OBJECTIVE—To gain structural insights into the autoantigenic properties of GAD65 in type 1 diabetes, we analyzed experimental epitope mapping data in the context of the recently determined crystal structures of GAD65 and GAD67, to allow “molecular positioning” of epitope sites for B- and T-cell reactivity.

RESEARCH DESIGN AND METHODS—Data were assembled from analysis of reported effects of mutagenesis of GAD65 on its reactivity with a panel of 11 human monoclonal antibodies (mAbs), supplemented by use of recombinant Fab to cross-inhibit reactivity with GAD65 by radioimmunoprecipitation of the same mAbs.

RESULTS—The COOH-terminal region on GAD65 was the major autoantigenic site. B-cell epitopes were distributed within two separate clusters around different faces of the COOH-terminal domain. Inclusion of epitope sites in the pyridoxal phosphate–and NH2-terminal domains was attributed to the juxtaposition of all three domains in the crystal structure. Epitope preferences of different mAbs to GAD65 aligned with different clinical expressions of type 1 diabetes. Epitopes for four of five known reactive T-cell sequences restricted by HLA DRB1*0401 were aligned to solvent-exposed regions of the GAD65 structure and colocalized within the two B-cell epitope clusters. The continuous COOH-terminal epitope region of GAD65 was structurally highly flexible and therefore differed markedly from the equivalent region of GAD67.

CONCLUSIONS—Structural features could explain the differing antigenicity, and perhaps immunogenicity, of GAD65 versus GAD67. The proximity of B- and T-cell epitopes within the GAD65 structure suggests that antigen-antibody complexes may influence antigen processing by accessory cells and thereby T-cell reactivity.

Type 1 diabetes is characterized by autoimmune reactivity to islet cell antigens that include the 65-kDa isoform of GAD (GAD65). Of the two isoforms of GAD, GAD67 and GAD65, only GAD65 is autoantigenic, in diseases that include particularly type 1 diabetes (1,2). Importantly, the specificity of antibodies for particular epitopes on GAD65 rather than their actual levels may be a better indicator of impending or actual destruction of islet β-cells. Thus in genetically prone individuals, changes in the focus of autoantibody responses to epitopes of GAD65, i.e., intramolecular epitope shifts during the period of pre-diabetic insulitis, herald the onset of overt type 1 diabetes (3).

B-cell/autoantibody epitopes on GAD65 engage conformational determinants that are widely distributed over the linear sequence of the three domains, NH2-terminal (amino acids 1–234), pyridoxal phosphate (PLP)–binding (235–442), and COOH-terminal (443–585) domains (4). Presently, unanswered questions include the precise location of epitope sites and critical binding residues for autoantibody recognition, the differing immune reactivity of the GAD65 versus the GAD67 isoform, and the failure of natural immune tolerance to GAD65 in the first place. These questions prompted the recent crystallographic structure determination of both GAD67 and GAD65 isoforms (5).

We have used the crystal structures for the “molecular positioning” of epitope sites for B- and T-cell reactivity by analysis of effects of mutagenesis and data from cross-blocking of monoclonal antibody (mAb) reactivity by radioimmunoprecipitation (RIP) with GAD65, using recombinant Fab (rFab) derived from various mAbs to inhibit the reactivity of a panel of mAbs. The findings delineate a major autoantigenic region on GAD65 at the COOH-terminal domain that structurally is highly flexible and in this respect differs markedly from the equivalent region in GAD67. Known T-cell epitope sequences mostly could be localized to the same region on the structure of GAD65. These findings may explain the unique immunogenicity of GAD65 versus GAD67.

mAbs to GAD65.

The panel of 11 human mAbs to GAD65 included M2, M3, M4, M5, M6, DPA, DPB, DPC, and DPD from two patients with type 1 diabetes (6,7) and b96.11 and b78 from a nondiabetic patient with autoimmune polyendocrine syndrome type 2 (8). Each of these patients shared the HLA DRB1*0401 allele associated with a high risk for diabetes. None of the mAbs reacts with GAD67. Data were available from published studies (4,911) on mutagenesis on the putative location of epitopes for these mAbs, as shown in Table 1.

Crystal structures of GAD65 and GAD67.

Details of the purification, crystallization, and structural determination of human GAD65 and GAD67 have been published previously (5). We used for crystallization an NH2-terminally truncated form of each isoform (hereinafter referred to as GAD67 and GAD65) that lacked the first 89 and 83 residues, respectively; the NH2-terminal truncation facilitated purification because this region is hydrophobic and highly susceptible to proteolysis and did not affect enzymatic properties (5) nor reactivity with specific antisera (1214). The proteins were expressed in Saccharomyces cerevisiae as fusions to a COOH-terminal hexahistidine tag and purified from the cell lysate by immobilized metal affinity chromatography and size exclusion chromatography in the presence of glutamate and PLP.

Location of epitopes on the structure of GAD65.

Analysis of structural differences between GAD65 and GAD67 and location of mutations shown to affect mAb binding were performed with the program Pymol (15). For each mAb to GAD65, the locations of mutations that either caused reduced reactivity by RIP or did not affect reactivity were mapped on the structure of dimeric GAD65; together, these data were used to delineate likely epitope regions. Structural alignments were performed with the program MUSTANG (16).

