects in antigen presentation, indicating a high degree of redundancy in the system. It has been particularly difficult to ascribe unique roles to individual proteases in the generation of defined peptideMHC-class II complexes because effects on the MHC-class II pathway may be caused by impaired invariant chain processing, impaired epitope generation, or impaired activation of proteases involved in these processes.
Autophagy and MHC-Class II Autophagy, which means to “eat oneself,” is the mechanism by which cells break down and recycle intracellular contents and is important for the maintenance of cellular homeostasis. Normally, autophagic activity is low, but during starvation, cellular stress, or infection, autophagy is upregulated and provides a source of peptides to the MHCclass II pathway. Autophagy is initiated by the recruitment of membrane, probably from the ER, to encircle a large volume of cytosol and its organellar contents. Initiation of this process in response to starvation is controlled by mTOR, which is released from its inhibitory complex when energy levels in the cell fall below a certain threshold. The recruited membrane then fuses to generate a doublemembrane vesicle that is targeted to the lysosomal pathway. Access to the lysosomal pathway not only allows the contents to be degraded into building blocks that can be used by the cell to form new proteins and lipids, but also delivers
SECTION | IX Handbook of Biologically Active Peptides: Immune/Inflammatory Peptides
them to the MHC-class II loading pathway described above. Autophagy by macrophages and DCs in response to infection can be mediated by pattern recognition receptors in the cytoplasm and at the cell surface. Almost all the toll-like receptors studied to date influence the autophagic pathway, and this represents an important defense mechanism for the cell to rid itself of intracellular bacteria and parasites that might otherwise evade detection.24
CONCLUDING REMARKS The MHC system and the peptides it presents to T-cells are at the heart of the adaptive immune response, and while we understand the basics of peptide processing and acquisition, there is much left to be learned. The outcome of a thorough and detailed understanding of these processes should provide the ability to manipulate the timing and location of peptide presentation. The ability to do this has implications in vaccine development, tumor immunity, transplant rejection, and autoimmunity.
REFERENCES 1. Anders AK, Call MJ, Schulze MS, Fowler KD, Schubert DA, Seth NP, et al. HLA-DM captures partially empty HLA-DR molecules for catalyzed removal of peptide. Nat Immunol 2011;12:54–61. 2. Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 1987;329: 512–8. 3. Blum JS, Cresswell P. Role for intracellular proteases in the processing and transport of class II HLA antigens. Proc Natl Acad Sci U S A 1988;85:3975–9. 4. Bouvier M, Wiley DC. Importance of peptide amino and carboxyl termini to the stability of MHC class I molecules. Science 1994;265:398– 402. 5. Brown JH, Jardetzky TS, Gorga JC, Stern LJ, Urban RG, Strominger JL, et al. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 1993;364:33–9. 6. Cascio P, Call M, Petre BM, Walz T, Goldberg AL. Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes. EMBO J 2002;21:2636–45. 7. Castellino F, Zappacosta F, Coligan JE, Germain RN. Large protein fragments as substrates for endocytic antigen capture by MHC class II molecules. J Immunol 1998;161:4048–57. 8. Denzin LK, Sant’Angelo DB, Hammond C, Surman MJ, Cresswell P. Negative regulation by HLA-DO of MHC class II-restricted antigen processing. Science 1997;278:106–9. 9. Dolan BP, Bennink JR, Yewdell JW. Translating DRiPs: progress in understanding viral and cellular sources of MHC class I peptide ligands. Cell Mol Life Sci 2011;68:1481–9. 10. Garrett TP, Saper MA, Bjorkman PJ, Strominger JL, Wiley DC. Specificity pockets for the side chains of peptide antigens in HLA-Aw68. Nature 1989;342:692–6. 11. Groll M, Ditzel L, Lowe J, Stock D, Bochtler M, Bartunik HD, et al. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 1997;386:463–71.
