- Email: [email protected]
Endosomal proteases and antigen processing
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TgBS 22 - OCTOBER 1997
Endoso i p r o t e a and antigen proc ing Beatrice Fineschi and Jim Miller T cells are activated by fragments of antigenic proteins bound to major histocompatibiaity complex (MHC) molecuges and displayed on the cell surface. MHC class H proteins scavenge processed protein antigens from within endosomal compartments. The antigenic peptides are generated within these and other intraceHular compartments using the array of proteolytic enzymes normally invoNed in terminal protein degradation. Antigen-presenting cells use diffe~ent mechanisms to expSoit and control the activity of these enzymes so as to ensure the generation of a wide variety of peptides, while preventing the destruction of antigenic epitopes by excessive proteolysis.
of pept~des derived from proteins produced by the antigen-presenting cell and targeted for degradation in lysosomes and from proteins that have been inter~ nalized or phagocytosed from extrace~lular sources. One of the stalking differences between MI-|C class 1 and class il is the length of the peptide-binding pocket (Fig. 1). Class I selectively associates with short peptides containing 8-11 amino acids and has binding pockets to accommodate the positively and negatively charged peptide termini. This is also the optimal length for transport across the ER by the TAP transporters (see Fig. 2) and so the system has evolved to selectively provide class I MHC with short peptides. The peptide-binding site of class II, however, is open at both ends and can accommodate longer peptides. Although the optimal length is 15-22 amino acid residues, class [I can associate with intact proteins as long as they contain a linear class II peptide-binding motif. This allows class II to associate with a more heterogeneous pool of peptides generated in endosomal compartments. However, it also creates a problem during class II biosynthesis in the ER. The ER is essentially a protein-folding compartment and contains many misfolded and partially folded proteins. To protect the open class B-binding site from being saturated with unfolded proteins in the ER. class ll associates with a nonpolymorphic protein called invariant chain 0i) (Fig. 2). lnvariant chain functions as a surrogate peptide, occupying the class II peptidebinding site and facilitating class I1 dimer assembly and folding in the ER. lnvariant chain also contains the endosomal localization signal that targets the class il-li complex from the transGolgi network CrGN) to endosomes. Once in endosomes, li must be removed to allow the class II peptide-binding site to load with antigenic peptides. This is accomplished through proteolysis of li and is facilitated by a cofactor, HLA-DM (Ref. 5). HLA-DMassociates with class ll in endosomes and appears to stabilize an open conformation, increasing the dissociation rate of peptides from class !!, allowing low-affinity peptides to be replaced with high-affinity peptides. By contrast to the protease sensitivity of It, class II has evolved to be extremely protease resistant, allowing class li to cycle through and collect peptides in acidic, protease-rich compartments. In this review, we will focus on the proteolytic processes involved in class 11 antigen processing and Ii degradation,
derived from self and from non-self proteins, of the approximately 1(1000(1 MHC molecules at the ceil surface only a small number will be associated with a specific antigenic peptide. T cells have evolved to respond to these small numbers and as little as ten to several hundred specific peptide-MHC complexes on the cell surface is sufficient to induce T-cell activationL Thus, although antigen processing is Largely a stochastic process, competition between peptides for MHC binding selects a subset of peptides for display at the cell surface, and the specificity and signaling thresholds of T cells allow for an immune response to small numbers of specific peptide-MHC complexes. There are two classes of MHC molecules, class I and class I!, each specialized at displaying at the cell surface peptides derived from different intracellular sources (Fig. 2; for more-detailed reviews of antigen presentation see Refs 2, 3). Class I molecules predominantly bind peptides during biosynthesis in the endoplasmic reticulum (ER). As such, most peptides are derived from proteins that are synthesized in the antigen-presenting cell, although there are several recent examples of proteins derived from exogenous sources gaining access to class ! molecules4. This bias for peptides derived from endogenously synthesiz,.d proteins focuses the class I antigen-presentation pathway to host cells that are infected with or harboring pathogenic organisms. By contrast, class 11molecules predomiB. Rneschi and J. Miller are in the Department nately bind peptides within endosomal/ of MolecularGeneticsand Cell Biologyand lysosomal compartments. Sampling pepthe Committeeon Cell Physic!ogy,University tides within this compartment allows of Chicago,Chicago,IL 60637, USA. class 11to associate with a broad array Email:[email protected] Copyright© 1997,ElsevierScienceLtd.Allrights reserved. 0968-000,~197/$!7.00 PII: S0968-0004(97)01116-X
THE ORCHF_STP,ATHON OF a complete immune response requires the simultaneous participation of several components of the immune system, including B lymphocytes and T lymphocytes. B cells produce antibodies that can recognize a broad range of chemically distinct molecules in their native form. T cells, by contrast, are limited in their mode of antigen recognition; their response is restricted to linear peptide sequences. For T-cell recognition, these peptides must be displayed at the cell surface in association with molecules encoded in the major histocompatibility complex (MHC). The peptides are generated within antigen-presenting cells through limited proteolysis and are then bound in a groove on the surface of MHC molecules (Fig. 1) and transported to the cell surface. In addition to peptides derived from foreign proteins (e.g. viral or bacterial proteins), MHC molecules also encounter and transport to the cell surface peptides derived from the intracellular processing of self proteins. This broad array of self and non-self peptides displayed on the plasma membrane of antigen-presenting cells is available for T-cell recognition. Most T cells that recognize peptides derived from self proteins are deleted during T-cell development, and so under normal circumstances only peptides derived from a foreign protein will elicit an immune response. Because antigen-presenting cells cannot discriminate between peptides
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and substrate, involving a covalent bond between the reduced sulfur of the cysteine in the active site of the enzyme and the carbonyl carbon of the scissile bond. The resulting acyl enzyme complex is resolved by acid hydrolysis. The basic mechanism for the aspartic proteases is similar, except that the carboxyl groups in the active site are not nucleophilic and rather use a hydrogen-bonded water molecule to attack the carbonyl carbon. The major cathepsins that have been implicated in antigen processing are the aspartic proteases cathepsins D and E, and the cysteine proteases cathepsins B, L and S. All of them are endopeptidases
with the exception of cathepsin B, which displays both endo. and exopeptidase activity. All of the known cathepsins are synthesized with an N-terminal pro-peptide, which is removed during maturation to yield the active form of the enzyme that ranges in molecular weight from 25-40 kDa. Figure 1 Most, but not all, of these enPeptlde-binding pockets of MHC class i and class II. zymes can also be cleaved Molecular surfaces of (a) MHC class I and (b) MHC asymmetrically to yield a class II are shown In white and the peptide in the double-chain form that does peptide-blndlng site is shown in color; carbon atoms not appear to differ in proteoare yellow, nitrogen atoms are blue and oxygen atoms lyric activity from the singleare red. The HLA.A2 (class I) structure bound to a peptide derived from influenza virus matrix protein 59 and chain enzyme Exceptions are the HLA.DR1 (class II) structure bound to peptide decathepsin S, which has been rived from influenza virus hemagglutinin6o are oriented observed only as a single so that the N-terminus of the peptide is on the left. chain, and cathepsin E, which This figure was producedand generously provided by exists as a homodimer. 1". Jardetzky (Northwestern University, Chicago, USA). These endosomal/lysoso. real enzymes function optiand on how antigen-presenting cells regu- mally at acidic pH. Members of the asparlate class il transport and antigen pro. tic proteases favor the most acidic cessing to allow class ii access to a broad conditions, with pH optima ranging bearray of potentially antigenic epitopes. tween 2.8 and 4. Cysteine proteases favor slightly less acidic conditions (pH 5-6) Endosomal proteam Involvedin antigen (Ref. 6 and references therein). Interestmceuing ingly, the ability of cathepsin B to func,antigen processing and Ii degradation tion either as an endo. or an exopeptidase have been shown to occur through the is at least in part determined by pH, valaction of a group of endosomalllysosomal ues less than pH 5.5 favoring its exopepenzymes termed cathepsins. Cathepsins tidase activity8. Most aspartic and cysbelong to two major families of enzymes, teine proteases are inactive at neutral cysteine and aspartic proteases, so classi- pH. One exceptiun is cathepsin S, which fied on the basis of their catalytic mecha- retains most of is proteolytic activity nism (see Refs 6, 7 and references even at pH 8 (Ref. 9). The optimal pH of therein). For cysteine proteases, cataly- each enzyme, however, can vary with sis occurs through the formation of an the species of origin and most dramatiintermediate complex between enzyme cally with the substrate used.
