Unique Epitopes of Glutamic Acid Decarboxylase Autoantibodies in ...

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The Journal of Clinical Endocrinology & Metabolism 88(10):4768 – 4775 Copyright © 2003 by The Endocrine Society doi: 10.1210/jc.2002-021529

Unique Epitopes of Glutamic Acid Decarboxylase Autoantibodies in Slowly Progressive Type 1 Diabetes TETSURO KOBAYASHI, SHOICHIRO TANAKA, MINORU OKUBO, KOJI NAKANISHI, TOSHIO MURASE, AND ÅKE LERNMARK Third Department of Internal Medicine (T.K., S.T.), Yamanashi Medical University, Tamaho, Yamanashi 409-3898, Japan; Department of Endocrinology and Metabolism (M.O., K.N.), Toranomon Hospital, Tokyo, 105-8470, Japan; Okinaka Memorial Institute for Medical Research (M.O., K.N., T.M.), Tokyo 105-8470, Japan; and Department of Medicine (Å.L.), University of Washington, Seattle, Washington 98195-7710 Disease-specific epitope profiles of glutamic acid decarboxylase (GAD)65 autoantibodies (GAD65Ab) were studied in slowly progressive type 1 (insulin-dependent) diabetes mellitus (SPIDDM) and acute onset type 1 (insulin-dependent) diabetes mellitus (AIDDM) using seven kinds of GAD65/67 chimeric molecules. Sera obtained from Japanese SPIDDM (n ⴝ 17) and AIDDM (n ⴝ 46) patients followed prospectively were analyzed by immunoprecipitation, ELISA, and Western blotting. GAD65Ab in all SPIDDM samples reacted specifically with an N-terminal linear epitope located on the membrane anchoring domain between amino acids 17–51 and C-terminal conformational epitope between amino acids 443–585 of

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UTOANTIBODIES TO GLUTAMIC acid decarboxylase (GAD) are detected in autoimmune diseases, including immune-mediated type 1 diabetes, stiff-man syndrome (SMS), and autoimmune polyendocrine syndrome type 1 (1–5). Autoimmune type 1 diabetic Japanese patients are composed of at least two clinical subtypes: acute onset form [acute onset type 1 (insulin-dependent) diabetes mellitus (AIDDM)] and slowly progressive form [slowly progressive type 1 (insulin-dependent) diabetes mellitus (SPIDDM)] (4 –9). The features of pancreatic autoantibodies, including GAD65 antibodies (GAD65Ab), insulinoma-associated protein 2/islet cell antigen 512 autoantibodies (IA2Ab), and insulin autoantibodies, differ between two forms of autoimmune type 1 diabetes. AIDDM tends to occur in the young associated with low-in-titer or negative GAD65Ab, whereas the titer of IA-2Ab is high (10). In contrast, SPIDDM occurs mostly in adult ages and progresses to the insulindependent stage during several years (5, 7–9), and GAD65Ab is characteristically high in titer and persist for a long time, whereas IA-2Ab is sometimes undetected or low in titer (6, 11, 12). These findings suggest that the nature of GAD65Ab and clinical phenotype differ between SPIDDM and AIDDM. In typical AIDDM, epitope regions of GAD65Ab reside on central and carboxy-terminal regions of the GAD65 molecule

Abbreviations: AIDDM, Acute onset type 1 (insulin-dependent) diabetes mellitus; AU, arbitrary units; GABA, ␥-aminobutyric acid; GAD, glutamic acid decarboxylase; GAD65Ab, glutamic acid decarboxylase 65 autoantibodies; HLA, human lymphocyte antigen; IA-2Ab, insulinomaassociated protein 2/islet cell antigen 512 autoantibodies; NS, not significant; SDS, sd score; SMS, stiff-man syndrome; SPIDDM, slowly progressive type 1 (insulin-dependent) diabetes mellitus.