Inhibition of mAb binding to full-length GAD65 using rFab.

Tests for colocalization of epitopes by cross-competition were performed using rFab from the mAbs b96.11, b78, DPA, DPC, DPD, M2, M3, and M4, prepared as previously described (17). The mAbs were tested at half-maximal binding concentrations, and results shown are from at least two separate experiments. Results were expressed as percent inhibition of full-length 35S-labeled GAD65 bound in the presence of rFab where percent inhibition = 100 − (counts per minute in the presence of mAb/counts per minute in the absence of mAb) × 100. Samples were analyzed in triplicate; the average intra-assay coefficient of variation was 5%, and the maximum value was 9%. A negative control rFab D1.3 specific to an irrelevant antigen, hen egg white lysozyme, was included at 5 μg/ml in each assay, and the cutoff for specifying blocking was conservatively set at >20% inhibition.

Flexibility in the COOH-terminal domain distinguishes GAD65.

The 2.3-Å crystal structures of GAD67 and GAD65 have been reported (5). Both isoforms adopt the same fold and formed obligate dimers from two monomeric units, each comprising the NH2-terminal, PLP-binding, and COOH-terminal domains (Fig. 1A and B). For each monomer, the NH2-terminal domain contained two parallel α-helices, helices 1 and 2, that were packed against the NH2-terminal and PLP-binding domains of the partner monomer. The PLP-binding domain contained nine α-helices, 3–11, that were surrounded by a seven-stranded mainly parallel β-sheet that adopted the type I PLP-dependent transferase-like fold. The COOH-terminal domain contained three α-helices, 13–15, and a short, buried, four-stranded anti-parallel β-sheet, s3C. This relative orientation of monomers within the dimer for both GAD65 and GAD67 was such that the domains from monomer A formed a contiguous surface patch with the domains of the partner monomer B (Fig. 1A). In addition, in both cases, the two active sites of the enzyme, located in the center of the PLP domain at the dimer interface, contained the cofactor PLP and the product γ-aminobutyric acid (GABA) (Fig. 1A and B), indicative of the active (holo-) state of the crystallized enzyme. Overall, of 41 nonconservative amino acid differences between the two isoforms, 32 were surface-exposed, and 9 were buried in the region structurally characterized. Differing surface-exposed residues between GAD67 and GAD65 were distributed over the entire structure of the molecule, with no obvious clustering apparent in any region (Fig. 1C).

There are two key differences between the two structures that have enzymatic and potentially antigenic consequences. First, for GAD67, each of the two active sites was substantially covered by a well-ordered catalytic loop comprising residues 432–443 contributed in trans from the partner monomer. Notably, for GAD65, this same catalytic loop, residues 422–433, was highly flexible and thus not visible in the electron density maps. This difference offers a structural rationalization for the contrasting enzymatic characteristics of the GAD isoforms and insight into the requirement of two GAD isoforms in mammals (5). Second, the two molecules differ markedly in their flexibility, with GAD65 being the more flexible, as indicated by the atomic temperature factors (B factors) (Fig. 1D). This difference is particularly pronounced in the COOH-terminal domain, in a surface-exposed loop (residues 518–520) that was too flexible to be resolved in the crystal structure of GAD65, in marked contrast to the well-defined structure of the corresponding sequence for GAD67.

Location of contact residues for human mAbs on GAD65 based on mutagenesis.

The known sites of point mutations or GAD67/65 sequence exchanges (chimeric molecules) that affected the binding of any of the panel of mAbs were mapped onto the structure of GAD65. The location of epitopes or epitope regions in the COOH-terminal domain, PLP domain, and the NH2-terminal domain are described below.

COOH-terminal domain.

Mutated residues that substantially decreased reactivity with five mAbs (M2, M3, M5, DPA, and b78) were heavily clustered in the region of GAD65 that surrounded the COOH-terminal flexible loop, residues 518–520, which differs markedly from the equivalent region of GAD67. Contact residues for the binding of these five mAbs were distributed over all four faces of the two autoantigenic COOH-terminal domains of dimeric GAD65, denoted as the α, β, γ, and δ faces (Fig. 2A and B), but two clusters of COOH-terminal contact residues (ctc1 and ctc2) could be clearly discerned. These were localized predominantly to opposing faces, α and δ, of the COOH-terminal domain, and could be represented by the epitope regions of two exemplary mAbs, M5 and M3 (Fig. 2A and B). Mutated residues in ctc1 that affected binding of mAbs M2, M5, and b78 were located in helix 14 (residues 522–540) and the adjacent COOH-terminal flexible loop in the α and β faces of the COOH-terminus of one GAD65 monomer and close to the catalytic loop of the partner monomer, and mutated residues that affected binding of M3 and DPA were localized to helices 13 and 15. (Fig. 2C and D). Notably, neither point mutation of residues nor insertions of GAD67 sequences on the α face had any effect on the binding of mAbs reactive with ctc2 on the opposite δ face of the COOH-terminal region and vice versa (Table 1).