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12. Hastings KT, Cresswell P. Disulfide reduction in the endocytic pathway: immunological functions of gamma-interferon-inducible lysosomal thiol reductase. Antioxid Redox Signal 2011;15:657–68. 13. Heemels MT, Schumacher TN, Wonigeit K, Ploegh HL. Peptide translocation by variants of the transporter associated with antigen processing. Science 1993;262:2059–63. 14. Honey K, Rudensky AY. Lysosomal cysteine proteases regulate antigen presentation. Nat Rev Immunol 2003;3:472–82. 15. Houde M, Bertholet S, Gagnon E, Brunet S, Goyette G, Laplante A, et al. Phagosomes are competent organelles for antigen cross– presentation. Nature 2003;425:402–6. 16. Kamphorst AO, Guermonprez P, Dudziak D, Nussenzweig MC. Route of antigen uptake differentially impacts presentation by dendritic cells and activated monocytes. J Immunol 2010;185:3426–35. 17. Kincaid EZ, Che JW, York I, Escobar H, Reyes-Vargas E, Delgado JC, et al. Mice completely lacking immunoproteasomes show major changes in antigen presentation. Nat Immunol 2011;13:129–35. 18. Kisselev AF, Akopian TN, Woo KM, Goldberg AL. The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes. Implications for understanding the degradative mechanism and antigen presentation. J Biol Chem 1999;274:3363–71. 19. Koch J, Tampe R. The macromolecular peptide-loading complex in MHC class I-dependent antigen presentation. Cell Mol Life Sci 2006;63:653–62. 20. Kochan G, Krojer T, Harvey D, Fischer R, Chen L, Vollmar M, et al. Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming. Proc Natl Acad Sci U S A 2011;108:7745–50. 21. McHeyzer-Williams LJ, McHeyzer-Williams MG. Antigen–specific memory B cell development. Annu Rev Immunol 2005;23:487–513. 22. Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature 2007;449:819–26. 23. Morris P, Shaman J, Attaya M, Amaya M, Goodman S, Bergman C, et al. An essential role for HLA-DM in antigen presentation by class II major histocompatibility molecules. Nature 1994;368:551–4. 24. Munz C. Enhancing immunity through autophagy. Annu Rev Immunol 2009;27:423–49. 25. Neefjes J. CIIV, MIIC and other compartments for MHC class II loading. Eur J Immunol 1999;29:1421–5. 26. Nitta T, Murata S, Sasaki K, Fujii H, Ripen AM, Ishimaru N, et al. Thymoproteasome shapes immunocompetent repertoire of CD8 + T cells. Immunity 2010;32:29–40. 27. Powis SJ, Townsend AR, Deverson EV, Bastin J, Butcher GW, Howard JC. Restoration of antigen presentation to the mutant cell line RMA-S by an MHC-linked transporter. Nature 1991;354:528–31. 28. Robinson J, Mistry K, McWilliam H, Lopez R, Parham P, Marsh SG. The IMGT/HLA database. Nucleic Acids Res 2011;39:D1171–6. 29. Roche PA, Cresswell P. Invariant chain association with HLA-DR molecules inhibits immunogenic peptide binding. Nature 1990;345: 615–8. 30. Stern LJ, Potolicchio I, Santambrogio L. MHC class II compartment subtypes: structure and function. Curr Opin Immunol 2006;18:64–9. 31. Tomasec P, Braud VM, Rickards C, Powell MB, McSharry BP, Gadola S, et al. Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science 2000;287:1031. 32. van Hall T, Oliveira CC, Joosten SA, Ottenhoff TH. The other Janus face of Qa-1 and HLA-E: diverse peptide repertoires in times of stress. Microbes Infect 2010;12:910–8.
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33. Villadangos JA, Schnorrer P. Intrinsic and cooperative antigen– presenting functions of dendritic-cell subsets in vivo. Nat Rev Immunol 2007;7:543–55. 34. Wearsch PA, Cresswell P. Selective loading of high-affinity peptides onto major histocompatibility complex class I molecules by the tapasin-ERp57 heterodimer. Nat Immunol 2007;8:873–81.
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35. Wiesner M, Stepniak D, de Ru AH, Moustakis AK, Drijfhout JW, Papadopoulos GK, et al. Dominance of an alternative CLIP sequence in the celiac disease associated HLA-DQ2 molecule. Immunogenetics 2008;60:551–5.
Peptide Loading of MHC Melissa J. Call
ABSTRACT The proteins of the major histocompatibility complex (MHC) are central to the ability of an organism to protect itself from foreign invasion, be it viral, bacterial, or parasitic. This “protection,” unfortunately for many, also mediates transplant rejection and, when mistakenly directed, results in autoimmune pathology. The two classes of MHC molecules source peptides in different ways and use different strategies to promote peptide loading. This chapter focuses on how immunogenic peptides are generated and the molecules that mediate peptide loading onto MHC proteins.
INTRODUCTION There are two main classes of MHC molecules that display peptide epitopes. Their roles in immunity are distinct and perhaps the simplest way to illustrate their function is to consider some of the roles each plays during the course of a viral infection. A viral infection begins when infectious viral particles penetrate protective barriers such as the skin or mucosal membranes. Once in contact with a cell that supports viral entry, invasion followed by viral replication within the cytoplasm can begin. Any given cell has many countermeasures available to respond to infection, but MHC-mediated responses are particularly important in efficient pathogen clearance and the generation of lasting immunity. In almost all cells of the body, MHCclass I molecules are constitutively expressed and loaded with peptides derived from proteins being synthesized and degraded within the cell, and thus, it is extremely challenging for a virus to replicate using host machinery without supplying viral peptides to the MHC-class I loading pathway. This provides an elegant system for cells to display a “snapshot” of the proteome at the cell surface for perusal by circulating lymphocytes. The role of CD8+ T-cells is to lyse cells that show signs of infection through the display of foreign peptides on surface MHC-class I molecules. Although this drastic response is extremely effective in limiting viral replication, it also has the potential to create catastrophic tissue damage if inappropriately deployed, and therefore, CD8+ T-cell responses are highly regulated. Before a CD8+ T-cell can kill an infected cell it Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00093-2 Copyright © 2013 Elsevier Inc. All rights reserved.