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None of the cathepsins studied appear to recognize a specific substrate sequence. In general, cathepsin E and D have similar substrate specificity (Ref. 10 and references therein) and both appear to prefer cleavage of peptide bonds between hydrophobic amino acids. At least one difference is the effect of proline at position P4*, which can favor cleavage by cathepsin E, while it has a negative effect on cathepsin D. Cathepsin D preferences can also include basic amino acids at positions P2 and P5. The primary specificity of cysteine proteases appears to be determined by the amino acids surrounding the scissile bond and in particular by the residues at positions P2 and P3 (Ref. 97. Cathepsin $ prefers smaller neuh'a~ or hydrophobic amino acids in the P2 and P3 sites while cathepsin L prefers substrates with bulky h~drophobic residues at these positions. Cathepsin B prefers to cleave peptides with two basic res~daes like arginine or lysine at the P1 and ?2 sites, although substrates with hydrophobic residues at position
P2 can also be efficiently cleaved. Most of the present knowledge on cleavage-site preferences derives from ~::~:..=e~~ :~r='a!lsynmetic substrates and the deduced motifs are only sometimes confirmed by the pattern of degradation of intact proteins. This is in part related to both enhancing and inhibitory effects of specific amino acid sidechains near the cleavage site, to non-specific proteasebinding sites and to conformational effects that modulate the sensitivity of individual cleavage sites. Therefore, for each substrate, the exact cleavage site will be determined by sequences surrounding the sdssile bond as well as by the overall structure of the substrate protein. The first studies that specifically implicated cathepsins in antigen processing made use of selective inhibitors of either cysteine or aspartic proteases. Various groups showed that inhibition of pro. teases belonging to either one of these classes abolished presentation of some antigenic epitopes and concluded that members of each family were necessary for antigen processing (see Refs 11, 12 and references therein). In these studies, different antigenic epitopes responded differently to treatment with aspartic or cysteine protease inhibitors, suggesting that proteases belonging to different *Each letter P represents a single aminoacid in the substrate polypeptidechain with P1 to PX proceeding towardthe N-terminusof the substrats and P'I to P'X proceedingtoward the C-terminussuch that: P3-P2-PI'P'I-P'2-P'3.
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Figure 2 Biosynthesis and peptide-loading pathways for MHC class I and class II. (a) A schematic representation of a cell showing the endoplasmic reticulum (ER), Goigi apparatus (GA), endosome and lysosome. Steps in class I biosynthesis are highlighted in green (1-4) and shown in greater detail in Ib}. Steps in class II biosynthesis and peptide loading are highlighted in pink and shown in greater detail if, (c) and (d). For class Irestricted antigen presentation, intact protein antigens are degraded in the cytosol primarily by the multisubunit proteosome, although other cytosolic proteases have also been recently implicated (step 1). The resulting peptides are transported across the membrane of the ER by the TAP transporter, a member of the ABC family of membrane transporters (£tep 2). On the lumenal side of the ER, newly synthesized class I[~2-microglobulin complexes associate with TAPtllrough the anr,illary proteins calreticulin (C) and tapasin (T) (step 3). Peptide loading onto class I releases it from the TAP complex, and the now fully assembled class I-peptide complex fellows the default transport pathway from the ER through the Golgi apparatus and onto the plasma membrane (step 4). For class H.restricted antigen presentation, shortly after biosynthesis, invariant chain (li) assembles into a homomeric trirner (step a) that sequentially loads with three class li el3 heterodimers to form a nine-chain complex (step b). Invariant chain functions in the ER as a surrogate peptide, facilitating class I! assembly and raiding and protecting the class il peptide-binding site from loading with peptides and unfolded proteins (step c). The class II-li complex exits the ER and transits through the Golgi apparatus (step a). In the transGolginetwork, intracellular sorting signals localized into the cytosolic tail of li divert the class II-li complex from the default pathway to the cell surface and into endosomal compartments (step e). The exact nature of these endosomal compartments and the specific events that take place in each compartment is not fully resolved, and in this figure is left as a single large vesicle (enaosome). Acid proteases (cathepsins) within the endosome degrade li, leaving class II associated with a peptide derived from li (CLIP) in the class II peptidebinding site (step f). The same set of proteases are involved in proteolyticp~'ocessingof intact protein antigens into smaller peptides (step g). Class II undergoes peptide exchange, releasing CLIP and binding peptides released from other proteins in a process that is facilitated by the class IIfamily member, HLA-DM (step h). The class II-peptide complex is now transported to the cell surface, where it is available for T-cell recognition.
classes may participate in the processing of aistinct antigenic epitopes. The interpretation of these inhibitor studies has been confounded by three features of antigen processing. First, with a few notable exceptions 13-~5,there are no biochemical reagents to detect specific peptide-MHC complexes. In most cases, the number of complexes generated during antigen processing is too few to
detect biochemically, even if the reagents were available. Therefore, most antigen processing studies rely on highly sensitive and highly specific T-cell assays, which as functional assays can be influenced by factors other than the number of peptide--MHC complexes. Second, as one might expect, in addition to being necessary for the generation of antigenic peptides, endosomal
proteases can also destroy antigenic epitopes. The destructive potential of some proteases was noted by Vidard et aL n, who found that inhibition of cysteine proteases enhanced the ability of a B-cell line to present a subset of antigenic epitopes. At least two of these cysteine proteases, cathepsias B and L, have been directly involved in the destruction of specific antigenic epitopes 16-18.