GAD65. The binding of GAD65Ab with N-terminal 83 residues in SPIDDM inversely correlated with the period in which insulin was not required. GAD65Ab in AIDDM did not react with N-terminal epitope located between amino acids 1– 83, irrespective of the titer of GAD65Ab. A novel epitope of GAD65Ab in AIDDM residing between amino acids 244 –360 was identified in 17% (8 of 46) of patients whose age of onset was younger than other AIDDM patients. In conclusion, GADAb in SPIDDM has unique N-terminal linear epitopes that are located on the anchoring domain of GAD65 molecules. Association is suggested between GAD65Ab targeted to this region and slowly progressive ␤-cell failure in SPIDDM. (J Clin Endocrinol Metab 88: 4768 – 4775, 2003)

(13–15). However, the epitopes and nature of GAD65Ab in SPIDDM are poorly defined. In the present study, we studied epitope regions of GAD65 in the sera of SPIDDM patients, using chimeric GAD65/GAD67 molecules and synthetic peptides. Longitudinal changes in intermolecular epitopes of GAD65Ab were also examined in SPIDDM and AIDDM. Materials and Methods Blood samples Serum samples were recruited from our consecutive samples, which were obtained during prospective studies from 1980 –1985 on pancreatic autoantibodies and ␤-cell function (5, 6, 8, 16) and were kept at ⫺80 C. The sera were obtained from 46 AIDDM [male/female, 24/22; mean age, 18 yr (range, 2– 61)] and 17 SPIDDM [male/female, 7/10; mean age, 48 yr (range, 20 – 68)] patients [mean duration before insulin treatment, 21 months (range, 3– 61)]. None of AIDDM patients or SPIDDM patients had autoimmune polyendocrine syndrome. All serum samples were collected between 3 months and 5 yr after the diagnosis of diabetes. Diagnostic criteria of type 1 diabetes were based on World Health Organization criteria (4). We define AIDDM in patients with acute clinical symptoms, including polyuria, polydipsia, and body weight loss and ketosis/ketoacidosis, and in whom insulin was started within 3 months of the diabetic symptoms. We define SPIDDM as: 1) diabetes with the initial clinical feature of non-insulin-dependency at least for 3 months after diagnosis of diabetes; 2) diabetes be treated with diet or oral hypoglycemic agents and eventually lapsing into an insulin-dependent state with complete absence of serum C-peptide response to oral glucose [we defined the time of insulin-dependency as that time when the integrated value of serum C-peptide at 0, 30, 60, 90, 120, and 180 min became less than 1.7 nm (Refs. 7–9)]; and 3) being persistently positive for GAD65Ab and islet cell autoantibodies at least 1 yr after diagnosis (5, 6). C-peptide response to 100-g oral glucose in 17 SPIDDM patients changed from 4.8 ⫾ 1.2 nm at baseline, which is when we began our observations of those patients, to 3.2 ⫾ 0.7 nm at 1 yr after we began observing those patients. At 2 yr after observation, those values were

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2.7 ⫾ 0.5 nm; and at 3 yr after observation, those values were 1.5 ⫾ 0.1 nm. Those values were 0.9 ⫾ 0.1 nm after 4 yr observation and 0.9 ⫾ 0.1 nm after 5 yr observation. After 6 yr of observation, those values were 0.8 ⫾ 0.1 nm. All patients progressed to an insulin-dependent sate 32 ⫾ 16 months (mean ⫾ sd; range, 6 – 60) after beginning of the observation. Mouse monoclonal GAD6 antibody against GAD65 (Roche Molecular Biochemicals Co., Indianapolis, IN) was also examined. The present study was approved by the Ethical Committee of the Toranomon Hospital and the Okinaka Memorial Institute for Medical Research.