must first become activated in the lymph node by dendritic cells (DCs). Conventional migratory DCs are found with high frequency in peripheral tissues near protective barriers to sense when these are disrupted and to direct T-cell responses accordingly.33 These sentinels straddle the innate and adaptive immune responses. DCs in the periphery are highly phagocytic and responsive to innate “danger signals”22 that warn of infection. Dying cells and virions are rich sources of antigen for DCs, and once activated by the danger signals, DCs undergo changes to initiate an immune response. They alter their chemokine and cytokine expression profiles, leading to the recruitment of other immune cells to the site of infection. They migrate to the draining lymph node and can pass antigen on to other specialized DC subsets that reside there or interact directly with a large population of naïve T-cells. Costimulatory molecules that are required for the productive stimulation of T-cells are upregulated. Antigen processing within the lysosomal pathway is also upregulated, as is MHC-class II, which is loaded with peptides from endocytic compartments before export to the cell surface. In a specialized mechanism of “crosspresentation” that occurs efficiently in certain DC subsets, the peptides from the phagocytic pathway are also loaded onto MHC-class I molecules. The end result of these processes yields a potent antigen-presenting cell that can present peptides to both CD4+ and CD8+ T-cells. Unlike CD8+ T-cells, CD4+ T-cells do not generally lyse the infected cells through direct cytotoxicity, but rather exert their important effects by directing the quality of the adaptive immune response. Through recognition of peptides displayed on MHC-class II molecules, CD4+ T-cells provide positive feedback in the form of cytokines to the DC, enhancing its ability to provoke long-lived and sustainable activation from naïve CD8+ T-cells, which then traffic to the site of infection to lyse infected cells. Activated CD4+ T-cells also promote antibody producing B-cell activation21 through recognition of MHC-class II-peptide complexes generated from processed antigen acquired through surface immunoglobulin receptors. Subsequent antibody class-switching and affinity maturation is critical to the ability of the host to generate sterilizing immunity. 687
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Although this simplistic overview of some MHCdirected processes only touches on a few of the events required for viral clearance, it highlights the features of the MHC system that share critical information among the many cell types that must cooperate to generate a successful immune response. In this context, MHC-class I is available to all cell types to display fragments of proteins currently being synthesized within the cell, while MHCclass II is reserved for the “professional” antigen-presenting cells and endows them with the special ability to display foreign epitopes without being directly infected. DCs are the most efficient antigen-presenting cells and have the ability to transfer the peptides derived from extracellular antigens into the MHC-class I pathway, representing an important link in the crosstalk between MHC-class I and MHC-class II mediated responses. In the following sections, I will go into more detail on how the peptides that are presented by MHC-class I and MHC-class II are generated and the sources from which they are derived.
THE MHC LOCUS The MHC locus, located on chromosome 6 in humans and chromosome 17 in mice, is a clustering of around 150 genes that are largely involved in immune regulation and encode many different proteins including both MHCclass I and MHC-class II molecules. In humans, the classical MHC-class I genes are extremely polymorphic and are encoded by HLA-A, HLA-B and HLA-C, the gene products of which dimerize with β2 microglobulin to form functional MHC-class I proteins. More than a thousand polymorphisms have been reported for each gene and many more will probably be found. There are currently 5400 distinct sequences reported for MHC-class I molecules. An individual possesses two alleles at each MHC-class I gene locus, one maternal and one paternal. Thus, an individual expresses up to six different classical MHC-class I gene products at the cell surface. MHC-class II proteins are formed by hetero-dimerization of α- and β-chains. Humans have three classical MHC-class II proteins, HLA-DR, HLA-DP, and HLADQ. The most polymorphic gene encodes the HLA-DR β-chain and the products of this gene combine with an invariant α-chain to make HLA-DR molecules. In HLADP and HLA-DQ both chains are polymorphic. There are currently around 1600 alleles reported for MHC-class II α- and β-chains, although the number of productive combinations that can form functional proteins is more difficult to estimate. Discussion of the nomenclature and diversity of MHC molecules is complex and beyond the scope of this chapter, and the reader is directed to this reference for further information.28
MHC-CLASS I The MHC-Class I Peptide-Binding Site MHC-class I gene products are type I membrane proteins. Each has a short cytoplasmic sequence, transmembrane domain, a membrane-proximal immunoglobulin domain and a membrane-distal peptide-binding groove. This groove is formed by an eight-strand antiparallel β-pleated sheet “floor” supporting two α-helices that flank the bound peptide. The folded protein is joined by β2-microglobulin which assumes a membrane-proximal position next to the class I immunoglobulin domain, underneath the floor of the peptide-binding groove2 (Fig. 1A). The α-helices that line the MHC-class I peptide-binding groove are closely apposed at both ends and MHC-class I molecules are therefore only able to “comfortably” bind peptides of 8–10 amino acids in length. The peptide is maintained in an extended conformation through a series of interactions. The N- and C-termini of the peptide are bound through a conserved network of hydrogen bonds, and key “anchor” residues have their side-chains buried in pockets found along the peptide-binding groove (Fig. 1B). The interactions mediated by the N- and C-termini represent the bulk of the peptide-binding energy4 and this sequenceindependent feature allows MHC-class I to bind extremely diverse peptides (each allele is estimated to have the ability to bind many thousands of different sequences). The series of pockets in the floor of the peptide-binding groove (Fig. 1C) provide distinct chemical environments that favor certain residues over others, and the polymorphisms in MHC-class I are concentrated in these regions to endow each MHC allele with a unique peptide specificity.10 These pockets place restrictions on size, charge, and polarity of anchor residues and can stabilize the peptides through hydrogen bonds, electrostatic interactions, hydrophobic packing, and van der Waals forces. Importantly, in most human MHC-class I alleles, the pocket that houses the peptide C-terminal residue favors hydrophobic residues and allows basic residues, but is incompatible with peptides that have an acidic residue at this position.