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and, more recently, catheps[n S in li degradation 2°;-~,27. In addition, it has been shown that cathepsins B and S can mimic natural steps of li degradation by removing |i from class ll-li complexes, freeing the class II peptide-binding site 2°,26. However, as in antigen processing, in vitro proteolysis of I[ is inefficient, suggesting that the sequential activity of several cathepsins may be involved. Antigen processing The intracellular localization of most
cathepsins to endolysosomal vesicles supports their role in the processing of endocytosed antigenic material. Two of these enzymes hi particular, cathepsins D and B, have been co-localized with class H in antigen-processing compartments 2~,29. By contrast, a clear endosomal localizLysosome ation for cathepsin E has not been established, lmmunofluorescence and biotrans-Golgi network chemical studies have localized these e~lzymes primarily to the ER of COS cells Figure 3 and to the cytosol of neutrophils, alIntracellular transport of cathepsins. Mannose-6-phosphate is added to cathepsins early in though cathepsin E has been localized biosynthesis and is recognized in the trans-Golginetwork (TGN)by the mannose phosphate to non-lysosomal cytoplasmic vesicles in receptor (MPR) (step 1). The cathepsin-MPR complex is transported directly from the TGN B cells (see Ref. 30 and references therein). to late endosomes (step 2). In the Iow-pH environment of the late endosomes, the cathepsin-MPR complex dissociates (step 3) and the empty MPR recycles back to the Golgi Cathepsins, like other acid hydro(step 4). The released cathepsins follow the default transport route from the late endolases, are transported to lysosomes seines into lysosomes, where they concentrate (step 5). Some of the MPRs are transthrough interaction with the mannose-6ported from the Golgi to the plasma membrane (step 6), where mis-sorted cathepsins and phosphate receptor (Fig. 3). Although cathepsins from the extracellular medium can be internalized into early endosomes the primary route of transport is from (step 7). This results in the presence of cathepsins throughout the endosomal pathway, the TGN to later endosomes, cathepsins with the highest concentration in lysosomes. have been found throughout the endosomai pathway, with the highest concenFinally, class Ii peptide binding de- more often the fragments are larger than tration in lysosomes. pends on the dissociation of the class ll- known minimal epitopes 1°.23-2s.InterestIn addition to proteoiysis, processing associated ii, a process that involves the [ngly,Van Noort et al. found that although of many antigens requires reduction of same proteases that have been impli- only cathepsin D could initiate the pro- disulfide bonds al and a CHO eel[ line decated In antigen processing. Studies using teolysis of myogiobin, cathepsin B could fective in disulfide-bond reduction has specific protease inhibltors have sug- trim the resulting fragments at their also been shown to be defective in pregested that aspartic proteases are in- C-termini23. senting antigens with disulfide bonds, volved in the initial stages of proteolysis 19 Together these data suggest that for but not those without them 3~.Lysosomes and that cysteine proteases are involved most antigens, processing occurs through are known to have a reducing environin the final steps of li degradation (see the sequential action of more than one ment 3~, but the relative reducing potenRefs 18--20 and references therein). enzyme. In particular, it is possible that tial of different endosomal compartments Thus, it has been difficult in these stud- the endopeptidase activity displayed by has not been determined. ies to assess whether the addition of all the cathepsins may initiate antigen Thus, intracellular degradative comprotease inhibitors had a direct or indi- processing and generate larger fragments partments comprise different populations rect effect on the processing and gener- containing antigenic epitopes. Subse- of organelles that display different pH, difation of specific antigenic peptides. quently, exopeptidases, including cathep- ferent proteolytic activities and possibly An alternative approach to examining sin B, may be responsible for trimming different reducing environments. Endothe role of specific enzymes in antigen the epitopes to their final size. Interest- cytosed material can potentially be proprocessing is to determine whether in ingly, for cathepsin B, the endopeptidase cessed in any of these proteolytic comvitro digestion with isolated enzymes is activity is less pH-dependent than the partments, having access initially to early sufficient to generate epitopes that could exopeptidase activity8, suggesting that endosomes and subsequently to compartbe presented without further processing cathepsin B could initiate antigen pro- ments of increasing proteolytic activity. to T cells. Using this approach cathep- cessing by protein cleavage in an early The search for the intracellular losins B, D and E have been implicated in compartment and trim the peptide in a cations that represent the major site(s) antigen processing ~0,u-z3. Although, in later, more-acidic compartment. of antigen processing suggests that later some cases, in vitro proteolysis of intact Similar studies on the in vitro degra- compartments play an important role in antigens can generate peptides similar in dation of li with isolated cathepsins antigen processing. This idea is supsequence to known minimal epitopes, have implicated cathepsin B, cathepsin D ported by the finding that the majority
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of crass 1i is localized to a specific late endosomal compartment called MilC (reviewed in Ref. 33). However, other studies suggest that class H might be found throughout the endosomal pathway, including in ~ysosomes 33'3~,and functional data support a ro~e for early compartments that are accessible by the transferdn receptor in the generation of some antigenic epitopes 35.36. The results suggest that more than one endosomal compartment might be i~wolved in antigen processing, and recent studies have confirmed that different antigenic epitopes are generated in different locations 37,38. Interestingly, class II may use different signals to access these compartments. Internalization of class H mediated by a signal encoded in the cytoso]ic domain of class lI itself provides access to early endosomal compartments. Deletion of this signal can inhibit presentation of a subset of antigenic epitopes. Presentation of other antigenic epitopes requires targeting of newly synthesized class ll-[i complexes to late endosomal compartments, a transport route that is mediated by signals within the cytosolic domain of li. The involvement of more than one compartment in ~ntigen processing was also directly demonstrated by Griffin et al. (Ref. 39) who showed that purified lowdensity endosomal fractions containing mild proteolytic activity, and high-density endosomal fractions containing high proteolytic activity, harbored functional class il molecules associated with distinct sets of peptides. By targeting class II molecules to different antigen-processing compartments, antigen-presenting cells can maximize the number of antigenic peptides that can be generated from the variety of antigens encountered. Some epitopes may be buried deep inside the structure of the native protein and may require the reducing environment and harsher conditions of later endosomal compartments in order to be released from the intact protein. Newly synthesized class II molecules still associated with li can be targeted to these compartments and may represent the pool of class ll required for the presentation of these epitopes. By contrast, other morelabile antigenic epitopes may be destroyed by those conditions and may require degradation in a milder environment accessible only by recycling class If. Interestingly, a correlation exists between the location of some of these epitopes within the native protein and their proteolytic requirements. The segment of hen egg lysozyme (amino acids 46-61)