Construction of chimeric GAD molecules Full-length human GAD65 cDNA inserted into the vector pcDNAII (Invitrogen Co., San Diego, CA), pEx9 (17), was used for GAD65 assay and chimeric construction. Full-length human GAD67 cDNA inserted into pcDNAI was donated by Dr. B. Michelsen (Hagedorn Research Institute, Gentofte, Denmark). Figure 1A shows a schematic representation of the chimeric molecules used in this study. The following primers were used for constructions of chimeric molecules: BamHI-S1: 5⬘-CCGGATCCCCGAGCTGATGGCGTCTTCGAC-3⬘, NarI-S2: 5⬘-CTCCTGGGGGCGCCATATCCAAC3⬘, BglII-S3: 5⬘-ATAAGATCTGGTTGCATGTCGATGCTGCCTG-3⬘, StuI-S4: 5⬘-ACAAGGCCTTTCAGTGTGGCCGCCACGTGGA-3⬘, BstXIS5: 5⬘AATCAAAGCCAGAATGATGGAGTCAGGTACGA-3⬘, NarI-A1: 5⬘-TTGGATATGGCGCCCCCAGGAGA-3⬘, BglII-A2: 5⬘-ACCAGATCTTATATTTCTCACATATATCTGC-3⬘, StuI-A3: 5⬘-GAAAGGCCTTGTCCCCGGTGTCGTAGGA-3⬘, XbaI-A4: 5⬘-CTATCTAGATTACAGATCCTGGCCCAGTCTT-3⬘. To construct chimera A, GAD65 (1–244)/GAD67 (253–369)/GAD65 (360–585), NarI/BglII sites were introduced at the appropriate location of GAD67 cDNA by PCR using NarI-S2 and BglII-A2 primers. The digested GAD67 fragment was exchanged with the corresponding fragment of the GAD65 cDNA using a native NarI/BglII site in the GAD65 cDNA. Likewise, to create chimera B, GAD65 (1–359)/GAD67 (370– 451)/GAD65 (443–585), BglII/StuI sites were introduced at the appropriate location of GAD67 cDNA by PCR using BglII-S3 and StuI-A3 primers. The digested GAD67 fragment was exchanged with a corresponding fragment of the GAD65 cDNA using BglII/StuI sites in the GAD65 cDNA. To create chimera C, GAD65 (1–244)/GAD67 (253–594), NarI/XbaI sites of GAD67 cDNA were amplified by PCR using NarI -S2 and XbaI-A4 primers, and the digested GAD67 fragment was exchanged with a corresponding fragment of the GAD65 cDNA using NarI/XbaI sites in the GAD65 cDNA. To create chimera D, GAD65 (1–244)/GAD67 (253– 451)/ GAD65 (443–585), Nar I/StuI sites of GAD67 cDNA were amplified by PCR using NarI-S2 and StuI-A3 primers, and the digested GAD67 fragment was exchanged with a corresponding fragment of the GAD65 cDNA using

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NarI/StuI sites in the GAD65 cDNA. To create chimera E, GAD65 (1– 442)/ GAD67 (452–594), StuI/XbaI sites of GAD67 cDNA were amplified by PCR using StuI-S4 and XbaI-A4 primers, and the digested GAD67 fragment was replaced with a corresponding fragment of the GAD65 cDNA using StuI/ XbaI sites in the GAD65 cDNA. To create chimera H, GAD67 (1– 451)/ GAD65 (443–585), BamHI/StuI sites were introduced at the appropriate location of GAD67 cDNA by PCR using BamHI-S1 and StuI-A3 primers. The digested GAD67 fragment was exchanged with a corresponding fragment of GAD65 cDNA using BamHI/StuI sites in the GAD65 cDNA. Chimera N, GAD65 (1– 83)/GAD67 (89–594), was donated by Dr. Falorni (University of Perugia) and was created as described previously (15). The DNA sequences of all the constructed chimeric molecules were confirmed by analysis using the Taq DyeDeoxy Terminator Cycle Sequencing kit and DNA sequencer 373 (Applied Biosystems, Foster City, CA).