The reactivity of M5, the prototypic mAb for ctc1, depended strongly on a nonconserved surface-exposed residue V532 located in the COOH-terminal end of helix 14 of GAD65 (Fig. 2C), and the binding of M5 required an additional five residues, with two located in the NH2- terminus of helix 14 (S524 and S527), two flanking the COOH-terminal unstructured loop (E517 and E521), and one within this loop (E520) (Fig. 2C). Combinations of these residues located in the most structural divergent region between GAD65 and GAD67 (Fig. 2C) affected the binding of the other four mAbs localized to ctc1 (Table 1). The important residues in ctc2 that engage mAbs M3 and DPA, distributed particularly on the δ face, lie close to the PLP domain, but this epitope region is remote from the catalytic loop. Reactivity of mAbs M3 and DPA, as judged by mutations, depended on residues of GAD65 located in helices 13 (N483) and 15 (H568), noting that mutation of N483 ablated reactivity only with M3, whereas mutation of H568 ablated reactivity with both mAbs (Fig. 2D).

PLP domain.

Epitopes on GAD65 for mAbs M4, M6, b96.11, and DPC have been mapped to the PLP domain by use of GAD65/67 sequence exchanges (Fig. 2E and F), but information on the exact location of contact sites is limited. However, point mutations of GAD65 did decrease reactivity for each of these mAbs. Epitope regions for both M4 and b96.11 were localized within residues 308–365, but the single point mutation shown to affect binding of M4 to GAD65 was that of K358, a residue that is surface exposed, but this of itself is insufficient data to define an epitope. For mAb b96.11, no single point mutation ablated reactivity, but several mutations that substantially reduced reactivity, including the PLP domain sequence 305DER307, were adjacent to the COOH-terminal epitope region ctc2. Thus, contact residues for b96.11 would lie at the junction of the PLP and COOH-terminal domains in the δ/β face of ctc2 (Fig. 2D). For mAb M6, the epitope region was localized within residues 242–282; binding to GAD65 was not affected by mutation of GAD65 E264T, but mutation of the equivalent residue of GAD67 T273E restored 50% of the reactivity with GAD65 (4). Notably, E264 lies on the interface of the dimer (Fig. 2E), within the previously identified 260PEVKEK265 region of identity with the P2-C protein of Coxsackie B virus (18), and close to residues 271–285, a major T-cell epitope restricted by HLA DR*0401 (19) (see below).

For mAb DPC, two distinct epitope regions were revealed by effects of sequence exchange: one involving residues 366–413 and one involving residues 134–242 overlapping the junction of the PLP and NH2-terminal domains. Within the crystal structure of GAD65, residues 366–413 in monomer B are positioned directly adjacent to the NH2-terminal residues 134–242 in the partner monomer A (Fig. 2E); both sequences contain structural and amino acid differences between GAD65 and GAD67. Also, for mAb DPC, an epitope site was defined by mutagenesis of residues P231 and S234, which are located on a well-defined loop in the PLP domain that differs structurally from the equivalent region of GAD67 (Fig. 2E and F). No mutagenesis data exist to identify the residues responsible for binding of mAb DPC within the NH2-terminal domain.

NH2-terminal domain.

Epitope regions for two mAbs, DPD and DPB, have been mapped exclusively to the NH2-terminal domain but only by use of GAD65/67 sequence exchanges. For mAb DPD, the epitope region is within residues 96–173, which lie precisely adjacent to the COOH-terminal end of helix 14 of the same monomer and include a structural difference between GAD65 and GAD67 created by a deletion of three amino acids in GAD65 (143Q—E144 vs. 149EGMEG153) (Fig. 1C). For mAb DPB, the epitope region is located within residues 1–96, which are lacking in the crystal structure of GAD65.

Competition by rFabs of mAb binding to GAD65.

Of rFab derived from mAbs to GAD65, there are three (M3, DPA, and b78) derived from mAbs that reacted with epitope regions at the COOH-terminus, three (b96.11, DPC, and M4) with the PLP domain, and two (DPC and DPD) with the NH2-terminal domain. When these rFab were tested for their capacity to block the reactivity by RIP between GAD65 and each of the mAbs (Table 2), each rFab (with one exception) strongly blocked the binding with GAD65 of the corresponding mAb. In general, the mAbs that reacted with one or the other of the two distinct COOH-terminal epitope clusters, ctc1 and ctc2, differed in susceptibility to cross-blocking by the rFab. For example, rFab from mAbs M3 and DPA (ctc2 epitopes, δ-face) of the COOH-terminus cross-blocked, but rFab from mAb b78 (ctc1 epitope, α-face) did not. Also, the binding of mAb M3 was blocked by rFab from mAb b96.11, consistent with the localization of the epitope for mAb b96.11 in the β and δ faces close to ctc2, although rFab from mAb M3, which localizes close to mAb b96.11, did not block binding to GAD65 of mAb b96.11. No other rFab tested blocked the binding of either M3 or DPA.