that requires targeting to later endosomai compartments for processing is buried inside the molecule, whereas two other determinants (amino acids 35-45 and 1~6-129), which are presented by recycling class H in early endosomaR compartments, are found in a moresuperficial position within the molecule3s. In addition to its role in class lI biosynthesis, li has also been implicated in modulating the proteolytic act!'Aty of endosomes. Presentation of some antigens was found to be selectively enhanced by synthesis of an alternatively spliced form of li, p41, while production of the major form of li, p31, has little or no effect on presentation of the same epitopes 4°. This was surprising because the 'business end' of li (endosomal sorting, class II association and inhibition of peptide loading) is shared between p31 and p41, and both forms can mediate these effects equally Recently, it has been found that the unique fragment of p41, encoded by the alternatively spliced exon, functions as a potent inhibitor of cathepsin L~8,4~ Together with previous reports that cathepsin L can destroy antigenic epitopes ~, these data suggest that p41 could enhance antigen presentation by preserving the integrity of cathepsin L-sensitive peptides. This model is supported by the finding that synthesis of p41 could be mimicked in p4l-negative cells by inhibition of cysteine proteases with synthetic compounds TM. Despite strong evidence suggesting a tunctional role for p41 in the presentation of some antigens, its exact role in vivo is not clear. Takaesu el al. :2 ~:ompared splenocytes from mice that synthesize only p31 or p41 and they did not find significa1~t differences in antigen presentation. One possible resolution of these findings is that p41 may function only in some populations of antigen-presenting cells, in this case, the inhibitory function of p41 may provide an example of a unique property of antigen-presenting cells, the ability to finely tune the proteolytic activity of the antigen-processing compartment in a manner that minimizes the chances of a peptide being destroyed by the very machinery necessary for its generation. The inhibitory activity of the p41 fragment is specific, being primarily restricted to cathepsins L, K and U and only partially inhibiting cathepsin H and papain. It has no effect on the proteolytic activity of the cysteine proteases cathepsin B and S, and it has no effect on proteases of other classes. In addition to the p41 fragment, other segments of the lumenal domain of li share some homology with
cystatins, a familyof less-specific cysteine protease inhibitors 4:~.Thus, li degradation in endosomal compartments may release subfragments that reduce the activity of endopeptidases in the class II peptidebinding compartment, stabilizing peptides for class II association. Cell-ty[~-st~©ifi©antigen p r ~ s i n g The repertoire of proteolytic activities involved in antigen processing is broadened by the fact that the immune system uses several different cell types to process and present antigen to T cells. Major differences in the cellular content of total and specific cathepsins have been observed in many laboratories and these differences may contribute to the ability of different antigen-presenting cell pqpulations, or even different lineages within the same population, to present different antigenic epitopes from intact antigenlZ,38,44.45. in one case, Barbey et al. 46 isolated lysosomes and endosomes from human antigen-presenting ceils derived from different individuals, and demonstrated directly that these differences in antigen processing correlated with differences in the proteolytic activity of the purified lysosomes. Because different antigenpresenting cells have different protease compositions, the appropriate intracellular compartment for antigen processing may differ according to cell type. McCoy et al. 36 noted that, whereas B cells can efficiently present pigeon cytochrome c targeted to an early compartment by the transferrin receptor, a macrophage cell line can only process this antigen if it is internalized by fluid-phase endocytosis. In addition to inherent cell-type differences in protease composition, the pattern of antigen processing by different antigen-presenting cells can be modulated by exposure to cytokines, such as interferon ~/(IFN-~/)47"49.Global effects of cytokines on antigen processing could reflect an increase in cathepsins; cathepsins B, S, L and, in some cases, D can be upregulated by IFN-~/and cathepsin E can be upregulated by the B-cell mitogen Staphylococcus aureus, in addition, granulocyte-macrophage colony-stimulating factor has been shown to increase the intracellular content of reducing agents, which may be important for denaturation of some antigens before proteolysis s°. The fate of antigen may also be determined by the route of internalization. Although many antigens may be presented after non-specific uptake by fluid-phase endocytosis, antigen-presenting cells can use specialized receptors or pathways
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for antigen internalization. Macrophages can engulf particulate antigen, including live pathogens, through phagocytosis, and dend~'itic cells and activated macrophages can internalize large volumes and particulates via a unique form of endocytosis termed macropinocytosis. Internalization through different routes may target antigen to different endosomal compartments that differ in protease composition. Receptor-mediated endocytosis through immunoglobulin receptors on B cells or Fc-receptors un macrophages and dendritic cells can dramatically increase the efficiency of antigen presentation by increasing the relative intracellular concentration of antigensL52. In addition, antibody binding before antigen processing can direct proteolytic events either enhancing the presentation of protected epitopes or suppressing the presentation of other epitopes spanning the same footprinted region (see Ref. 53 and references therein).
the antigen-processing compartment, class 11 molecules function like scavengers that load a sampling of the available peptides and carry them to the cell surface for presentation to T cells. Loading of class 11 with antigenic peptides is aided by the ancillary proteins, li and HLA-DM, which facilitate the generation of class ll-peptide complexes at different stages during biosynthesis and transport to the plasma membrane. The final array of class ll-peptide complexes displayed at the cell surface represents a random sampling of fragments derived from foreign pathogens as well as fragments derived from a cell's own proteins. T cells that recognize MHC-peptide complexes containing peptides derived from self proteins are deleted or inactivated during T-cell development. Thus, the specificity of the immune response is determined not by antigen processing, but rather by T cells that recognize and respond only to class II molecules bound to peptides of unique size and sequence. Changes in the array of peptides that are produced during the course of an immune response could result in the production of variant peptides that can antagonize an ongoing response56.s7.Or alternatively, alterations in the pattern of processing [or self proteins may unmask potent self epitopes and induce autoimmune responses 58. in this regard, consistency in antigen processing is more important than specificity.
CIm II-peptide association The antigenic peptides presented by class II molecules are normally 15-22 amino acid residues in length. Because the peptide-binding groove is open at both ends (see Fig, 1), class I| can accom':.,,,.~-]:~e longer peptides and even denatured intact proteins. The ability of unfolded proteins or large peptides to bind to class !! before proteolysis is complete may play an important role in preserving the Integrity of antigenic epi- A©knowiodgements We thank A. Sant [or heh)|ul suggestopes. Peptlde binding to class ll has been shown in vitro to protect the pep- tions and C. Watts [or sharing data before publication. tide [tom proteolysis ~4. In addition, the ability of class I! to binu to antigen before complete proteo- References 1 Viola, A. and Lanzavecchia, A. (1996) Science iysis may modulate the pattern of anti273, 104-106 gen degradation. By selecting a region of 2 Pieters, J. (1997) Curr. Opin. ImmunoL 9, 89-96 the antigen before all the fragments are 3 York, I. and Rock, K. (1996) Annu. Rev. ImmunoL 14, 369-396 released, MHC-guided processing may 4 Rock, K. (1996) Immunol. Today17, 131-137 determine the efficiency of presentation of 5 Kropshopfer, H., Hammerling, G. and Vogt, A. B. certain epitopes. Because many peptides (1997) Immunol. Today 18, 77-82 are competing for class il binding, the abil- 6 Kirschke,H. and Barrett, A. J. (1987)in Lysosornes: ity of class Uto capture a specific epitope Their Role in Protein Breakdown(Glaumann,H. and Ballard, F. J., eds), pp. 193-238, Academic Press before proteolysis may give that peptide 7 RawUngs, N. D. and Barrett, A. J. (1994) a competitive advantage over other pepMethods Enzymol. 244, 461-511 tides in the same polypeptide. This role of 8 Takahashi, T. et al. (1986) J. Biol. Cllem. 261, class li in selecting specific peptides for 9375-9381 presentation may contribute to the ob9 Brornme,D. et al. (1989) Biochem.1 264, 475-481 served dominance of immune responses 10 Hewitt, E. et al. J. ImmunoL (in press) 11 Vidard, L., Rock, K. L. and Benacerraf, B. against a limited number of epitopes 55. Conclusion Antigen processing is a stochastic process whereby antigen-presenting cells use the normal proteolytic machinery to generate a variety of different fragments from exogenous antigens and self peptides, in
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587-591 26 Roche, P. and Cresswell, P. (1991) Proc. Natl. Acad. ScL U. S. A. 88, 3150-3154 27 Reyes,V. E et at. (1991)J. ImmunoL•46, 3877-3880 28 GuagUardi,L. et al. (1990) Nature 343,133-139 29 Kleijmeer, M. J. et aL (1995) J. Immunol. 154,