MHC-Class I Peptide-Loading Complex MHC-class I proteins are directed to the endoplasmic reticulum (ER) on synthesis for folding and secretion to the cell surface. MHC-class I molecules are unable to reach the cell surface until they are loaded with peptide. Immediately after synthesis, MHC-class I heavy chain associates with calnexin, a molecular chaperone that resides in the ER (Fig. 2a). Calnexin is associated with ERp57, a protein thiol oxoreductase that influences disulfide bond formation in the nascent MHC-class I heavy chain. Through interaction with these proteins, the heavy chain is folded
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(A)
(B)
(C)
FIGURE 1 (A) Side view of MHC-class I (left) and MHC-class II (right) showing the peptide-binding site at the top and the membrane proximal immunoglobulin domains at the bottom. (B) Looking down on the peptide-binding site, which is formed by α-helices that flank the peptide and supported by an eight-strand β-sheet. Conserved hydrogen bonds that stabilize the peptide backbone are indicated as dashed lines in each structure. Note that in MHC-class II, the α- and β-chains together combine to form the groove, and peptide extends out of the peptide-binding site. The peptide N- and C-termini are indicated with N and C respectively. (C) The same view as (B), but in surface rendering with the peptide removed. The pockets that serve as anchor points for peptide side chains are labeled A–F for MHC-class I and P1–P9 for MHC-class II. PBD files 1S9Y and 1DLH were used to generate this figure. See color plate 23.
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a llowing recruitment of β2-microglobulin, signaling that the MHC-class I molecule is ready for peptide loading (Fig. 2b). In humans, the folded MHC-class I – β2-microglobulin heterodimer then dissociates from calnexin and joins the peptide-loading complex containing calreticulin, transporter associated with antigen processing (TAP), tapasin, and ERp57 (Fig. 2c).19 ERp57 is covalently linked to tapasin through a disulfide bond and this complex bridges
the empty MHC heavy chain to TAP and calreticulin. In addition to its structural role in the peptide-loading complex, biochemical studies suggest that tapasin-ERp57 can modulate the quality of the peptide repertoire by favoring the loading of high-affinity peptides and promoting dissociation of prebound, low affinity peptides.34 TAP is an ATP-dependent transporter that delivers peptides from the cytosol to the ER lumen and comprises two Key
(A) Classical MHC class I Pathway Extracellular
d.
Sec61 chanel + protein Folding MHC class I, calnexin and ERp57
AA A AA AA A AA AA AA
e.
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mRNA, ribosome and nascent protein
β2 microglobulin
Cytosol
A AA AA A AA
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(B) Classical MHC class II Pathway
Translating invariant chain
Extracellular j.
Translating MHC alpha chain Translating MHC beta chain
MVB
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MHC class II, invariant chain trimer
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h. A AAA AA
Cytosol
MHC class II molecule Protein antigen
A AA AA AA AA
AA A AA AA
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MHC class II-CLIP
FIGURE 2 (A) MHC-class I heavy chains are synthesized in the ER (a) and then partially folded with assistance from calnexin and ERp57 (b). After β2 microglobulin joins the MHC-class I heavy chain the heterodimer is incorporated into the peptide-loading complex and loaded with peptides transported from the cytoplasm to the ER lumen by TAP and trimmed to the correct size by ERAP family proteases (c). Peptide-loaded MHC-class I is then transported via the Golgi to the cell surface (d). Proteasomes cleave proteins in to peptides, which can be selected by TAP for transport into the ER (e). (B) MHC-class II chains and invariant chain are synthesized in the ER (f) and fold together into a nonameric complex with 3 invariant chains, and 3 MHC-class II molecules (g). A region of the invariant chain (CLIP) occupies the peptide-binding groove of MHC-class II. This complex is exported via the Golgi to multivesicular bodies in the endocytic compartment (h). Proteolytic cleavage of invariant chain occurs to release MHC-class II molecules and allow HLA-DM mediated release of CLIP and replacement with peptides from endocytozed proteins (i). MHC-class II molecules are transported to the cell surface for display to CD4+ T-cells (j). See color plate 24.
