Autoimmune epitopes: autoepitopes

Autoimmune epitopes: autoepitopes

Autoimmunity Reviews 3 (2004) 487 – 492 www.elsevier.com/locate/autrev Autoimmune epitopes: autoepitopes Ian R. Mackay*, Merrill J. Rowley Department...

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Autoimmunity Reviews 3 (2004) 487 – 492 www.elsevier.com/locate/autrev

Autoimmune epitopes: autoepitopes Ian R. Mackay*, Merrill J. Rowley Department of Biochemistry and Molecular Biology, Monash University Clayton, 3800, Victoria Australia Available online 21 August 2004

Abstract The identity of reactants for autoantibodies has been successively refined from whole cellular organelles (immunofluorescence), identified molecules (immunoblot; gene expression libraries), epitope regions (truncated cDNAs; peptide scanning) to contact residues, as described here. Most autoantibodies react with conformational epitopes, in which amino acids distant in the linear sequence come into contiguity by protein folding. Identification of contact sites with the antibody paratope requires particular technologies, crystallography, or antibody screening of phage-displayed random peptide libraries. The latter is illustrated by our studies on the autoepitope for anti-PDC-E2 (AMA) in primary biliary cirrhosis (PBC), anti-GAD65 in type 1 diabetes, and anti-C1 of type II collagen in collagen-induced arthritis. More precise definition of the structure of conformational autoepitopes could (a) clarify controversial aspects of autoimmunity including epitope mimicry, epitope spreading, and molecular spatial relationships between B and T cell autoepitopes, and (b) impact on novel diagnostic and therapeutic (vaccine) molecules. D 2004 Published by Elsevier B.V. Keywords: Autoimmunity; Epitope; Pyruvate dehydrogenase E2; Glutamic acid decarboxylase; Type II collagen

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Historical background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mapping of autoepitopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pyruvate dehydrogenase complex E2: autoantigen for PBC . . . . . . . . . . . Glutamic acid decarboxylase (GAD) 65 kDa—autoantigen for Type 1 diabetes . Type II collagen (CII) : autoantigen for inflammatory arthritis. . . . . . . . . . T cell autoepitopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Victoria, Australia. Tel.: +61 3 96820717; fax: +61 3 99054699. E-mail address: [email protected] (I.R. Mackay). 1568-9972/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.autrev.2004.07.011

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Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The many fascinating aspects to autoimmune epitopes include the key question on why these are so uniquely selected in autoimmunity from among the myriads of possible molecular configurations expressed in the organism. The answer could well reveal the solution to genesis of autoimmunity. Questions of interest are whether autoimmune epitopes constitute just a few dominant or multiple disparate sequences on an autoantigen, the frequency and significance of spreading (recruitment) of epitopes, relationships between epitope locations for B and T lymphocytes, and whether molecular specification of epitopes can provide support for epitope mimicry as a trigger for autoimmunity. In this synoptic review, we consider the analysis of autoepitopes in three selected diseases, primary biliary cirrhosis (PBC), autoimmune Type 1 diabetes mellitus, and rheumatoid arthritis (RA).

2. Historical background Burnet [1] in his classic 1957 monograph on clonal selection, in recounting his version of bThe Facts of ImmunityQ, depicts a representative antigenic molecule showing the entire surface occupied by potential

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antigenic determinants (Fig. 1). Just a few of these, A–D, are specified as being bactiveQ in a capacity to elicit a corresponding antibody population a–d, with the remainder being binactiveQ by reason of their identity with a self motif. Burnet cited Kabat in proposing that an antigenic determinant might comprise as few as two to four critical amino acids. Jerne [2] in 1960 introduced epitope in the context of a variety of synonyms to denote an antigenic determinant, and paratope as the site on the antibody molecule that engaged the epitope; this became the accepted terminology. Contemporaneously, in the early 1960s, indirect immunofluorescence (IIF) became widely adopted to detect the presence in tissues of antigenic reactants with suspected autoimmune sera. Although numerous autoantigenic reactivities did become recognized, the readouts could be described only in terms of gross cellular organelles, i.e., as antibodies to nuclei (ANA), mitochondria (AMA), microsomes, etc., or as whole cytoplasmic specificities, such as thyroid, gastric, or pancreatic islet cell cytoplasms. It was not until the 1980s that analytical and molecular techniques were to provide insights into the identity of reactants revealed by the fluorescence microscope. Primary biliary cirrhosis (PBC) was one of the earliest diseases to be studied in this context when AMA-positive sera were studied by immunoblotting on fractionated

Fig. 1. Burnet’s diagram in 1959 of antigenic determinants (epitopes) with his legend stating: bNote that of the large number of potential antigenic determinants only those not represented in the components of the reacting animal are active.Q At that time, autoimmunity had not become widely accepted, autoantigens not visualized, T and B cells not discovered, and the nature of protein structure not known.