5715-5724 30 Finley, E. M. and Kornfeld, S. (1994) J. Biol. Chem. 49, 31259-31266 31 Collins, D. S., Unanue, E. R. and Harding, C. V. (1991) J. ImmunoL 147, 4054-4059 32 Merkel, B. J., Mandet, R., Ryser, H. J-P. and McCoy, K. (1995) J. ImmunoL 154, 128-136 33 Harding, C. (1996) Crit. Rev. Immunol. 16, 13-29 34 Castellino, F, and Germain, R. N. (1995) Immunity 2, 73-88 35 McCoy, K. L., Noone, M., Inman, J. K. and Stutzman, R. (1993) J. ImmunoL 150,1691-1704 36 McCoy, K. L. et aL (1993) J. ImmunoL 151,
6757-6768 37 Pinet, V. et al. (1995) Nature 375, 603-606 38 Zhong, G., Romagnoli, P. and Germain, R. N. (1997) J. Exp. Meal. 185, 429-438 39 Griffin, J. P., Chu, R. and Harding, C. V. (1997) J. ImmunoL 158, 1523-1532 40 Peterson, M. and Miller, J. (1992) Nature 357,
596-598 Bevec,T. et at. (1996) J. Exp.Med. /83,1331-1338 Takaesu,N. T. et al. (1997)J. ImmunoL158,187-199 Katunuma,N. et al. (1994) EEBSLett. 349, 265=269 Demotz, S. et aL (1989) J. ImmunoL 143, 3881-3886 45 Michalek, M. T. et at. (1989) Proc. Natl. Acad. ScL U. S. A, 86, 3316-3320 46 Barbey, C., Watts, C. and Corradtn, G. (1995) Eur. J. ImmunoL 25, 30-36 47 Frosch, S. et al. (1993) Int. ImmunoL 5, 1551-1558 48 Nadler,S. et at. (1994) Eur.J. tmmunoL24, 3124-3130 49 8iegrist, C. A., Martinez-Soria,E., Kern, I. and Mach, B. (1995)I. Exp. Med. 182, 1793-1799 50 Frosch, $. et aL (1993) Eur.J. ImmunoL23, 1430-1434 51 Lanzavecchia,A. (1996) Curr. Opin. Immunol. 8, 348.354 52 Watts, C. (1997) Annu. Rev. ImmunoL 15, 821-850 53 Simitsek, P. D. et aL (1995) J. Exp. Med. 181, 1957-1963 54 Mourtisen, S. et aL (1992) J. Immunol. 149, 1987-1993 55 Sercarz, E. E. et al. (1993) Annu. Rev. Immunol. 11, 729-766 56 Sloan-Lancaster,J. and Allen, P. (1996) Annu. Rev. ImmunoL 14, 1-27 57 Vidal, K. and Allen, P. (1996) Semin. Immunol. 8, 117-122 58 Lanzaveccllia, A. (1995) J. Exp. Med. 181, 1945-1948 59 Madden, D. R., Garboczi, D. N. and Wiley, D. C. (1993) Cell 75, 693-708 60 Stern, L. J. et al. (1994) Nature 368, 215-221 41 42 43 44
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Endoso i p r o t e a and antigen proc ing Beatrice Fineschi and Jim Miller T cells are activated by fragments of antigenic proteins bound to major histocompatibiaity complex (MHC) molecuges and displayed on the cell surface. MHC class H proteins scavenge processed protein antigens from within endosomal compartments. The antigenic peptides are generated within these and other intraceHular compartments using the array of proteolytic enzymes normally invoNed in terminal protein degradation. Antigen-presenting cells use diffe~ent mechanisms to expSoit and control the activity of these enzymes so as to ensure the generation of a wide variety of peptides, while preventing the destruction of antigenic epitopes by excessive proteolysis.
of pept~des derived from proteins produced by the antigen-presenting cell and targeted for degradation in lysosomes and from proteins that have been inter~ nalized or phagocytosed from extrace~lular sources. One of the stalking differences between MI-|C class 1 and class il is the length of the peptide-binding pocket (Fig. 1). Class I selectively associates with short peptides containing 8-11 amino acids and has binding pockets to accommodate the positively and negatively charged peptide termini. This is also the optimal length for transport across the ER by the TAP transporters (see Fig. 2) and so the system has evolved to selectively provide class I MHC with short peptides. The peptide-binding site of class II, however, is open at both ends and can accommodate longer peptides. Although the optimal length is 15-22 amino acid residues, class [I can associate with intact proteins as long as they contain a linear class II peptide-binding motif. This allows class II to associate with a more heterogeneous pool of peptides generated in endosomal compartments. However, it also creates a problem during class II biosynthesis in the ER. The ER is essentially a protein-folding compartment and contains many misfolded and partially folded proteins. To protect the open class B-binding site from being saturated with unfolded proteins in the ER. class ll associates with a nonpolymorphic protein called invariant chain 0i) (Fig. 2). lnvariant chain functions as a surrogate peptide, occupying the class II peptidebinding site and facilitating class I1 dimer assembly and folding in the ER. lnvariant chain also contains the endosomal localization signal that targets the class il-li complex from the transGolgi network CrGN) to endosomes. Once in endosomes, li must be removed to allow the class II peptide-binding site to load with antigenic peptides. This is accomplished through proteolysis of li and is facilitated by a cofactor, HLA-DM (Ref. 5). HLA-DMassociates with class ll in endosomes and appears to stabilize an open conformation, increasing the dissociation rate of peptides from class !!, allowing low-affinity peptides to be replaced with high-affinity peptides. By contrast to the protease sensitivity of It, class II has evolved to be extremely protease resistant, allowing class li to cycle through and collect peptides in acidic, protease-rich compartments. In this review, we will focus on the proteolytic processes involved in class 11 antigen processing and Ii degradation,
derived from self and from non-self proteins, of the approximately 1(1000(1 MHC molecules at the ceil surface only a small number will be associated with a specific antigenic peptide. T cells have evolved to respond to these small numbers and as little as ten to several hundred specific peptide-MHC complexes on the cell surface is sufficient to induce T-cell activationL Thus, although antigen processing is Largely a stochastic process, competition between peptides for MHC binding selects a subset of peptides for display at the cell surface, and the specificity and signaling thresholds of T cells allow for an immune response to small numbers of specific peptide-MHC complexes. There are two classes of MHC molecules, class I and class I!, each specialized at displaying at the cell surface peptides derived from different intracellular sources (Fig. 2; for more-detailed reviews of antigen presentation see Refs 2, 3). Class I molecules predominantly bind peptides during biosynthesis in the endoplasmic reticulum (ER). As such, most peptides are derived from proteins that are synthesized in the antigen-presenting cell, although there are several recent examples of proteins derived from exogenous sources gaining access to class ! molecules4. This bias for peptides derived from endogenously synthesiz,.d proteins focuses the class I antigen-presentation pathway to host cells that are infected with or harboring pathogenic organisms. By contrast, class 11molecules predomiB. Rneschi and J. Miller are in the Department nately bind peptides within endosomal/ of MolecularGeneticsand Cell Biologyand lysosomal compartments. Sampling pepthe Committeeon Cell Physic!ogy,University tides within this compartment allows of Chicago,Chicago,IL 60637, USA. class 11to associate with a broad array Email:[email protected] Copyright© 1997,ElsevierScienceLtd.Allrights reserved. 0968-000,~197/$!7.00 PII: S0968-0004(97)01116-X
THE ORCHF_STP,ATHON OF a complete immune response requires the simultaneous participation of several components of the immune system, including B lymphocytes and T lymphocytes. B cells produce antibodies that can recognize a broad range of chemically distinct molecules in their native form. T cells, by contrast, are limited in their mode of antigen recognition; their response is restricted to linear peptide sequences. For T-cell recognition, these peptides must be displayed at the cell surface in association with molecules encoded in the major histocompatibility complex (MHC). The peptides are generated within antigen-presenting cells through limited proteolysis and are then bound in a groove on the surface of MHC molecules (Fig. 1) and transported to the cell surface. In addition to peptides derived from foreign proteins (e.g. viral or bacterial proteins), MHC molecules also encounter and transport to the cell surface peptides derived from the intracellular processing of self proteins. This broad array of self and non-self peptides displayed on the plasma membrane of antigen-presenting cells is available for T-cell recognition. Most T cells that recognize peptides derived from self proteins are deleted during T-cell development, and so under normal circumstances only peptides derived from a foreign protein will elicit an immune response. Because antigen-presenting cells cannot discriminate between peptides
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and substrate, involving a covalent bond between the reduced sulfur of the cysteine in the active site of the enzyme and the carbonyl carbon of the scissile bond. The resulting acyl enzyme complex is resolved by acid hydrolysis. The basic mechanism for the aspartic proteases is similar, except that the carboxyl groups in the active site are not nucleophilic and rather use a hydrogen-bonded water molecule to attack the carbonyl carbon. The major cathepsins that have been implicated in antigen processing are the aspartic proteases cathepsins D and E, and the cysteine proteases cathepsins B, L and S. All of them are endopeptidases