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proteins, TAP1 and TAP2.27 Like MHC-class I, TAP exhibits selectivity for peptides that have hydrophobic or basic C-terminal residues and are between 8 and 16 amino acids in length.13 TAP has the ability to transport peptides that are longer than those preferred by MHC-class I, and proteases that trim the N-terminus are present in the ER lumen to ensure peptides of optimal length can be generated. In humans, ERAP1 and ERAP2 fulfill this role. Studies with ERAP1 have determined that this protease exhibits selectivity for peptides between 9 and 16 amino acids in length and displays little activity for peptides longer or shorter than this. A model has been proposed that suggests the peptide C-terminal residue is bound by ERAP1 in a pocket distinct from the active site and that the distance between these two sites determines the selectivity of ERAP1 for peptide length. If accurate, it would suggest that peptide trimming occurs before loading into MHC-class I.20
The Classical MHC-Class I Pathway The source of peptides displayed by classical MHC-class I molecules and the pathways that generate them have been topics of intense research. Initially, it was assumed that peptides were simply “borrowed” from natural turnover processes, but because proteins often have half-lives of hours to days within a cell, the kinetics of this process did not seem to be congruous with the observation that viral peptides could be displayed by MHC-class I molecules in less than an hour after infection. Although natural turnover processes do account for some of the peptides displayed by MHC-class I, it is now thought that the bulk of peptides are acquired through the cleanup of incomplete synthesis products. Estimates indicate that up to 40% of the products generated by protein synthesis are rapidly degraded because of errors that might include early termination, misfolding, missplicing, and frameshift errors. Whether protein synthesis is simply a messy business or whether these errors are made specifically to generate peptides for the MHC-class I pathway is not known and remains a matter of debate.9 Proteins are targeted to the proteasome after becoming tagged with poly-ubiquitin chains, the reasons for which are diverse and include normal protein turnover, the destruction of improperly synthesized proteins described above, as well as regulatory proteins controlling cell cycle progression and intracellular signaling. The proteasome is a multisubunit complex11 with a cylindrical catalytic core that is gated at each end by regulatory cap particles that are defined by their sedimentation values. The addition of the 19S regulatory caps to the 20S proteasomal core constitutes the 26S proteasome. Poly-ubiquitin chains of the proteins to be degraded are recognized by proteins in the 19S regulatory cap and, in an ATP-dependent process, ubiquitin is removed and the protein unwound to allow entry into the 20S proteasomal core. It is here that cleavage occurs, generating
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peptide fragments between 2 and 25 amino acids in length18 (Fig. 2e). In humans the cylindrical 20S proteasomal core is made up of four heptameric rings. The alpha subunits, α1–-7, flank each end of the cylinder and form narrow openings that are modulated by the 19S regulatory cap. Sandwiched between the α rings are two antiparallel β rings made up of β1–β7 subunits.11 Only three of these contain catalytically active sites, but between them they are able to cut after almost any amino acid: β5 is chymotrypsin-like, cleaving after bulky hydrophobic residues; β2 is trypsin-like and prefers to cleave after basic residues; and β1, which is caspase-like, cleaves after acidic and branched amino acids. The pattern of cleavage for a particular protein is not random, and the same fragments are generated each time a protein is processed because the activity of the catalytic triad in each β subunit is sensitive to the sequence context around a cleavage site. The combination of limited time within the proteasome chamber and preferred sequences determining initial cleavage sites results in consistent, though difficult to predict, peptide products for any given protein. Peptides that are longer than seven amino acids in length are potential ligands for MHC-class I molecules, but most do not survive exposure to cytosolic proteases. A nine amino acid peptide in the cytosol has a half-life of less than 10 s, and therefore only peptides that are rapidly transported into the ER lumen have the opportunity to fold with MHCclass I heavy chains awaiting export to the cell surface.
The Immunoproteasome Almost all cells in the body have the ability to express interferon (IFN) α/β in response to infection. This hallmark of stress, among others, is recognized by natural killer (NK) cells, which in turn secrete IFNγ. IFNγ has potent effects on antigen processing in both the MHC-class I and II pathways and is particularly important in antiviral responses. Under normal cellular conditions, the proteasome is actually quite poor at generating peptides suitable for MHC-class I binding because most products are smaller than the minimal length required for ER import and MHCclass I loading. Additionally, all MHC-class I molecules studied to date prefer hydrophobic or basic anchors at the peptide C-terminus and acidic residues in particular seem to be excluded. This means that peptides generated by β1 cleavage, which occurs after acidic residues, are essentially lost from the MHC-class I repertoire. However, IFNγ exposure causes expression of three new β subunits, β1i, β2i, and β5i, which replace β1, β2, and β5 in the 20S proteasome catalytic core and all but eliminate the caspase-like activity. Thus most peptides processed by this alternative proteasome, termed the “immunoproteasome,” contain suitable C-terminal anchor residues for MHC-class I binding. In addition to replacement of three β subunits, the immunoproteasome also uses an alternative regulatory cap known
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as PA28, REG, or 11S Activator, which is IFNγ-inducible and significantly increases the maximal velocity of peptide hydrolysis compared with the normal 26S proteasome.6 Interestingly, although the expression of the immunoproteasome is IFNγ dependent in most cell types, the alternative subunits are constitutively expressed in some cells of hematopoietic origin, including DCs and lymphoid cells, emphasizing their specialization for antigen processing and presentation. Mice lacking all three alternative catalytic subunits exhibited alterations in the MHC-class I peptide repertoire that were significant enough to cause the rejection of splenocytes transferred from wild type mice despite being matched at all MHC loci.17
A Specialized Thymic Proteasome Within the thymus, the organ where T-cells reactive to the self are deleted to prevent autoimmunity, the cells that mediate this “negative selection” express both the proteasome and the immunoproteasome. Interestingly, there is a third “thymoproteasome” that uses an alternative β5 subunit in a distinct population of thymic stromal cells responsible for “positive selection,” a process that ensures T-cells receive tonic stimulation by MHC for survival in the periphery. The thymoproteasome generates peptides that display significantly reduced affinity for MHC-class I, and it has been noted that mice deficient in this alternative β5 subunit have severe defects in positive selection.26 The mechanism behind this observation is not yet understood, but it has been proposed that unstable MHC-class I peptide complexes may be somehow required for this process or that the thymoproteasome generates unique stimulatory peptides to drive positive selection.