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mitochondria, thereby revealing specific reactivity with components of ~70 and ~48 kDa, leading to the consideration of the identity of the actual autoepitope [3]. Then came autoantibody screening of gene expression libraries in the bacteriophage Egt11 grown in E. coli; amplification of antibody-reactive colonies and sequencing of cDNA inserts allowed for deduction of protein sequences and thus the identity of the autoimmune reactant [4]. Again, one of foremost of the diseases studied by this technology was PBC, with eventual revelation of the reactant, initially detected by complement fixation in 1958, by immunofluorescence as AMA in 1965, and by immunoblot (70 kDa) in 1987, as the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2; [5,6]). Thereafter, numerous other autoantigens similarly became identified.

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itive identification of epitope residues in actual contact with the antibody paratope requires the crystallographic solution of complexes of antigenic molecules with antibody, but antibody screening of random peptide libraries displayed on coat proteins of bacteriophages can often be used to identify sufficient contact residues to localise a conformational epitope or to derive a mimotope which reflects the conformational features of the epitope. The technique involves biopanning with the autoantibody and comparison of peptide inserts of the selected phage with primary sequence data, and, if possible, with a structural model of the antigen. Phage library screening has particular advantages and limitations, as recently reviewed by Rowley et al. [11]. We here describe the utility of phage display to map conformational autoepitopes based on three exemplary autoantigens.

3. Mapping of autoepitopes The question that then arose was the precise location on the autoantigen of the reactant, i.e., the actual autoepitope. Various strategies were developed based on the supposition that an antibody epitope could be defined in terms of a linear amino acid sequence. The techniques included expression of proteins from an enzymatically truncated cDNA that encoded the autoantigen [7], use of cDNAs to create hybrid molecules or swap mutants at specified locations [8], molecular genetic insertion of short cassettes representing candidate epitopes into a homologous but nonautoantigenic host molecule [9], and testing of short synthetic peptides spanning the sequence of the autoantigen [10]. However, informative as these techniques were, the shortcomings were that antibody epitopes mostly could be mapped only to sequences of some 100 or more residues. The greatly decreased reactivity of most autoantibodies with short linear synthetic peptides versus the intact molecule would suggest that such peptides were merely components of a complete conformational epitope. Indeed, it is now recognised that most epitopes are conformational, with 15–22 protein residues in contact with the combining site of the antibody, and a subset of critical contact residues that contribute most of the free binding energy. These residues constitute the functional epitope and can be scattered over two or more discontinuous polypeptide segments [11]. Pos-

4. Pyruvate dehydrogenase complex E2: autoantigen for PBC As described above, the traditional AMA (anti-M2) in the PBC sera reacts selectively with E2 subunits of one or more of the 2-oxo-acid dehydrogenase complexes (2-OADC), most often pyruvate dehydrogenase complex E2 (PDC-E2). The autoepitope for PDCE2 was initially localized on the basis of reactivity across species, and homology among the E2 subunits of the other reactive 2-OADC enzymes, to a short sequence of highly conserved amino acids that form the attachment site of the lipoyl cofactor [5]. Further epitope mapping using truncated fragments of PDCE2 located a major epitope within the inner lipoyl domain of PDC-E2, but the epitope region could not be defined by less than 93 residues, 128 to 221 [7], and the antigenic reactivity of synthetic linear peptides in the epitope region was significantly weaker than that of the whole PDC-E2 protein [5]. To further examine the conformational structure of the antibody epitope, we used IgG from PBC sera that contained high titers of anti-PDC-E2 to screen phagedisplayed random peptide libraries. The phage clones selected were tested for reactivity with affinity purified anti-PDC-E2, and peptide inserts from reactive phage were aligned with a published NMR structure of the inner lipoyl domain. The epitope was deemed to require two relatively distant motifs on the linear

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sequence of PDC-E2 brought into contiguity by protein folding, 131HM132 and 178F(E)V180 [12]. It is notable that none of the reactive phagotopes contained a sequence containing lysine, despite K173 (and the attached lipoyl group) appearing from earlier studies to constitute part of the epitope. Interpretations on the conformational epitope structure of PDC-E2 were confirmed in two ways: first, by the creation of alanine mutants of the implicated residues of PDC-E2, which showed that a quadruple mutant 132HYA, 133MYA, 178FYA, 180VYA, had only 18% of the reactivity of the parent molecule; and second, by the demonstration that rabbits immunized with one of the most reactive phage clones produced antibodies that reacted with intact PDC-E2 [13].