Cross-competition studies provided important evidence that other epitopes that could not be mapped to the COOH-terminal domain by mutagenesis did in fact lie close to either ctc1 or ctc2. Thus two mAbs that reacted with NH2-terminal epitopes, DPD and DPC, gave reactivity with GAD65 that was strongly blocked by rFab from mAb b78, which engages epitopes in ctc1. With the exception of rFab from DPD, there was strong mutual cross-competition among all three mAbs, suggesting that epitopes for each overlapped, and thus each could be considered as a component of ctc1 (Fig. 2E and F). Also consistent with the location of contact residues, the two mAbs for which epitopes mapped to the PLP domain, DPC and b96.11, were not cross-competitive, and the cross-competition that was demonstrable between mAbs DPC and b78 (see above) indicates that the epitope for DPC lies close to ctc1 epitopes, whereas the epitope for b96.11 lies close to ctc2 epitopes. The single mAb tested by cross-competition for which an epitope could not be localized to either ctc1 or ctc2 was M4, which maps to the PLP domain; rFab from M4 blocked the binding to GAD65 of mAbs DPC, DPD and b78 (ctc1) and also of mAb b96.11 (ctc2). The single amino acid K358, which has been shown by mutagenesis to be required for the binding to GAD65 of M4, lies on the face of the GAD65 dimer between ctc1 and ctc2 (Fig. 2E).

T-cell epitopes on GAD65.

To examine human T-cell epitopes in the context of the crystal structure of GAD65, we selected five particular immunodominant T-cell epitopes of GAD65 identified in several studies (1921), designated T1-T5, each restricted by HLA DRB1*0401 (Table 3). The COOH-terminal domain contained three epitopes, T3 (residues 481–495), T4 (residues 511–525), and T5 (residues 551–585); the PLP domain contained one, T2 (residues 270–286); and the NH2-terminal domain contained one, T1 (residues 116–130). Positioning of these on the crystal structure of dimeric GAD65 showed that T1, T3, T4, and T5 were closely associated (Fig. 3). T5 comprised part of helix 15 and the buried COOH-terminal β-sheet s3C, with its surface-exposed component located between two other epitopes, T1 and T3, indicative of strong interactions with both regions (Fig. 3B and D). T1 corresponds to helix 1 in the NH2-terminus, residues 116–130. T3 localizes at the COOH-terminal end of helix 13 and its adjacent loop and also is spatially contiguous with the T4 epitope region (residues 511–525) that corresponds to the highly flexible COOH-terminal loop for which structure is lacking. Taken together, there are four T-cell epitopes that form a contiguous linear patch on the surface of the GAD dimer located within the two major B-cell epitope regions ctc1 and ctc2 and another, T2, with a different location (residues 271–285), close to the 260PEVKEK265 sequence of similarity with the Coxsackie B virus protein 2C (18) and mainly buried within the PLP domain.

The analysis of crystal structures of both NH2-terminally truncated GAD65 and GAD67 has provided a molecular basis for epitope locations, assisted by data derived from several sources: binding assays using a unique panel of human mAb to GAD65, point and sequence exchange mutagenesis of GAD65, and cross-competition experiments with GAD65-specific monoclonal rFab. Notably, we identified a major region of antigenic activity for GAD65 predominantly centered on a flexible and structurally undefined region in the COOH-terminal domain that differs strikingly between GAD65 and GAD67 and including also conformationally contiguous parts of the PLP and NH2-terminal domains. Within this region, two independent clusters of B-cell epitopes, ctc1 and ctc2, were positioned on opposing faces of the COOH-terminal domain as judged by reactivity of mAbs to GAD65 with particular mutants of GAD65, and blocking studies using human rFab. For T-cells, four of five previously recognized DRB1*0401-restricted epitopes on GAD65 formed a contiguous surface-exposed patch between the two B-cell epitope clusters.

The division of the COOH-terminal epitope region of GAD65 into two epitope clusters, ctc1and ctc2, on opposing faces (α and δ) of the molecule was based on cross-reactivity of mAbs, although boundaries between the two regions were not entirely distinct, because rFab from mAb b78, which engaged residues in ctc1, caused partial inhibition (63%) of the binding to GAD65 of mAb b96.11, which engaged residues in ctc2, although not vice versa. One explanation would be that the epitopes partially overlap, with that for mAb b78 located on the α and β face of the COOH-terminal domain and that for mAb b96.11 located on the δ and β faces. The mAbs that engaged ctc1 in the present study included M2, M5, b78, and DPD, and published data indicate that additional human mAbs (M7, M8, and M9) engage epitopes in ctc1, because these mAbs are cross-competitive, and also block binding of M2 and M5 but not binding of mAbs M1 and M3, which engage residues in ctc2 (4). Previous studies have defined epitopes for anti-GAD65 simply according to their location in domains based on the linear sequence, NH2-terminal, PLP, or COOH-terminal. This is superseded by the structural data, because some epitopes hitherto assigned to the PLP domain are either contiguous with or even part of ctc1, e.g., mAb DPC, or of ctc2, e.g., mAb b96.11, and all mAbs for which epitopes apparently include an NH2-terminal component, such as mAbs DPD, DPC, M8, and M9, engage epitopes in ctc1.

Although different mAbs from a single anti-GAD+ patient could be reactive with either ctc1 or ctc2 epitopes (Table 1), published data suggest that a response biased toward one or the other of the two epitope regions aligns with differing clinical expressions of type 1 diabetes. Thus, anti-GAD65 reactive with ctc1, as defined by blocking by rFab from mAbs b78, DPD, and DPC, was associated with enzyme inhibitory antibodies, whether from patients with Stiff Person Syndrome (22) or a subset of ketosis-prone type 1 diabetes with a higher β-cell functional reserve and a more benign clinical course (23). Also, anti-GAD65 reactive with ctc1/NH2-terminal epitopes was associated with slowly progressive type 1 diabetes, i.e., latent autoimmune diabetes of adults (24,25). In contrast, anti-GAD65 reactive with ctc2 epitopes as judged by inhibition by rFab from mAbs b96.11 and DPA was associated with high-risk HLA-DQ alleles and rapidly progressive diabetes (3). Thus our data now structurally establish the epitope preference of autoantibodies to GAD65 that may dictate differing clinical expressions of type 1 diabetes.