with the exception of cathepsin B, which displays both endo. and exopeptidase activity. All of the known cathepsins are synthesized with an N-terminal pro-peptide, which is removed during maturation to yield the active form of the enzyme that ranges in molecular weight from 25-40 kDa. Figure 1 Most, but not all, of these enPeptlde-binding pockets of MHC class i and class II. zymes can also be cleaved Molecular surfaces of (a) MHC class I and (b) MHC asymmetrically to yield a class II are shown In white and the peptide in the double-chain form that does peptide-blndlng site is shown in color; carbon atoms not appear to differ in proteoare yellow, nitrogen atoms are blue and oxygen atoms lyric activity from the singleare red. The HLA.A2 (class I) structure bound to a peptide derived from influenza virus matrix protein 59 and chain enzyme Exceptions are the HLA.DR1 (class II) structure bound to peptide decathepsin S, which has been rived from influenza virus hemagglutinin6o are oriented observed only as a single so that the N-terminus of the peptide is on the left. chain, and cathepsin E, which This figure was producedand generously provided by exists as a homodimer. 1". Jardetzky (Northwestern University, Chicago, USA). These endosomal/lysoso. real enzymes function optiand on how antigen-presenting cells regu- mally at acidic pH. Members of the asparlate class il transport and antigen pro. tic proteases favor the most acidic cessing to allow class ii access to a broad conditions, with pH optima ranging bearray of potentially antigenic epitopes. tween 2.8 and 4. Cysteine proteases favor slightly less acidic conditions (pH 5-6) Endosomal proteam Involvedin antigen (Ref. 6 and references therein). Interestmceuing ingly, the ability of cathepsin B to func,antigen processing and Ii degradation tion either as an endo. or an exopeptidase have been shown to occur through the is at least in part determined by pH, valaction of a group of endosomalllysosomal ues less than pH 5.5 favoring its exopepenzymes termed cathepsins. Cathepsins tidase activity8. Most aspartic and cysbelong to two major families of enzymes, teine proteases are inactive at neutral cysteine and aspartic proteases, so classi- pH. One exceptiun is cathepsin S, which fied on the basis of their catalytic mecha- retains most of is proteolytic activity nism (see Refs 6, 7 and references even at pH 8 (Ref. 9). The optimal pH of therein). For cysteine proteases, cataly- each enzyme, however, can vary with sis occurs through the formation of an the species of origin and most dramatiintermediate complex between enzyme cally with the substrate used.
375
None of the cathepsins studied appear to recognize a specific substrate sequence. In general, cathepsin E and D have similar substrate specificity (Ref. 10 and references therein) and both appear to prefer cleavage of peptide bonds between hydrophobic amino acids. At least one difference is the effect of proline at position P4*, which can favor cleavage by cathepsin E, while it has a negative effect on cathepsin D. Cathepsin D preferences can also include basic amino acids at positions P2 and P5. The primary specificity of cysteine proteases appears to be determined by the amino acids surrounding the scissile bond and in particular by the residues at positions P2 and P3 (Ref. 97. Cathepsin $ prefers smaller neuh'a~ or hydrophobic amino acids in the P2 and P3 sites while cathepsin L prefers substrates with bulky h~drophobic residues at these positions. Cathepsin B prefers to cleave peptides with two basic res~daes like arginine or lysine at the P1 and ?2 sites, although substrates with hydrophobic residues at position
P2 can also be efficiently cleaved. Most of the present knowledge on cleavage-site preferences derives from ~::~:..=e~~ :~r='a!lsynmetic substrates and the deduced motifs are only sometimes confirmed by the pattern of degradation of intact proteins. This is in part related to both enhancing and inhibitory effects of specific amino acid sidechains near the cleavage site, to non-specific proteasebinding sites and to conformational effects that modulate the sensitivity of individual cleavage sites. Therefore, for each substrate, the exact cleavage site will be determined by sequences surrounding the sdssile bond as well as by the overall structure of the substrate protein. The first studies that specifically implicated cathepsins in antigen processing made use of selective inhibitors of either cysteine or aspartic proteases. Various groups showed that inhibition of pro. teases belonging to either one of these classes abolished presentation of some antigenic epitopes and concluded that members of each family were necessary for antigen processing (see Refs 11, 12 and references therein). In these studies, different antigenic epitopes responded differently to treatment with aspartic or cysteine protease inhibitors, suggesting that proteases belonging to different *Each letter P represents a single aminoacid in the substrate polypeptidechain with P1 to PX proceeding towardthe N-terminusof the substrats and P'I to P'X proceedingtoward the C-terminussuch that: P3-P2-PI'P'I-P'2-P'3.
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Figure 2 Biosynthesis and peptide-loading pathways for MHC class I and class II. (a) A schematic representation of a cell showing the endoplasmic reticulum (ER), Goigi apparatus (GA), endosome and lysosome. Steps in class I biosynthesis are highlighted in green (1-4) and shown in greater detail in Ib}. Steps in class II biosynthesis and peptide loading are highlighted in pink and shown in greater detail if, (c) and (d). For class Irestricted antigen presentation, intact protein antigens are degraded in the cytosol primarily by the multisubunit proteosome, although other cytosolic proteases have also been recently implicated (step 1). The resulting peptides are transported across the membrane of the ER by the TAP transporter, a member of the ABC family of membrane transporters (£tep 2). On the lumenal side of the ER, newly synthesized class I[~2-microglobulin complexes associate with TAPtllrough the anr,illary proteins calreticulin (C) and tapasin (T) (step 3). Peptide loading onto class I releases it from the TAP complex, and the now fully assembled class I-peptide complex fellows the default transport pathway from the ER through the Golgi apparatus and onto the plasma membrane (step 4). For class H.restricted antigen presentation, shortly after biosynthesis, invariant chain (li) assembles into a homomeric trirner (step a) that sequentially loads with three class li el3 heterodimers to form a nine-chain complex (step b). Invariant chain functions in the ER as a surrogate peptide, facilitating class I! assembly and raiding and protecting the class il peptide-binding site from loading with peptides and unfolded proteins (step c). The class II-li complex exits the ER and transits through the Golgi apparatus (step a). In the transGolginetwork, intracellular sorting signals localized into the cytosolic tail of li divert the class II-li complex from the default pathway to the cell surface and into endosomal compartments (step e). The exact nature of these endosomal compartments and the specific events that take place in each compartment is not fully resolved, and in this figure is left as a single large vesicle (enaosome). Acid proteases (cathepsins) within the endosome degrade li, leaving class II associated with a peptide derived from li (CLIP) in the class II peptidebinding site (step f). The same set of proteases are involved in proteolyticp~'ocessingof intact protein antigens into smaller peptides (step g). Class II undergoes peptide exchange, releasing CLIP and binding peptides released from other proteins in a process that is facilitated by the class IIfamily member, HLA-DM (step h). The class II-peptide complex is now transported to the cell surface, where it is available for T-cell recognition.
classes may participate in the processing of aistinct antigenic epitopes. The interpretation of these inhibitor studies has been confounded by three features of antigen processing. First, with a few notable exceptions 13-~5,there are no biochemical reagents to detect specific peptide-MHC complexes. In most cases, the number of complexes generated during antigen processing is too few to
detect biochemically, even if the reagents were available. Therefore, most antigen processing studies rely on highly sensitive and highly specific T-cell assays, which as functional assays can be influenced by factors other than the number of peptide--MHC complexes. Second, as one might expect, in addition to being necessary for the generation of antigenic peptides, endosomal
proteases can also destroy antigenic epitopes. The destructive potential of some proteases was noted by Vidard et aL n, who found that inhibition of cysteine proteases enhanced the ability of a B-cell line to present a subset of antigenic epitopes. At least two of these cysteine proteases, cathepsias B and L, have been directly involved in the destruction of specific antigenic epitopes 16-18.