In vitro transcription/translation and immunoprecipitation of GAD65, GAD67, and chimeric GAD molecules Plasmid cDNA coding for GAD65, GAD67, or GAD65/67 chimera molecules described above were labeled by coupled in vitro transcription/translation (18). Briefly, 2 ␮g plasmid cDNA was incubated with [35S]methionine (10 mCi/ml, ⬎1,000 Ci/mmol; Amersham Ltd., Amersham, Buckinghamshire, UK) and the reticulocyte lysate system according to the manufacturer’s instructions (TNT SP6 coupled reticulocyte lysate system; Promega, Madison, WI). Incorporation of radioactivity in recombinant proteins was monitored by precipitation with trichloroacetic acid. A total of 10,000 cpm in vitro synthesized GAD and 4 ␮l serum diluted in 50 ␮l buffer A (20 mm Tris, 150 nm NaCl, pH 7.4 with 0.15% BSA, 0.5% Tween-20) were mixed in 96-well microtiter plates (Greiner, Nurtingen, Germany). After overnight incubation on a rotating platform, 20 ␮l Protein A Sepharose (Amersham PharmaciaBiotech UK Ltd., Buckinghamshire, UK) (50% vol/vol) was added for 2 h, followed by the transfer of the samples into prewashed 96-well filtration plates (Multiscreen BV 1.2 ␮m; Millipore, Bedford, MA). Plates were extensively washed five times in buffer A (150 ␮l), and precipitates were punched out into 5-ml scintillation vials (Multiple 8-punch system; Millipore) to count bound proteins in a liquid scintillation counter. In each experiment, the same positive and negative standard sera were included in duplicate. Binding of GAD65Ab with GAD65 (1–585) was expressed as an index, with arbitrary units (AU) calculated as follows: AU ⫽ [cpm (test serum) ⫺ cpm (negative standard serum)]/[cpm (positive standard serum) ⫺ cpm (negative standard serum)]. One positive serum from a type 1 diabetic subject and two negative control sera from

FIG. 1. Immnoprecipitation of chimeric GAD65/67 constructs by the sera from SPIDDM. Immune complexes were precipitated by protein A-Sepharose. Schematic representation of seven kinds of chimeric molecules (E, B, A, D, H, C, and N) used in the study are shown in panel A. Numbers on the constructs indicate amino acid positions in human GAD65 and GAD67. All sera from the patients with SPIDDM reacted with GAD65/67 chimeras (E, B, A, D, H, C, and N) (panel B). The reactivity with GAD65/67 chimeras (H, C, N, and GAD67) was significantly decreased, compared with that of GAD65. Results are expressed as mean SD score (SDS) ⫾ SEM. a, P ⬍ 0.01 vs. binding GAD65 (1–585).

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healthy subjects were used in each assay. Intraassay and interassay coefficients of variation of the GADAb assay were 7.7% (n ⫽ 9) and 10.9% (n ⫽ 9), respectively. Each serum was analyzed in duplicate in each immunoprecipitation assay, and the mean of three independent assays was used for analysis. Our laboratory participated in the second GADAb workshop sponsored by the Immunology of Diabetes Workshop (19), and our laboratory scored 80% sensitivity and 100% assay specificity on the workshop sera. The cutoff levels for positive GAD65Ab and reactivity with chimeric molecules were set at mean ⫹ 3 sd of the levels in 100 normal control sera. The upper levels of normal AU were 0.04 for GAD65Ab; 0.03 for both GAD chimera A and chimera B; 0.02 for chimera C; 0.03 for chimera D; 0.04 for chimera E, chimera H, and chimera N; and 0.03 for GAD67Ab assays. For comparing the bindings of autoantibodies to the chimeric GAD 65/67 constructs, sd score (SDS) values (20) were calculated for all sera based on mean value and sd of indexes with 100 normal control sera [SDS ⫽ (antibody index of test serum-mean index of healthy control sera)/sd of the indexes of healthy control sera]. Positive was defined as an SDS of 3 or more. For all assays, sera from the same group of normal controls were used to determine the cut-off values.