The identification of an immunodominant B-cell epitope region in the COOH-terminal region of GAD65 conforms with known features of the normal antibody response. Immunodominant regions of antigens comprise sites on the antigenic molecule that are strongly recognized by most immunized individuals, although there are differences among individuals that reflect the summated reactivity of multiple B-cell clones that contribute to the response. Affinity maturation of an ongoing antibody response is associated with both “epitope focusing” to provide for selection of higher affinity antibodies and also “epitope spreading” to enable the immune response to encompass wider regions of the antigenic molecule. In type 1 diabetes specifically, there is evidence that the autoantibody response may first involve the COOH-terminal domain, with later spreading to the PLP and NH2-terminal domains (3,26).

Notably, four of five major T-cell epitopes restricted by the high-risk HLA allele DRB1*0401 could be localized on the structure of GAD65 within the same region as the immunodominant B-cell epitope regions ctc1 and ctc2, and the fifth epitope was close to E264, which defines the M6 epitope in the PLP domain. Moreover, as CD8+ T-cells restricted by HLA class 1 appear to be more directly relevant to the effector stage of pathogenesis, it is of interest that the class II CD4+ T-cell epitope (T1, residues 115–130) overlaps a major class I CD8+ T-cell epitope (residues 113–124) restricted by HLA A2 (27). The finding that T-cell epitopes often occur close to B-cell epitopes, for both extrinsic and autoantigens (28), directs attention to the role of B-cells in modulating T-cell responses (2934). In antigen-presenting cells, antigen-antibody complexes can remain intact after the internalization and fragmentation by proteases along the antigen-processing pathway (32,33), thus protecting residues from proteolysis and modulating presentation of peptides and ensuing T-cell responses. Studies on B-cell modulation of the T-cell response to GAD65 indicate that B-cell clones producing mAbs DPA and DPD (but not DPC) can present the T1 (residues 115–130), T2 (residues 270–283), and T5 (residues 556–575) peptide epitope sequences to DRB1*0401-restricted T-cell clones (30).

An enduring question for autoimmunity is why only a limited proportion, ∼2–3% (35), of all human proteins become selected as autoantigens and why only limited sites on such molecules serve as autoepitopes. Among possible explanations, structural characteristics are cited (35), albeit in the absence of knowledge of the actual protein structure of most autoantigenic molecules. This can now be examined in the case of GAD in the light of the contrasting antigenicity of the two isomers, GAD65 and GAD67, together with the substantial information derived from mutants of GAD65 based on sequence exchanges or single point mutations and the reactivity of such mutants with human mAbs exclusively to GAD65. We have already commented on distinctive features of epitope distribution on GAD65, notably the clear association of epitopes with regions of increased flexibility of protein structure of GAD65 versus GAD67 (5). Structural features associated with antigenicity for other proteins include high flexibility, loops, and protrusion from the antigen surface, and charged amino acids (3639), all features of the COOH-terminal domain of GAD65. Comparably, structural flexibility was described for the immunodominant epitope of the human autoantigen thyroid peroxidase (40), although not for epitope regions on the crystal structure of the type 1 diabetes autoantigen IA-2 (41). Finally, if the engagement of epitopes of ctc2 were to be predictive of progression of individuals at risk to overt type 1 diabetes, knowledge of the clustering of epitopes for anti-GAD65 at the COOH- terminus and the existence of two separate COOH-terminal epitope clusters could allow the development of assay systems to exploit this. Also, if GAD65-based immunotherapies were to appear promising in retarding progression in at risk individuals to overt type 1 diabetes, consideration could be given to vaccine constructs based on epitopes associated with disease progression.

FIG. 1.

A: The crystal structure of dimeric GAD65 showing assembly of NH2-terminal, PLP, and COOH-terminal domains of monomers A and B. The catalytic loop (red lines, residues 431–441) and the COOH-terminal loop (red dotted lines, residues 518–520) are highly mobile and thus were not visible in the structure, and the cofactor PLP and the product GABA are shown in red and yellow spheres, respectively. B: The monomeric structure of GAD65 within the asymmetric unit of the crystal with secondary structure elements of GAD65 monomer B labeled accordingly. Also shown are the α-helices in the NH2-terminal, PLP, and COOH-terminal domains colored according to A; the PLP and COOH-terminal β-sheets colored in red and yellow, respectively; the catalytic loop and the COOH-terminal mobile loops represented by red solid and dotted lines, respectively; and the 260PEVKEK265 region of sequence identity with P2C of Coxsackie B virus. C: Molecular surface of GAD65 dimer colored according to sequence conservation between GAD67 and GAD65, with green representing conserved residue and wheat nonconserved residues. Mutations that decreased mAb binding are outlined (black dotted lines), and a box indicates the location of a three–amino acid deletion in GAD65 (see results). D: Molecular surfaces of GAD65 and GAD67 colored according to flexibility/mobility as measured by atomic temperature (B) factors. The flexibility of the GAD isoforms is represented as a gradient, blue (low, ordered) to red (high, mobile). Residues 518–520, which are too mobile to be visualized in the crystal structure, are represented as red dotted lines. GAD65 residues 434–437 (red) are a flexible sequence flanking the COOH-terminal end of the unstructured catalytic loop.