Crosspresentation MHC-class I is expressed ubiquitously and, for the most part, displays peptides derived from intracellular sources, with the notable exception of certain specialized cell types such as CD8αα+ DCs that have the ability to load proteins derived from extracellular sources onto MHC-class I. This specialized pathway, termed crosspresentation, allows priming of cytotoxic CD8+ T-cells by professional antigenpresenting cells that are not directly infected. Endocytic receptors seem to be critical regulators of crosspresentation, with antigens targeted to DEC205, for instance, being efficiently processed for MHC-class I loading, while those targeted to DCIR2 are not. Although it was initially assumed that these markers simply distinguished cells with different inherent abilities to crosspresent, follow-up experiments determined that introduction of DEC205 into the CD8− DC subset that does not normally cross-present, is sufficient to enable cross-presentation to occur.16 The evidence suggests that crosspresentation is proteasome dependent, and therefore a mechanism by which antigen can escape the
Chapter | 93 Peptide Loading of MHC
phagolysosome to reach the cytoplasm must exist. It is considered unlikely that antigens access the cytoplasm through permeabilized phagosomal membranes and research has instead focused on channels that have the ability to translocate proteins to the cytoplasm. The process of ER associated protein degradation (ERAD), which translocates misfolded proteins from the ER to the cytoplasm for degradation by the proteasome provides an attractive solution to this problem, but requires either the recruitment of ER membrane, or more controlled delivery of ERAD components to the phagosomal pathway. Once antigen has reached the cytoplasm it may intersect with the classical MHC-class I loading pathway, but evidence suggests that peptide loading during crosspresentation may actually occur in the phagocytic compartment itself, with the recruitment of proteasomes and peptide-loading machinery directly to the vesicles.15 Although the mechanics of cross-presentation remain controversial, it is clear that this process is extremely important for CD8+ T-cell function.
Nonclassical MHC-Class I Although the products of the classical MHC-class I alleles HLA-A, -B, and -C have a crucial role in binding peptides for display to cytotoxic T-cells, the nonclassical HLA-E, -F, and -G alleles have more poorly defined roles. These proteins do bind peptide, but they are much less polymorphic than HLA-A, -B, and -C, and this is reflected in their restricted peptide repertoire. The best studied of these alleles is HLA-E, which is recognized by the CD94/NKG2 receptor system and seems to have a major role in regulating NK cell activity.32 HLA-E primarily binds peptides that are derived from the signal sequences of other MHC-class I molecules and is believed to convey information about normal MHC biosynthesis to NK cells. NK cells are reactive to alterations in the MHC-class I pathway that are commonly caused by viral proteins evolved to inhibit MHC-mediated immune responses. Normal operation of the pathway results in peptide-loaded HLA-E binding to inhibitory receptors on NK cells and suppression of cytotoxicity, but loss of this signal in virally infected cells can trigger killing. In a striking evolutionary “tit-for-tat,” human cytomegalovirus encodes a homolog of the MHC-class I leader peptide to maintain HLA-E at the cell surface while other virulence factors downregulate classical MHC-class I expression.31
MHC-CLASS II MHC-Class II Peptide-Binding Site Nascent MHC-class II molecules form from α- and β-chains that are assembled in the ER. Like the MHC-class I heavy chain, the β-chain forms a membrane-proximal immunoglobulin domain, but unlike its class I counterpart,
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only contributes to half of the peptide-binding domain. The α-chain helps complete the peptide-binding domain and also contributes a membrane-proximal immunoglobulin domain so that, unlike MHC-class I, MHC-class II does not require β2 microglobulin (Fig. 1). Although the overall fold of MHC-class II molecules resembles that of MHC-class I, there are significant differences that endow these closely related structures with very different peptidebinding properties.5 The peptide is accommodated in the groove in an extended conformation similar to that of a type II poly-proline helix and importantly, because the helices in MHC-class II molecules do not close together at each end of the peptide-binding groove as with MHC-class I proteins, MHC-class II molecules can bind much longer peptides. This means that polar contacts cannot always be made to free N- and C-termini of the peptide and interactions are instead dominated by a conserved network of hydrogen bonds to polar backbone atoms along the entire length of the peptide (Fig. 1B). Structural and biochemical studies indicate that the strongest binding energy is contributed by hydrogen bonds concentrated at the N-terminus of the bound peptide. These sequence-independent interactions are further complemented by anchoring pockets that, like MHC-class I, favor peptides with compatible amino acids (Fig. 1C). The register that any given peptide binds to MHC class II is defined by the residue that binds in the P1 pocket. The peptide residues are sequentially numbered in an N- to C-terminal direction. P1, P4, P6, and P9 residues interact with the major pockets in MHC, while P2, P5, and P8 are major TCR contact points. The P1 and P4 pockets of HLA-DR molecules contribute the most to peptide stability. A natural polymorphism at residue 86 of the HLA-DR β-chain influences the size of the P1 pocket. A glycine at this position provides a large pocket that can accommodate bulky hydrophobic residues while a valine at this position presents a shallower pocket that prefers smaller aliphatic residues.