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and, more recently, catheps[n S in li degradation 2°;-~,27. In addition, it has been shown that cathepsins B and S can mimic natural steps of li degradation by removing |i from class ll-li complexes, freeing the class II peptide-binding site 2°,26. However, as in antigen processing, in vitro proteolysis of I[ is inefficient, suggesting that the sequential activity of several cathepsins may be involved. Antigen processing The intracellular localization of most
cathepsins to endolysosomal vesicles supports their role in the processing of endocytosed antigenic material. Two of these enzymes hi particular, cathepsins D and B, have been co-localized with class H in antigen-processing compartments 2~,29. By contrast, a clear endosomal localizLysosome ation for cathepsin E has not been established, lmmunofluorescence and biotrans-Golgi network chemical studies have localized these e~lzymes primarily to the ER of COS cells Figure 3 and to the cytosol of neutrophils, alIntracellular transport of cathepsins. Mannose-6-phosphate is added to cathepsins early in though cathepsin E has been localized biosynthesis and is recognized in the trans-Golginetwork (TGN)by the mannose phosphate to non-lysosomal cytoplasmic vesicles in receptor (MPR) (step 1). The cathepsin-MPR complex is transported directly from the TGN B cells (see Ref. 30 and references therein). to late endosomes (step 2). In the Iow-pH environment of the late endosomes, the cathepsin-MPR complex dissociates (step 3) and the empty MPR recycles back to the Golgi Cathepsins, like other acid hydro(step 4). The released cathepsins follow the default transport route from the late endolases, are transported to lysosomes seines into lysosomes, where they concentrate (step 5). Some of the MPRs are transthrough interaction with the mannose-6ported from the Golgi to the plasma membrane (step 6), where mis-sorted cathepsins and phosphate receptor (Fig. 3). Although cathepsins from the extracellular medium can be internalized into early endosomes the primary route of transport is from (step 7). This results in the presence of cathepsins throughout the endosomal pathway, the TGN to later endosomes, cathepsins with the highest concentration in lysosomes. have been found throughout the endosomai pathway, with the highest concenFinally, class Ii peptide binding de- more often the fragments are larger than tration in lysosomes. pends on the dissociation of the class ll- known minimal epitopes 1°.23-2s.InterestIn addition to proteoiysis, processing associated ii, a process that involves the [ngly,Van Noort et al. found that although of many antigens requires reduction of same proteases that have been impli- only cathepsin D could initiate the pro- disulfide bonds al and a CHO eel[ line decated In antigen processing. Studies using teolysis of myogiobin, cathepsin B could fective in disulfide-bond reduction has specific protease inhibltors have sug- trim the resulting fragments at their also been shown to be defective in pregested that aspartic proteases are in- C-termini23. senting antigens with disulfide bonds, volved in the initial stages of proteolysis 19 Together these data suggest that for but not those without them 3~.Lysosomes and that cysteine proteases are involved most antigens, processing occurs through are known to have a reducing environin the final steps of li degradation (see the sequential action of more than one ment 3~, but the relative reducing potenRefs 18--20 and references therein). enzyme. In particular, it is possible that tial of different endosomal compartments Thus, it has been difficult in these stud- the endopeptidase activity displayed by has not been determined. ies to assess whether the addition of all the cathepsins may initiate antigen Thus, intracellular degradative comprotease inhibitors had a direct or indi- processing and generate larger fragments partments comprise different populations rect effect on the processing and gener- containing antigenic epitopes. Subse- of organelles that display different pH, difation of specific antigenic peptides. quently, exopeptidases, including cathep- ferent proteolytic activities and possibly An alternative approach to examining sin B, may be responsible for trimming different reducing environments. Endothe role of specific enzymes in antigen the epitopes to their final size. Interest- cytosed material can potentially be proprocessing is to determine whether in ingly, for cathepsin B, the endopeptidase cessed in any of these proteolytic comvitro digestion with isolated enzymes is activity is less pH-dependent than the partments, having access initially to early sufficient to generate epitopes that could exopeptidase activity8, suggesting that endosomes and subsequently to compartbe presented without further processing cathepsin B could initiate antigen pro- ments of increasing proteolytic activity. to T cells. Using this approach cathep- cessing by protein cleavage in an early The search for the intracellular losins B, D and E have been implicated in compartment and trim the peptide in a cations that represent the major site(s) antigen processing ~0,u-z3. Although, in later, more-acidic compartment. of antigen processing suggests that later some cases, in vitro proteolysis of intact Similar studies on the in vitro degra- compartments play an important role in antigens can generate peptides similar in dation of li with isolated cathepsins antigen processing. This idea is supsequence to known minimal epitopes, have implicated cathepsin B, cathepsin D ported by the finding that the majority
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of crass 1i is localized to a specific late endosomal compartment called MilC (reviewed in Ref. 33). However, other studies suggest that class H might be found throughout the endosomal pathway, including in ~ysosomes 33'3~,and functional data support a ro~e for early compartments that are accessible by the transferdn receptor in the generation of some antigenic epitopes 35.36. The results suggest that more than one endosomal compartment might be i~wolved in antigen processing, and recent studies have confirmed that different antigenic epitopes are generated in different locations 37,38. Interestingly, class II may use different signals to access these compartments. Internalization of class H mediated by a signal encoded in the cytoso]ic domain of class lI itself provides access to early endosomal compartments. Deletion of this signal can inhibit presentation of a subset of antigenic epitopes. Presentation of other antigenic epitopes requires targeting of newly synthesized class ll-[i complexes to late endosomal compartments, a transport route that is mediated by signals within the cytosolic domain of li. The involvement of more than one compartment in ~ntigen processing was also directly demonstrated by Griffin et al. (Ref. 39) who showed that purified lowdensity endosomal fractions containing mild proteolytic activity, and high-density endosomal fractions containing high proteolytic activity, harbored functional class il molecules associated with distinct sets of peptides. By targeting class II molecules to different antigen-processing compartments, antigen-presenting cells can maximize the number of antigenic peptides that can be generated from the variety of antigens encountered. Some epitopes may be buried deep inside the structure of the native protein and may require the reducing environment and harsher conditions of later endosomal compartments in order to be released from the intact protein. Newly synthesized class II molecules still associated with li can be targeted to these compartments and may represent the pool of class ll required for the presentation of these epitopes. By contrast, other morelabile antigenic epitopes may be destroyed by those conditions and may require degradation in a milder environment accessible only by recycling class If. Interestingly, a correlation exists between the location of some of these epitopes within the native protein and their proteolytic requirements. The segment of hen egg lysozyme (amino acids 46-61)