FIG. 1.

A: The crystal structure of dimeric GAD65 showing assembly of NH2-terminal, PLP, and COOH-terminal domains of monomers A and B. The catalytic loop (red lines, residues 431–441) and the COOH-terminal loop (red dotted lines, residues 518–520) are highly mobile and thus were not visible in the structure, and the cofactor PLP and the product GABA are shown in red and yellow spheres, respectively. B: The monomeric structure of GAD65 within the asymmetric unit of the crystal with secondary structure elements of GAD65 monomer B labeled accordingly. Also shown are the α-helices in the NH2-terminal, PLP, and COOH-terminal domains colored according to A; the PLP and COOH-terminal β-sheets colored in red and yellow, respectively; the catalytic loop and the COOH-terminal mobile loops represented by red solid and dotted lines, respectively; and the 260PEVKEK265 region of sequence identity with P2C of Coxsackie B virus. C: Molecular surface of GAD65 dimer colored according to sequence conservation between GAD67 and GAD65, with green representing conserved residue and wheat nonconserved residues. Mutations that decreased mAb binding are outlined (black dotted lines), and a box indicates the location of a three–amino acid deletion in GAD65 (see results). D: Molecular surfaces of GAD65 and GAD67 colored according to flexibility/mobility as measured by atomic temperature (B) factors. The flexibility of the GAD isoforms is represented as a gradient, blue (low, ordered) to red (high, mobile). Residues 518–520, which are too mobile to be visualized in the crystal structure, are represented as red dotted lines. GAD65 residues 434–437 (red) are a flexible sequence flanking the COOH-terminal end of the unstructured catalytic loop.

FIG. 2.

A and B: Views of the surface structure of dimeric GAD65 with monomers A and B in dark blue and cyan, respectively, showing two COOH-terminal clusters of epitopes (ctc1 and ctc2) on opposing faces of the COOH-terminal domain. Epitopes for mAbs M5 and M3 represent ctc1 and ctc2, respectively. In B, the molecule is rotated 180° along the vertical axis, and different faces of the COOH-terminal domain are shown as α, β, γ, and δ faces. The M5 epitope resides primarily on the α and β faces and the M3 epitope on the δ and γ faces, with broad epitope locations mapped using GAD67/65 sequence exchanges shown in yellow and single contact sites for mAb binding in red. The disordered catalytic loop and the COOH-terminal flexible loop are represented by red dotted lines. C and D: Superposition of the GAD65 (dark blue) and GAD67 (green) COOH-terminal domains in the ctc1 and ctc2 regions, with broad epitope locations mapped using GAD67/65 sequence exchanges (514–528, and 532–540) in yellow, single contact sites for mAb binding shown as red sticks, and corresponding side chains of residues in the nonantigenic structure of GAD67 as green sticks. Notably, residues critical for binding of mAbs M8 and M9 (R536 and Y540) in α-helix 14 (C), identified by point mutations (9), are contiguous to NH2-terminal domain residues (131–140) that also are required for binding of mAbs M8 and M9 according to epitope mapping data using GAD67/65 chimeras (residues 96–242 and 532–540 in the NH2- and COOH-termini of GAD65, respectively) (9). E and F: Views of the surface of GAD65 showing epitope regions within the PLP domain mapped using GAD67/65 sequence exchanges (yellow) and contact sites for mAbs by point mutations (red). E: The epitope recognized by mAb DPC lies within the PLP domain of monomer B and the NH2-terminal domain of monomer A. Single contact sites for two other mAbs, M6 and M4, which can be mapped to the PLP domain are shown, with evident proximity of E264 to the catalytic loop (solid red line). F: The epitope region of mAb DPD lies within the NH2-terminal domain, residues 96–173, as mapped using chimeric GAD67/65 molecules but with no point mutation data available. Epitope regions for mAbs DPC and DPD overlap in the NH2-terminus of GAD65 and lie close to residues in the COOH-terminal domain that affect binding of mAb b78, consistent with the strong cross-inhibition obtained using the three rFab.

FIG. 2.