Western blotting and blocking study Recombinant human full-length GAD65 and GAD67 were gifts from Dr. R. B. Smith (FIRS Laboratories, Cardiff, UK). Western blotting analysis was done as described (21). Briefly, recombinant GAD65 or GAD67 were subjected to sodium dodecyl sulfate gel electrophoresis on 7% polyacrylamide gels (10 ␮l/lane). After electrophoresis and electrophoretic transfer, the nitrocellulose membrane (Millipore) was cut into strips for immunoblotting. The strips were blocked for 1 h at room temperature with Block Ace (Yukijirushi, Osaka, Japan) containing 5% nonfat-milk/Tris-buffered saline and incubated overnight at 4 C with patients’ sera or the GAD6, specific monoclonal antibody to GAD65. After washing with 0.3% Tween-20 in Tris-buffered saline (pH 7.4), strips were incubated for 4 h at room temperature with 1:125 to 1:500 diluted patient’s serum. After washing, blots were incubated for 60 min at room temperature with peroxidase-labeled rabbit antihuman IgG or goat antimouse IgG (1:1000) (MBL, Tokyo, Japan). After rinsing, the strips were developed with 0.01% diaminobenzidine substrate (Sigma Chemical Co., St. Louis, MO). Unlabeled chimeric GAD65/67 molecules were prepared by in vitro transcription/translation on a reticulocyte lysate system as described above except that amino acid mixture contained unlabeled methionine rather than [35S]methionine. For the blocking studies, unlabeled GAD chimeric protein (representing about 20% of an in vitro translation with 1 ␮g GAD RNA) was incubated with 20 ␮l diluted serum (1:100) overnight at 4 C. This incubated mixture of serum and unlabeled GAD chimeric protein was rotated at 100,000 ⫻ g, and supernatant was used for Western blotting.


absorbance at 450 nm was measured with an automatic ELISA plate reader (Molecular Devices Co., Sunnyvale, IL).

Human lymphocyte antigen (HLA)-DQ genotyping HLA-DQA1 and -DQB1 genotyping was performed by a previously described method (23).

Statistical analysis Mann-Whitney’s U test was used for evaluating the difference of binding of sera with GAD65, GAD67, chimeric GAD molecules, or synthetic GAD peptides. Spearman’s test was used for analyzing correlation between binding of GAD65, GAD67, or chimeric GAD molecules with patients’ sera and the period before insulin treatment in SPIDDM patients. Fisher’s exact test was used for the comparison of the frequencies of HLA-DQ genotypes. The frequencies of all data were expressed as mean ⫾ sem.

Results SPIDDM antibodies bind to a novel GAD65 N-terminal epitope region

The SPIDDM sera were all positive for GAD65Ab but negative for GAD67Ab (Fig. 1). The binding assessed by SDS remained unaffected by chimeras E, B, and A when compared with the binding to GAD65 (Fig. 1), suggesting that most of the SPIDDM GAD65Ab binding would reside on the N-terminal and middle parts of GAD65. The binding to chimera D was insignificantly reduced, suggesting the presence of GAD65Ab that would recognize the N-terminal end of GAD65. This was verified by the analysis of chimeras C and N (Fig. 1). All 17 SPIDDM sera bound to N-terminal GAD chimeric molecules N [GAD65 (1– 83)/GAD67 (89 –594)] and C [GAD65 (1–244)/GAD67 (253–594)] (Fig. 1). The binding of SPIDDM sera with chimeric molecules N and C were comparable, indicating that the epitope resides at amino acids 1– 83 of GAD65 molecule (Fig. 1). There was no correlation between GAD65Ab titers and the binding with GAD chimera N, [GAD65 (1– 83)/GAD67 (89 – 594)] in AIDDM or SPIDDM sera (Fig. 2). All 17 SPIDDM sera bound with chimera H [GAD67 (1– 451)/GAD65 (443–585)]