5. Glutamic acid decarboxylase (GAD) 65 kDa—autoantigen for Type 1 diabetes The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is a major autoantigen in Type 1 diabetes. Antibody epitopes on GAD65 initially were localized by expression of truncated proteins from deletion mutants of cDNARs or by creation of swap mutants in which sequences of GAD65 were replaced by homologous sequences of the nonautoantigenic isoform, GAD67 [8]. Two main epitope regions of GAD65 for diabetes sera, and for various monoclonal islet cell antibodies (MICAs), were located, one in the midregion, in the vicinity of the attachment site for the pyridoxal phosphate (PLP) cofactor, and one in the Cterminal region; but again, the lengths of these epitope regions indicated a conformational structure. In the most detailed study [14], a panel of MICAs was tested against a large panel of chimeric GAD65/67 mutants or GAD65 mutants created by substitution of single residues of GAD67. Interpretations were made based on homology models of the middle and C-terminal regions of GAD derived from the crystal structure of the related enzyme, ornithine decarboxylase. In this study, no single epitope(s) could be defined, and the range of autoreactivity of the human mAb to GAD65 was sufficiently broad as to suggest a distribution of epitopes over the entire external surface of the mid- and C-terminal regions of the GAD65 molecule. In our laboratory, we have used phage display to examine conformational epitopes on GAD65. Random

peptide phage displayed libraries screened with the human mAbs, MICA3 and MICA4, and prevalent motifs from reactive clones were aligned to a homology model of the PLP-binding domain of GAD65 [15]. The MICA3-derived phage clones identified a conformational structure comprising three surface-exposed loops, residues 262–270, residues 285–296, and residues 315–334, with all being accommodated within ˚ diameter of an antibody paratope; notably, the 25-A residues 262–270 contained the PEVKEK sequence that is implicated as a site of epitope mimicry with Coxackie B virus coat protein [15]. Deletion of the entire PEVKEK loop greatly decreased the reactivity of GAD65 with both MICA3 and MICA4, and also with Type 1 diabetes sera, providing further evidence for a major conformational epitope in this region [16]. Thus, use in combination of panels of human mAbs, phage display, alanine mutagenesis of bparentalQ GAD65, and serological testing suggests that autoantigens contain a few immunodominant regions, within which are epitope variants that reflect affinity maturational changes in V-genes encoding products of B cells. In this respect, autoantibody responses are similar to those to a foreign antigen.

6. Type II collagen (CII) : autoantigen for inflammatory arthritis CII is a credible candidate as the primary autoantigen for rheumatoid arthritis by reason of its high representation in articular cartilage, the presence particularly in early RA of autoantibodies to CII, and the resemblance to RA of the experimental model, collagen-induced arthritis (CIA). Mice with CIA have been a source of numerous mAbs that react with native but not denatured CII, certain of which readily transfer arthritis to naRve recipients. These mAbs have been mapped to particular fragments of CII derived by cleavage with cyanogen bromide, particularly CB11 and CB10, and detailed epitope mapping on native triple helical collagen has been performed by insertion of fragments of CII as short cassettes into the nonautoantigenic collagen type X [9], thus creating sets of chimeric molecules that could be tested against mAbs from mice with CIA. Various epitope regions were identified along the native CII molecule, particularly the C1 epitope in CB11 (residues 359–363), recognized

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by various mAbs, including CII-C1 [9], and also by sera from patients with RA [17]. According to these studies, the minimum primary sequence for reactivity of mAb CII-C1 with CII was ARGLT. In our laboratory, antibody screening by phage display using CII-C1 and a second mAb CB268 reactive with precisely the same epitope identified a particular motif, basic, basic, hydrophobic, often RRL, that was consistent with this, and with the known requirement for the construction of a conformational epitope, with contributions from residues on separate alpha chains of the native CII model [18,19]. Notably, CII-C1 and certain other autoepitope-specific mAbs to CII have arthritogenic effects on passive transfer to naive mice and, correspondingly, disruptive effects on collagenous matrix when added to cultured chondrocytes [20].