A and B: Views of the surface structure of dimeric GAD65 with monomers A and B in dark blue and cyan, respectively, showing two COOH-terminal clusters of epitopes (ctc1 and ctc2) on opposing faces of the COOH-terminal domain. Epitopes for mAbs M5 and M3 represent ctc1 and ctc2, respectively. In B, the molecule is rotated 180° along the vertical axis, and different faces of the COOH-terminal domain are shown as α, β, γ, and δ faces. The M5 epitope resides primarily on the α and β faces and the M3 epitope on the δ and γ faces, with broad epitope locations mapped using GAD67/65 sequence exchanges shown in yellow and single contact sites for mAb binding in red. The disordered catalytic loop and the COOH-terminal flexible loop are represented by red dotted lines. C and D: Superposition of the GAD65 (dark blue) and GAD67 (green) COOH-terminal domains in the ctc1 and ctc2 regions, with broad epitope locations mapped using GAD67/65 sequence exchanges (514–528, and 532–540) in yellow, single contact sites for mAb binding shown as red sticks, and corresponding side chains of residues in the nonantigenic structure of GAD67 as green sticks. Notably, residues critical for binding of mAbs M8 and M9 (R536 and Y540) in α-helix 14 (C), identified by point mutations (9), are contiguous to NH2-terminal domain residues (131–140) that also are required for binding of mAbs M8 and M9 according to epitope mapping data using GAD67/65 chimeras (residues 96–242 and 532–540 in the NH2- and COOH-termini of GAD65, respectively) (9). E and F: Views of the surface of GAD65 showing epitope regions within the PLP domain mapped using GAD67/65 sequence exchanges (yellow) and contact sites for mAbs by point mutations (red). E: The epitope recognized by mAb DPC lies within the PLP domain of monomer B and the NH2-terminal domain of monomer A. Single contact sites for two other mAbs, M6 and M4, which can be mapped to the PLP domain are shown, with evident proximity of E264 to the catalytic loop (solid red line). F: The epitope region of mAb DPD lies within the NH2-terminal domain, residues 96–173, as mapped using chimeric GAD67/65 molecules but with no point mutation data available. Epitope regions for mAbs DPC and DPD overlap in the NH2-terminus of GAD65 and lie close to residues in the COOH-terminal domain that affect binding of mAb b78, consistent with the strong cross-inhibition obtained using the three rFab.

FIG. 3.

A--D: Sites of established of autoantibody (A) and T-cell determinants (B--D) on the structure of dimeric GAD65. A: Three views of the surface structure of GAD65 showing B-cell epitope regions ctc1 and ctc2, and single contact sites in the COOH terminus for mAb binding in red and site in the PLP domain in yellow. B: Three views of the surface structure of GAD65 showing T-cell epitope sequences in orange (T1), green (T3), red (T4), and yellow (T5). These T-cell sequences are mostly surface exposed and form a contiguous patch on the surface of GAD65 in the same region as most B-cell epitopes. C: A cartoon representation of the GAD65 structure showing the single T-cell epitope sequence (T2) in the PLP domain located on the dimer interface and not surface exposed but close to E264, which is a component of the epitope for mAb M6. D: Cartoon representation of the structure of GAD65 showing T-cell epitope sequences T1, T3, T4, and T5.

FIG. 3.

A--D: Sites of established of autoantibody (A) and T-cell determinants (B--D) on the structure of dimeric GAD65. A: Three views of the surface structure of GAD65 showing B-cell epitope regions ctc1 and ctc2, and single contact sites in the COOH terminus for mAb binding in red and site in the PLP domain in yellow. B: Three views of the surface structure of GAD65 showing T-cell epitope sequences in orange (T1), green (T3), red (T4), and yellow (T5). These T-cell sequences are mostly surface exposed and form a contiguous patch on the surface of GAD65 in the same region as most B-cell epitopes. C: A cartoon representation of the GAD65 structure showing the single T-cell epitope sequence (T2) in the PLP domain located on the dimer interface and not surface exposed but close to E264, which is a component of the epitope for mAb M6. D: Cartoon representation of the structure of GAD65 showing T-cell epitope sequences T1, T3, T4, and T5.

TABLE 1

Panel of 11 human mAbs to GAD65 derived from two patients with type 1 diabetes and one with autoimmune polyendocrine syndrome type 2 with corresponding epitope data

mAbSource*Epitope regionMutants of GAD65 that affect reactivity
COOH-terminus    
    M3 Type 1 diabetes i 483–499, 556–585 N483A, H568Q 
    DPA Type 1 diabetes ii 483–499, 556–585 H568Q 
    M2 Type 1 diabetes i 514–528 E517P, E520P 
    M5 Type 1 diabetes i 512–540 E517P, E520P, E521Q, S524E, S527H, V532K 
    b78 APS-2 532–540, 514–528 V532K, S524A/R525A/L526A§ 
NH2-terminus    
    DPD Type 1 diabetes ii 96–173 No data 
    DPB Type 1 diabetes ii 1–102 No data 
PLP domain    
    b96.11 APS-2 308–365 R357A/K376A, F344A, D305A/E306A/R307A, K498A/P499A/Q500A 
    M4 Type 1 diabetes i 308–365 K358N 
    M6 Type 1 diabetes i 242–282 E264 
PLP and NH2-terminus    
    DPC Type 1 diabetes ii 134–242, 366–413 P231S/S234D 
mAbSource*Epitope regionMutants of GAD65 that affect reactivity
COOH-terminus    
    M3 Type 1 diabetes i 483–499, 556–585 N483A, H568Q 
    DPA Type 1 diabetes ii 483–499, 556–585 H568Q 
    M2 Type 1 diabetes i 514–528 E517P, E520P 
    M5 Type 1 diabetes i 512–540 E517P, E520P, E521Q, S524E, S527H, V532K 
    b78 APS-2 532–540, 514–528 V532K, S524A/R525A/L526A§ 
NH2-terminus    
    DPD Type 1 diabetes ii 96–173 No data 
    DPB Type 1 diabetes ii 1–102 No data 
PLP domain    
    b96.11 APS-2 308–365 R357A/K376A, F344A, D305A/E306A/R307A, K498A/P499A/Q500A 
    M4 Type 1 diabetes i 308–365 K358N 
    M6 Type 1 diabetes i 242–282 E264 
PLP and NH2-terminus    
    DPC Type 1 diabetes ii 134–242, 366–413 P231S/S234D 
*