Synthetic GAD peptides and ELISA Five kinds of GAD peptides (overlapping 19-mer) corresponding to the sequence of human GAD65 amino acids 1– 83 were synthesized and supplied at more than 80% purity, with HPLC (QIAGEN, Tokyo, Japan). These peptides include: 1) amino acids 1–19: MASPGSGFWSFGSEDGSGD; 2) amino acids 17–35: SGDSENPGTARAWCQVAQK; 3) amino acids 33–51: AQKFTGGIGNKLCALLYGD; 4) amino acids 49 – 67: YGDAEKPAESGGSQPPRAA; and 5) amino acids 65– 83: RAAARKAACACDQKPCSCS. Bindings of each peptide to the sera from the patients with SPIDDM (n ⫽ 17) and AIDDM (n ⫽ 46) and the nondiabetic control subjects (n ⫽ 100) were measured by an ELISA system as described previously (22). In brief, 50 ␮l of 1 ␮m of each peptide diluted in PBS was used to coat individual wells in a 96-well plate overnight at 4 C. After five washes with PBS containing 0.05% Tween 20, the wells were incubated with PBS containing 25% Block Ace (Yukijirushi) and 1% rabbit serum overnight at 4 C. After five washes, the wells were coated with 1:4 dilution of Block Ace and 1:100 dilution of sera overnight at 4 C. After five washes, the wells were coated with 1:10 dilution of Block Ace and 1:6000 dilution of peroxidase-conjugated rabbit antihuman IgG and incubated for 1 h at room temperature. The wells were washed five times before adding Slow TMB (Pierce, Rockford, IL) as substrate. The plates were kept at room temperature for 10 min. After adding 1 m H2SO4,

FIG. 2. Correlation between GAD65Ab titers and the bindings to N-terminal GAD65 chimera N [GAD65 (1– 83)/GAD67 (89 –594)] in SPIDDM (F) and AIDDM (E). Dotted lines, Mean ⫹ 3 SD in nondiabetic sera: SDS, SD score.



(Fig. 1), indicating a presence of another epitope at the GAD65 COOH-terminal region [IDDM-E2 by Daw et al. (13)]. The 17 SPIDDM sera were therefore next assayed for binding to GAD65 by immunoblotting. GAD65Ab epitope in SPIDDM, which resides at N-terminal region, is a linear epitope

The reactivity of antibodies in AIDDM and SPIDDM against linear epitope(s) was studied by Western blotting with recombinant human GAD65 (Fig. 3). Positive reactivity was detected in all SPIDDM sera with GAD65, whereas none of the sera from AIDDM patients showed a positive reactivity with GAD65 (Fig. 3). The positive reactivity of SPIDDM sera with denatured GAD65 in Western blotting was neutralized after preincubation with chimera N [GAD65 (1– 83)/GAD67 (89 –594)]. The reactivity of SPIDDM sera with denatured GAD65 was unchanged after preincubation with chimera H [GAD67 (1– 451)/GAD65 (443–585)]. Positive reaction was not detected with Western blotting using GAD67 and sera from AIDDM or SPIDDM. N-terminal GAD65Ab epitope in SPIDDM examined by ELISA using synthetic GAD peptides

The reactivity to GAD peptides (2) and (3), which were corresponding to the sequence of human GAD65 (amino acids 17–51) (Fig. 4) of the sera from SPIDDM patients, was significantly higher than that of normal controls. The reactivity of the sera from AIDDM was insignificant when compared with normal controls. N-terminal binding correlated with the period before insulin treatment in SPIDDM

The binding of the sera from SPIDDM patients with GAD chimera N [GAD65 (1– 83)/GAD67 (89 –594)] correlated well with the period in which insulin was not required to control hyperglycemia (r ⫽ ⫺0.884, P ⬍ 0.0001) (Fig. 5A). In contrast, no correlation was observed between binding of the sera from SPIDDM patients with chimera H [GAD67 (1– 451)/

FIG. 3. Analysis by Western blotting on the reactivity of the sera from SPIDDM (lanes 1–3) and acute-onset type 1 diabetes (AIDDM) (lanes 4 – 6), GAD6 (lane 7), and normal control (lane 8) with denatured GAD 65 protein. Positive staining was observed from lanes 1–3 and lane 7.

FIG. 4. Peptide reactivity of the GAD65Ab-positive sera from SPIDDM (f), AIDDM (p), and controls (䡺) assayed by ELISA. *, P ⬍ 0.0147; **, P ⬍ 0.0001 vs. controls.