that requires targeting to later endosomai compartments for processing is buried inside the molecule, whereas two other determinants (amino acids 35-45 and 1~6-129), which are presented by recycling class H in early endosomaR compartments, are found in a moresuperficial position within the molecule3s. In addition to its role in class lI biosynthesis, li has also been implicated in modulating the proteolytic act!'Aty of endosomes. Presentation of some antigens was found to be selectively enhanced by synthesis of an alternatively spliced form of li, p41, while production of the major form of li, p31, has little or no effect on presentation of the same epitopes 4°. This was surprising because the 'business end' of li (endosomal sorting, class II association and inhibition of peptide loading) is shared between p31 and p41, and both forms can mediate these effects equally Recently, it has been found that the unique fragment of p41, encoded by the alternatively spliced exon, functions as a potent inhibitor of cathepsin L~8,4~ Together with previous reports that cathepsin L can destroy antigenic epitopes ~, these data suggest that p41 could enhance antigen presentation by preserving the integrity of cathepsin L-sensitive peptides. This model is supported by the finding that synthesis of p41 could be mimicked in p4l-negative cells by inhibition of cysteine proteases with synthetic compounds TM. Despite strong evidence suggesting a tunctional role for p41 in the presentation of some antigens, its exact role in vivo is not clear. Takaesu el al. :2 ~:ompared splenocytes from mice that synthesize only p31 or p41 and they did not find significa1~t differences in antigen presentation. One possible resolution of these findings is that p41 may function only in some populations of antigen-presenting cells, in this case, the inhibitory function of p41 may provide an example of a unique property of antigen-presenting cells, the ability to finely tune the proteolytic activity of the antigen-processing compartment in a manner that minimizes the chances of a peptide being destroyed by the very machinery necessary for its generation. The inhibitory activity of the p41 fragment is specific, being primarily restricted to cathepsins L, K and U and only partially inhibiting cathepsin H and papain. It has no effect on the proteolytic activity of the cysteine proteases cathepsin B and S, and it has no effect on proteases of other classes. In addition to the p41 fragment, other segments of the lumenal domain of li share some homology with
cystatins, a familyof less-specific cysteine protease inhibitors 4:~.Thus, li degradation in endosomal compartments may release subfragments that reduce the activity of endopeptidases in the class II peptidebinding compartment, stabilizing peptides for class II association. Cell-ty[~-st~©ifi©antigen p r ~ s i n g The repertoire of proteolytic activities involved in antigen processing is broadened by the fact that the immune system uses several different cell types to process and present antigen to T cells. Major differences in the cellular content of total and specific cathepsins have been observed in many laboratories and these differences may contribute to the ability of different antigen-presenting cell pqpulations, or even different lineages within the same population, to present different antigenic epitopes from intact antigenlZ,38,44.45. in one case, Barbey et al. 46 isolated lysosomes and endosomes from human antigen-presenting ceils derived from different individuals, and demonstrated directly that these differences in antigen processing correlated with differences in the proteolytic activity of the purified lysosomes. Because different antigenpresenting cells have different protease compositions, the appropriate intracellular compartment for antigen processing may differ according to cell type. McCoy et al. 36 noted that, whereas B cells can efficiently present pigeon cytochrome c targeted to an early compartment by the transferrin receptor, a macrophage cell line can only process this antigen if it is internalized by fluid-phase endocytosis. In addition to inherent cell-type differences in protease composition, the pattern of antigen processing by different antigen-presenting cells can be modulated by exposure to cytokines, such as interferon ~/(IFN-~/)47"49.Global effects of cytokines on antigen processing could reflect an increase in cathepsins; cathepsins B, S, L and, in some cases, D can be upregulated by IFN-~/and cathepsin E can be upregulated by the B-cell mitogen Staphylococcus aureus, in addition, granulocyte-macrophage colony-stimulating factor has been shown to increase the intracellular content of reducing agents, which may be important for denaturation of some antigens before proteolysis s°. The fate of antigen may also be determined by the route of internalization. Although many antigens may be presented after non-specific uptake by fluid-phase endocytosis, antigen-presenting cells can use specialized receptors or pathways
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for antigen internalization. Macrophages can engulf particulate antigen, including live pathogens, through phagocytosis, and dend~'itic cells and activated macrophages can internalize large volumes and particulates via a unique form of endocytosis termed macropinocytosis. Internalization through different routes may target antigen to different endosomal compartments that differ in protease composition. Receptor-mediated endocytosis through immunoglobulin receptors on B cells or Fc-receptors un macrophages and dendritic cells can dramatically increase the efficiency of antigen presentation by increasing the relative intracellular concentration of antigensL52. In addition, antibody binding before antigen processing can direct proteolytic events either enhancing the presentation of protected epitopes or suppressing the presentation of other epitopes spanning the same footprinted region (see Ref. 53 and references therein).
the antigen-processing compartment, class 11 molecules function like scavengers that load a sampling of the available peptides and carry them to the cell surface for presentation to T cells. Loading of class 11 with antigenic peptides is aided by the ancillary proteins, li and HLA-DM, which facilitate the generation of class ll-peptide complexes at different stages during biosynthesis and transport to the plasma membrane. The final array of class ll-peptide complexes displayed at the cell surface represents a random sampling of fragments derived from foreign pathogens as well as fragments derived from a cell's own proteins. T cells that recognize MHC-peptide complexes containing peptides derived from self proteins are deleted or inactivated during T-cell development. Thus, the specificity of the immune response is determined not by antigen processing, but rather by T cells that recognize and respond only to class II molecules bound to peptides of unique size and sequence. Changes in the array of peptides that are produced during the course of an immune response could result in the production of variant peptides that can antagonize an ongoing response56.s7.Or alternatively, alterations in the pattern of processing [or self proteins may unmask potent self epitopes and induce autoimmune responses 58. in this regard, consistency in antigen processing is more important than specificity.
CIm II-peptide association The antigenic peptides presented by class II molecules are normally 15-22 amino acid residues in length. Because the peptide-binding groove is open at both ends (see Fig, 1), class I| can accom':.,,,.~-]:~e longer peptides and even denatured intact proteins. The ability of unfolded proteins or large peptides to bind to class !! before proteolysis is complete may play an important role in preserving the Integrity of antigenic epi- A©knowiodgements We thank A. Sant [or heh)|ul suggestopes. Peptlde binding to class ll has been shown in vitro to protect the pep- tions and C. Watts [or sharing data before publication. tide [tom proteolysis ~4. In addition, the ability of class I! to binu to antigen before complete proteo- References 1 Viola, A. and Lanzavecchia, A. (1996) Science iysis may modulate the pattern of anti273, 104-106 gen degradation. By selecting a region of 2 Pieters, J. (1997) Curr. Opin. ImmunoL 9, 89-96 the antigen before all the fragments are 3 York, I. and Rock, K. (1996) Annu. Rev. ImmunoL 14, 369-396 released, MHC-guided processing may 4 Rock, K. (1996) Immunol. Today17, 131-137 determine the efficiency of presentation of 5 Kropshopfer, H., Hammerling, G. and Vogt, A. B. certain epitopes. Because many peptides (1997) Immunol. Today 18, 77-82 are competing for class il binding, the abil- 6 Kirschke,H. and Barrett, A. J. (1987)in Lysosornes: ity of class Uto capture a specific epitope Their Role in Protein Breakdown(Glaumann,H. and Ballard, F. J., eds), pp. 193-238, Academic Press before proteolysis may give that peptide 7 RawUngs, N. D. and Barrett, A. J. (1994) a competitive advantage over other pepMethods Enzymol. 244, 461-511 tides in the same polypeptide. This role of 8 Takahashi, T. et al. (1986) J. Biol. Cllem. 261, class li in selecting specific peptides for 9375-9381 presentation may contribute to the ob9 Brornme,D. et al. (1989) Biochem.1 264, 475-481 served dominance of immune responses 10 Hewitt, E. et al. J. ImmunoL (in press) 11 Vidard, L., Rock, K. L. and Benacerraf, B. against a limited number of epitopes 55. Conclusion Antigen processing is a stochastic process whereby antigen-presenting cells use the normal proteolytic machinery to generate a variety of different fragments from exogenous antigens and self peptides, in
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