Derivation of mAbs according to Richter et al. (6) for type 1 diabetes i, Madec et al. (7) for type 1 diabetes ii, and Tremble et al. (8) autoimmune polyendocrine syndrome type 2 (APS-2). From data on epitope regions on linear sequence of GAD65 and GAD65 mutants according to

Schwartz et al. (4),

Powers et al. (9),

§

O'Connor et al. (10), and

Fenalti et al. (11).

Binding not affected by mutation of GAD65 E264T, but mutation of GAD67 T273E conferred 50% reactivity (4).

TABLE 2

Cross-competition of binding of human mAb to GAD65 by rFab

mAb, KmM3*DPA*b78, 1 × 10−10DPD, 0.8 × 10−8DPC, 4.7 × 10−9b96.11, 3.4 × 10−9M4*
M3 rFAB 47 75 19 36 ND 13 ND 
DPA rFAB 45 65 15 
b78 rFAB 20 84 75 77 63 24 
DPD rFAB 23 24 49 21 32 ND 
DPC rFAB 71 85 92 23 
b96.11 rFAB 64 17 17 36 93 ND 
M4 rFAB 36 76 66 75 86 73 
mAb, KmM3*DPA*b78, 1 × 10−10DPD, 0.8 × 10−8DPC, 4.7 × 10−9b96.11, 3.4 × 10−9M4*
M3 rFAB 47 75 19 36 ND 13 ND 
DPA rFAB 45 65 15 
b78 rFAB 20 84 75 77 63 24 
DPD rFAB 23 24 49 21 32 ND 
DPC rFAB 71 85 92 23 
b96.11 rFAB 64 17 17 36 93 ND 
M4 rFAB 36 76 66 75 86 73 

Data are percent blocking of binding by rFab at the highest concentration of rFab used.

*

Not determined (ND).

rFab from DPD blocked GAD65 binding of mAb DPD by only 21% and did not block the reactivity of several other mAbs from which the corresponding rFab strongly blocked the binding of DPD with GAD65.

TABLE 3

Reported antigenic T-cell peptides on GAD65 restricted by HLA DR*0401

Epitope positionT-cell epitopes*Reactive peptides
T1 116–130 MNILLQYVVKSFDRS 115–127 
  VD---N--R-T---- 116–130 
T2 271–286 PRLIAFTSEHSHFSL 274–286 
  -K-VL----Q--Y-L 266–280 
   271–285 
   276–290 
T3 481–495 LYNIIKNREGYEMVF 481–495 
  --AK-----EF----  
T4 511–525 PSRLTLEDNEERMSR 511–525 
  Q---GVP-SPQ-REK  
T5 551–585 GDKVNFFRMVISNPAA 551–565 
  ---A---N-------- 556–570 
  THQDIDFLIEEIERLGQDL 566–580 
  --QS--------------- 553–572§ 
   555–567§ 
   569–585§ 
   554–570 
Epitope positionT-cell epitopes*Reactive peptides
T1 116–130 MNILLQYVVKSFDRS 115–127 
  VD---N--R-T---- 116–130 
T2 271–286 PRLIAFTSEHSHFSL 274–286 
  -K-VL----Q--Y-L 266–280 
   271–285 
   276–290 
T3 481–495 LYNIIKNREGYEMVF 481–495 
  --AK-----EF----  
T4 511–525 PSRLTLEDNEERMSR 511–525 
  Q---GVP-SPQ-REK  
T5 551–585 GDKVNFFRMVISNPAA 551–565 
  ---A---N-------- 556–570 
  THQDIDFLIEEIERLGQDL 566–580 
  --QS--------------- 553–572§ 
   555–567§ 
   569–585§ 
   554–570 
*

Sequence of T-cell epitopes in GAD65 and below the corresponding sequence of GAD67 showing differences; bold sequences indicate reported core immunogenic sequences, alternate immunogenic sequences are underlined.

Wicker et al. (22).

Patel et al. (20).

§

Nepom et al. (21).

Published ahead of print at http://diabetes.diabetesjournals.org on January 2008. DOI: 10.2337/db07-1461.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

G.F. has received financial support for his PhD candidature from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Foundation, subordinated to the Ministry of Education, Brazil. J.C.W. is a National Health and Medical Research Council (NHMRC) Principal Research Fellow and Monash University Senior Logan Fellow. A.M.B. is a NHMRC Senior Research Fellow. This study has received funding and support from the NHMRC of Australia, the Australian Research Council, and the Australian Synchrotron Research Program.

We thank the Advanced Photon Source and Industrial Macromolecular Crystallography Association Collaborative Access Team (IMCA-CAT) from synchrotron facilities.

Part of this work was presented at the 9th International Congress of the Immunology of Diabetes Society and American Diabetes Association Research Symposium, Nov 14–18 2007.

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