GAD65 (443–585)], GAD65, and GAD67 and the period before insulin treatment (Fig. 5, B–D). Binding of AIDDM sera with GAD65 middle and COOHterminal regions

The binding of eight of 46 (17%) AIDDM sera assayed by immunoprecipitation with GAD chimera A [GAD65 (1– 244)/GAD67 (253–369)/GAD65 (360 –585)] as well as GAD chimera D [GAD65 (1–244)/GAD67 (253– 451)/GAD65 (443– 585)] was not significantly different from normal sera (Fig. 6, A and B). These data indicate that some AIDDM sera recognized a novel epitope region of amino acids 244 –360 residues named as IDDM-E3. The ages of the AIDDM patients were younger (mean, 7 yr; range, 2–11) than other AIDDM patients (mean, 19 yr; range, 8 – 61, P ⬍ 0.05). In 14 of 46 (31%) sera from AIDDM patients, significant binding was observed with chimera A [GAD65 (1–244)/ GAD67 (253–369)/GAD65 (360 –585)] but not with chimera D [GAD65 (1–244)/GAD67 (253– 451)/GAD65 (443–585)], indicating the presence of a middle epitope at 244 – 443 amino acids [similar to IDDM-E1 (240 – 435 amino acids) by Daw et al. (13)] (Fig. 6, A and C). The remaining 24 of 46 (52%) sera bound GAD chimera H [GAD67 (1– 451)/GAD65 (443–585)] along with significant 244 – 443 amino acids epitope, indicating that these sera have GADAb binding to both the COOH-terminal [similar to IDDM-E2 by Daw et al. (13)] and the middle epitope regions (Fig. 6, A and D). None of the AIDDM sera immunoprecipitated the N-terminal epitope GAD chimeras N and C of GAD65 and the GAD67 (Fig. 6, B–D). Monoclonal antibody GAD6 bound chimeras B, A, D, and H but did not bind with chimera E [GAD65 (1– 442)/GAD67 (452–594)]. These results are in accordance with the previous results that showed a presence of epitope region amino acids 529 –585 of GAD65 in GAD6 antibody (24).

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FIG. 5. Correlation between the period in which insulin was not required to control hyperglycemia in SPIDDM and the binding with N-terminal GAD65/67 chimera N [GAD65 (1– 83)/ GAD67 (89 –594)] (A), binding with C-terminal GAD65/67 chimera H [GAD67 (1– 451)/GAD65 (443–585)] (B), level of GAD65Ab (C), and level of GAD67Ab (D). All samples were negative for GAD67Ab. SDS, SD score.

Binding of SPIDDM and AIDDM sera with pyridoxal phosphate binding site

The binding of GADAb in SPIDDM sera to chimeric GAD molecules, in which pyridoxal phosphate binding site of GAD65 (K396) was replaced with GAD67 (370 – 451) (chimera B), was similar to those of GAD65 (Fig. 1, A and B). In contrast, the binding of AIDDM sera to chimera B was lower, compared with GAD65 (1–585) (Fig. 6, A–D). Longitudinal changes of the epitopes in SPIDDM and AIDDM

In SPIDDM, the binding of GAD65Ab to the N-terminal epitope region [chimera N, GAD65 (1– 83)/GAD67 (89 –594)] was 1.40 ⫾ 0.32 AU at onset and did not change after 5 yr of onset of diabetes [1.35 ⫾ 0.41 AU, not significant (NS), n ⫽ 17)]. The binding of other chimeric molecules, including chimeras A, B, C, D, E, and H with SPIDDM sera, after 5 yr, did not show significant change (data not shown). The average titer of GAD65Ab in SPIDDM patients was 4.63 ⫾ 0.43 AU at onset and remained unchanged (at 4.51 ⫾ 0.56 AU) 5 yr after onset. In 14 of 24 (58%), AIDDM sera reacted with both the middle and COOH-terminal epitopes at onset; there was a reduced reactivity with COOH-terminal epitope 5 yr after diagnosis. The titer of GAD65Ab deceased significantly from 3.72 ⫾ 0.71 AU at onset and 1.63 ⫾ 0.51 AU at 5 yr after diagnosis (P ⬍ 0.01 vs. value at onset) in the AIDDM patients.