7. T cell autoepitopes Identification of T-cell autoepitopes is in some respects simpler, but in others more complex, than for antibody autoepitopes; simpler because such epitopes are short linear peptides that are accommodated within the antigen-binding groove of MHC molecules, and more complex because of (a) the relatively low representation of reactive T cells in blood versus the affected tissue [21], (b) the polymorphic nature of MHC molecules resulting in differing selection, for any given autoimmune disease, of T-cell epitopes among different individuals or inbred mouse strains [22], and (c) the apparent redundancy or polyreactivity, in some circumstances, of peptide recognition by the T-cell receptor [23]. As an intuitive expectation, based on bassociative recognitionQ of antigen by B and T cells, there should be some contiguity of the B and T cell epitopes, but this is yet to be clearly shown for most autoimmune reactivities, although in the case of the autoantigens discussed here, there is some evidence for coassociation. Thus, for PBC, T-cell epitopes have been identified using T-cell lines derived from blood mononuclear cells from patients with PBC tested against sequential 14-mer peptides, reviewed in Ref. [21]. Epitopes were identified in the inner (and outer) lipoyl domains of PDC-E2, with a critical motif ExDK at positions 170–173, that included the lipoyl-binding lysine. Notably, this T-cell epitope is located in the primary sequence of PDC-E2 within the two particular

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regions, residues 132–133 and 178–180, that we have identified as parts of a conformational B cell epitope. For type 1 diabetes, one T cell epitope (among others) has been localized to the PEVKEK region of GAD65 [24], and for both CIA and RA, an immunodominant T cell epitope has been identified at positions 263–270, within the CB11 fragment, in which is located the C1 antibody epitope [25].

8. Conclusions Knowledge of autoepitopes is crucial for the understanding of autoimmunity including interactions between T and B lymphocytes. Screening of phagedisplayed peptide libraries now allows for precision in mapping of antibody epitopes, including likely contact residues in a conformational epitope region. Rewards could include insights into autoimmune induction and mechanisms of injury, and improved diagnostics and vaccine-type immunotherapies.

Acknowledgement We thank Duncan Crombie for designing Fig. 1.

Take-home messages ! Autoantibodies mostly engage conformational epitopes, wherein spatially disparate motifs are brought into contiguity by protein folding. ! Current techniques for defining a reactive molecular sequence (autoepitope) on an autoantigenic molecule do not identify critical contact residues for an autoantibody or an autoimmune T cell receptor. ! Phage display provides a novel technique that can be used to identify such critical contact residues and to localize conformational epitopes in the absence of a crystal structure. ! Rewards of precise contact residue-based epitope mapping could include better understanding of the genesis of autoimmune disease (epitope mimicry, epitope spreading nature of B and T cell interactions) and also provision of improved diagnostic and therapeutic (vaccine) molecules.

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References [1] Burnet FM. The Clonal Selection Theory of Acquired Immunity. Cambridge7 University Press; 1959. p. 29 – 48. Chap. 3. [2] Jerne NK. Immunological speculations. Annu Rev Microbiol 1960;14:341 – 58. [3] Mackay IR, Frazer IH, McNeilage J, Whittingham S. Autoepitopes and autoimmune disease. Ann N Y Acad Sci 1986; 475:59 – 65. [4] Gershwin ME, Mackay IR, Sturgess A, Coppel RL. Identification and specificity of a cDNA encoding the 70 kD mitochondrial antigen recognized in primary biliary cirrhosis. J Immunol 1987;138:3525 – 31. [5] Yeaman SJ, Fussey SP, Danner DJ, James OF, Mutimer DJ, Bassendine MF. Primary biliary cirrhosis: identification of two major M2 mitochondrial autoantigens. Lancet 1988;1: 1067 – 70. [6] Van de Water J, Gershwin ME, Leung P, Ansari A, Coppel RA. The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase. J Exp Med 1988;167: 1791 – 9. [7] Surh CD, Coppel R, Gershwin ME. Structural requirement for autoactivity on human pyruvate dehydrogenase-E2, the major autoantigen of primary biliary cirrhosis. J Immunol 1990;144: 3367 – 74. [8] Daw K, Powers AC. Two distinct glutamic acid decarboxylase autoantibody specificities in IDDM target different epitopes. Diabetes 1995;44:216 – 20. [9] Schulte S, Unger C, Mo JA, Wendler O, Bauer E, Frischols S, et al. Arthritis-related B cell epitopes in collagen II are conformation-dependent and sterically privileged in accessible sites of cartilage collagen fibrils. J Biol Chem 1998;273:1551 – 6. [10] Mackay IR, Whittingham SF, Fida S, Myers M, Ikuno N, Gershwin ME, et al. The peculiar immunity of primary biliary cirrhosis. Immunol Rev 2000;174:226 – 37. [11] Rowley MJ, O’Connor K, Wijeyewickrema L. Phage display for epitope determination: a paradigm for identifying receptor ligand interactions. Biotechnol Annu Rev 2004;10:151 – 87. [12] Rowley MJ, Scealy M, Whisstock JC, Jois JA, Wijeyewickrema LC, Mackay IR. Prediction of the immunodominant epitope of the pyruvate dehydrogenase complex E2 in primary biliary cirrhosis using phage display. J Immunol 2000;164:3413 – 9. [13] Scealy M, Mackay IR, Rowley MJ. Indentification by phage display and site directed mutagensis of amino acids critical for binding of an autoantibody to a conformational epitope. 2004 [submitted for publication]. [14] Schwartz HL, Chandonia JM, Kash SF, Kanaani J, Tunnell E, Domingo A, et al. High resolution epitope mapping and structural modelling of the 65 kDa form of human glutamic acid decarboxylase. J Mol Biol 1999;287:983 – 99.