Invariant Chain Processing and HLA-DM-Mediated Peptide Exchange Each MHC-class II heterodimer assembles in the ER with the invariant chain, a trimeric chaperone that is exported through the Golgi and trans-Golgi network to intersect with the endosomal pathway in multivesicular antigen-processing compartments (Fig. 2f–h).30 The invariant chain serves a number of purposes. A fragment of the invariant chain is embedded in the peptide-binding site to prevent opportunistic binding of proteins/peptides in the ER29 and to stabilize the MHC-class II peptide-binding site, which is prone to collapse and aggregation if left unoccupied. The invariant chain cytoplasmic domain also contains trafficking signals that target MHC-class II to the appropriate subcellular location for peptide loading to occur.
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As the invariant chain occupies the peptide-binding groove, it must be dislodged from MHC-class II to allow new peptide acquisition, and two important processes ensure that this is achieved. First, proteolytic degradation of invariant chain occurs which releases MHC-class II from all but the invariant-chain-derived fragment protecting the peptide-binding site. This fragment is termed CLIP and typically corresponds to residues approximately 80–105 depending on the fine mapping of protease cleavage sites. Degradation of invariant chain involves a number of proteases that are sensitive to the protease inhibitor leupeptin,3 and although the complete pathway has not been elucidated, two proteases in particular play nonredundant roles in the latter steps of invariant chain processing: these are cathepsin S (in the periphery) and cathepsin L (in the thymus).14 Attempts to identify proteases that have a nonredundant role in early steps of invariant chain processing have failed, and this no doubt reflects the susceptibility of the invariant chain to cleavage by multiple enzymes. HLA-DM ensures that the protective CLIP peptide is then exchanged for exogenously derived peptides from the endocytic pool. HLA-DM is a heterodimeric MHC-class II protein that does not itself bind peptides.23 HLA-DM resides in late endosomal compartments and is responsible for “editing” the peptide repertoire to promote formation of high-affinity complexes with very stably bound peptides. To achieve this, HLA-DM binds to MHC-class II molecules that contain suboptimal P1 anchor residues and stabilizes the empty groove to encourage bound peptides to leave and give new peptides access to sample the site. Peptides that supply optimal P1 anchor residues then cause HLA-DM to dissociate from MHC-class II and are protected from further editing by stable P1 interactions between peptide and MHC that block HLA-DM binding.1 HLA-DM is negatively regulated by HLA-DO in a process that is thought to be mediated by competitive binding to HLA-DM in a way that prohibits interaction with MHC-class II complexes. HLA-DO expression depends on cell type and activation state, and is thought to be an important regulatory pathway in B-cells.8
Properties of CLIP The CLIP peptide binds promiscuously to many MHC-class II alleles, albeit with widely variable affinity. For instance, the half-life of a MHC-class II allele that is associated with susceptibility to rheumatoid arthritis (DRB1*0401) in complex with CLIP is in the order of minutes, while another common allele (DRB1*0101) forms complexes that are stable for days. These differences in stability are unsurprising given that MHC-class II is highly polymorphic and those polymophisms are concentrated within the peptide-binding groove. In this context, perhaps what is more surprising is that there exists a single peptide that seems to interact
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with most of the MHC-class II molecules. Only one crystal structure of an MHC-class II molecule in complex with CLIP has been determined. In this structure, methionine 90 of the invariant chain occupies the P1 pocket of the class II molecule HLA-DR3 (DRB1*0301). Almost all of the MHC-class II alleles analyzed thus far through mutagenesis studies appear to bind CLIP in this register, with the notable exceptions of two HLA-DQ alleles associated with celiac disease in which threonine 94 occupies the P1 pocket.35 The peptide-binding site of MHC-class II molecules is open at each end, giving peptides the ability to shift register in the binding site. This, no doubt, allows different alleles to select different segments of the CLIP sequence according to variable motif preferences.
Generation of Peptides for MHC-Class II Loading Antigen that is taken up by phagocytosis, receptor-mediated endocytosis or pinocytosis must traverse the endocytic pathway before loading onto MHC-class II molecules can be accomplished. The endocytic pathway has distinct compartments. Early endosomes are the principal sorting organelles where decisions are made as to whether molecules are exported back to the cell surface (recycling endosomes) or delivered to late endosomes. Early endosomes are mildly acidic and while MHC peptide complexes exhibit extraordinarily long half-lives, under acidic conditions peptide release is accelerated and therefore recycling endosomes are a possible site of peptide exchange. Late endosomes, however, are the principle location of MHC-class II loading (pH ~5.5) (Fig. 2h). There is some debate as to whether there are distinct late endosomes that are specialized for MHC-class II loading (typically referred to as MIIC) or whether the components required for loading are distributed throughout all late endosomes in antigen-presenting cells.25 The final destination in the endocytic pathway is the lysosome. Lysosomes are highly acidified and even MHC-class II molecules, which are acid resistant, are unable to survive for long in this environment. In immature DCs, MHC-class II is targeted to lysosomes for degradation until maturation signals are encountered. MHC-class II is then directed to the late endosomes for peptide loading and then to the cell surface for peptide display. The proteases with the largest role in generating antigenic peptide fragments for MHC-class II are the cathepsins. Cathepsins are primarily cysteine proteases with pH- and redox-dependent activity and are distributed throughout the endosomal compartment.14 Expressed as proforms, they require proteolytic cleavage themselves to become activated. Although some cathepsins can accomplish their own activation through autocatalytic proteolysis, others require a second protease, which itself might display pH dependence. In this manner, the activity of certain cathepsins can
Chapter | 93 Peptide Loading of MHC
be restricted to different parts of the endocytic pathway, and this is believed to influence the rate of proteolysis to ensure large enough protein fragments reach the compartments where peptide loading takes place. MHC-class II molecules bind peptides in extended conformations, and for this to occur, denaturation of secondary structure must take place. In the endosomal pathway proteins are denatured by a combination of events: acidification interferes with salt bridges and hydrogen bonds; IFNγ-inducible lysosomal thiol reductase (GILT) reduces cysteine-mediated disulfide bonds;12 and endoproteases such as cathepsins B, D, F, G, H, K, S, or asparagine endopeptidase (AEP) cleave exposed flexible areas of proteins to cause catastrophic destabilization.14 It is probable that MHC-class II molecules then bind to large polypeptide fragments, which are trimmed from the C- and N-termini by cathepsins with carboxy and amino proteolytic activity, respectively. This allows MHC-class II, which is assisted by HLA-DM, to bind securely to a peptide epitope and protect it from further proteolysis.7 Model antigens have been used in an attempt to determine the relative contribution of proteases to the generation of epitopes recognized by T-cells. Each cathepsin’s activity destroys some epitopes while creating others, but mice that are deficient in individual cathepsins (with the exception of Cathepsin S and L, which are required for invariant chain processing) show only minor defects in antigen presentation, indicating a high degree of redundancy in the system. It has been particularly difficult to ascribe unique roles to individual proteases in the generation of defined peptideMHC-class II complexes because effects on the MHC-class II pathway may be caused by impaired invariant chain processing, impaired epitope generation, or impaired activation of proteases involved in these processes.
Autophagy and MHC-Class II Autophagy, which means to “eat oneself,” is the mechanism by which cells break down and recycle intracellular contents and is important for the maintenance of cellular homeostasis. Normally, autophagic activity is low, but during starvation, cellular stress, or infection, autophagy is upregulated and provides a source of peptides to the MHCclass II pathway. Autophagy is initiated by the recruitment of membrane, probably from the ER, to encircle a large volume of cytosol and its organellar contents. Initiation of this process in response to starvation is controlled by mTOR, which is released from its inhibitory complex when energy levels in the cell fall below a certain threshold. The recruited membrane then fuses to generate a doublemembrane vesicle that is targeted to the lysosomal pathway. Access to the lysosomal pathway not only allows the contents to be degraded into building blocks that can be used by the cell to form new proteins and lipids, but also delivers
SECTION | IX Handbook of Biologically Active Peptides: Immune/Inflammatory Peptides
them to the MHC-class II loading pathway described above. Autophagy by macrophages and DCs in response to infection can be mediated by pattern recognition receptors in the cytoplasm and at the cell surface. Almost all the toll-like receptors studied to date influence the autophagic pathway, and this represents an important defense mechanism for the cell to rid itself of intracellular bacteria and parasites that might otherwise evade detection.24
CONCLUDING REMARKS The MHC system and the peptides it presents to T-cells are at the heart of the adaptive immune response, and while we understand the basics of peptide processing and acquisition, there is much left to be learned. The outcome of a thorough and detailed understanding of these processes should provide the ability to manipulate the timing and location of peptide presentation. The ability to do this has implications in vaccine development, tumor immunity, transplant rejection, and autoimmunity.
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