[15] Myers MA, Davies JM, Tong JC, Whisstock J, Scealy M, Mackay IR, et al. Conformational epitopes on the diabetes autoantigen GAD65 identified by phage display and molecular modeling. J Immunol 2000;165:3830 – 8. [16] Tong JC, Myers MA, El Kabanni O, Mackay IR, Zimmet PZ, Rowley MJ. The PEVKEK region of the pyridoxal phosphate binding domain of GAD65 expresses a dominant B cell epitope for Type 1 diabetes sera. Ann N Y Acad Sci 2002; 958:182 – 9. [17] Burkhardt H, Koller T, Engstrfm A, Nandakumar KS, Turnay J, Kraetsh HG, et al. Epitope specific recognition of Type 2 collagen by rheumatoid arthritis antibodies is shared by recognition by antibodies that are arthritogenic in collagen induced arthritis in the mouse. Arthritis Rheum 2002;46: 2339 – 48. [18] Davies JM, Rowley MJ, Mackay IR. Phagotopes derived by antibody screening of phage-displayed random peptide libraries vary in immunoreactivity: studies using an exemplary monoclonal antibody, CII-C1 to type II collagen. Immunol Cell Biol 1999;77:483 – 90. [19] Xu Y, Ramsland PA, Davies JM, Scealy M, Nandakumar KS, Holmdahl R, et al. Two monoclonal antibodies to precisely the same epitope of type II collagen select non-cross reactive phage clones by phage display: implications for autoimmunity and molecular mimicry. Mol Immunol 2004;41:411 – 9. [20] Amirahmadi SF, Pho MH, Gray RE, Crombie DE, Whittingham SF, Biurrun Zuasti B, et al. An arthritogenic monoclonal antibody to type II collagen, CII-C1, impairs cartilage formation by cultured chondrocytes. Immunol Cell Biol 2004;82:427 – 34. [21] Gershwin ME, Nishio A, Ishibasi H, Lindor KD. Primary biliary cirrhosis. In: Gershwin ME, Vierling JM, Manns MP, editors. Liver Immunology. Philadelphia7 Hanley & Belfus; 2003. p. 311 – 27. [22] Endl J, Otto H, Jung G, Dreisbusch B, Donie F, Stahl P, et al. Identification of naturally processed T cell epitopes from glutamic acid decarboxylase presented in the context of HLA DR alleles by T lymphocytes of recent onset diabetes patients. J Clin Invest 1997;99:2405 – 15. [23] Wucherpfenning KW, Strominger JL. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin protein. Cell 1995;80:695 – 705. [24] Rharbaoui F, Mayer A, Granier C, Bouanani M, Thivolet C, Pau B, et al. T cell response pattern to glutamic acid decarboxylase 65 (GAD65) peptides of newly diagnosed type 1 diabetic patients sharing susceptible HLA haplotypes. Clin Exp Immunol 1999;117:30 – 7. [25] Backlund J, Carlsen S, Hfger T, Holm B, Fugger L, Kihlberg J. Predominant selection of T cells specific for the glycosylated collagen type II epitope (263–270) in humanized transgenic mice and in rheumatoid arthritis. Proc Natl Acad Sci 2002;99: 9960 – 5.