The remaining 10 of 24 (42%) AIDDM sera, which initially had reactivity with both middle and COOH-terminal epitopes, had both middle and COOH-terminal epitopes even after 5 yr. All AIDDM sera, which initially reacted only with middle (IDDM-E1) epitope (n ⫽ 14) or the unique 244 – 360 (IDDM-E3) epitope (n ⫽ 8), did not show any significant change 5 yr after onset, whereas the titer of GAD65Ab decreased significantly (data not shown). HLA-DQA1, and -DQB1 genotypes and genotypic combinations of HLA-DQA1-DQB1 haplotypes in SPIDDM and AIDDM

HLA-DQA1*0303-DQB1*0401 haplotype was more common in SPIDDM than in control subjects (Table 1). In contrast, AIDDM had significantly high prevalence of HLADQA1*0302-DQB1*0303 haplotype in a homozygous manner or in a heterozygous manner with HLA-DQA1*0303DQB1*0401 haplotype (Table 1). Discussion

We demonstrate that SPIDDM patients in Japan are characterized by GAD65Ab that binds to the N-terminal region of GAD65 defined by residues 1– 83. We could narrow down the N-terminal region of GAD65 (amino acids 17–51) by an ELISA system using synthesized GAD peptides. These autoantibodies against GAD65 were unique because they were also detected by Western blotting. We could not find any



FIG. 6. Binding of GAD65/67 chimeric molecules by the sera from acute-onset type 1 diabetes (AIDDM). Schematic representation of seven kinds of chimeric molecules (E, B, A, D, H, C, and N) used in the study (panel A). Numbers on the constructs indicate amino acid positions in human GAD65 and GAD67. The binding pattern of the sera from 17% (8 of 46) AIDDM patients show a presence of epitope specifically limited residues 253–369 of GAD65 molecules (panel B). The binding pattern of the sera from 31% (14 of 46) AIDDM patients shows a presence of epitope of residues 253– 451 (panel C). The binding pattern of the sera from 52% (24 of 46) AIDDM patients shows a presence of epitopes of residues 253– 451 and 443–585 (panel D). Data are expressed as the mean SDS ⫾ SEM. All sera from the patients with AIDDM do not bind with N-terminal GAD65/67 chimeras (C and N). The amount of GAD65/67 chimera molecules immunoprecipitated is expressed in terms of SDS. a, P ⬍ 0.01 vs. binding GAD65 (1–585). SDS, SD score. TABLE 1. Frequencies of the combinations of HLA-DQA1-DQB1 haplotypes in SPIDDM and AIDDM

DQA1*-DQB1*/DQA1*-DQB1*

0303-0401/0303-0401 0303-0401/0302-0303 0302-0303/0302-0303 0303-0401/X 0302-0303/X

SPIDDM (n ⫽ 17)

AIDDM (n ⫽ 43)

Nondiabetic controls (n ⫽ 84)

P value vs. controls (and relative risk)

n (%)

n (%)

n (%)

SPIDDM

AIDDM

1 (6) 0 (0) 0 (0) 8 (47) 3 (18)

1 (2) 8 (19) 9 (21) 4 (9) 12 (28)

1 (1) 0 (0) 2 (2) 15 (18) 12 (14)

NS NS NS 0.0218 (4.1)b NS

NS 0.0001 (19.2) 0.0010 (10.9)a NS NS

NS, Not significant; X ⫽ 0302-0303 or 0303-0401. a P ⫽ 0.0497 for the comparison with SPIDDM. b P ⫽ 0.0024 for the comparison with AIDDM.

change in intermolecular epitope at onset and 5 yr after onset in SPIDDM sera. This may suggest that the N-terminal epitope region of GAD65 observed in SPIDDM is unique and

is not the consequence of epitope spreading from certain initial epitope(s). Our findings in SPIDDM contrasted with previous studies in a Caucasian IDDM patient, in which no

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N-terminal epitope was demonstrated (13–15). The Nterminal epitope region of GAD65 was reported in nondiabetic, first-degree relatives in a Caucasian population (25). The pathogenetic significance remains speculative. The Nterminal region of GAD65 molecules involves the membrane anchoring site that is hydrophobic in nature (26, 27), and is associated with the sorting of GAD65 molecules to synapticlike microvesicular structures in ␤-cells. GAD65 is potentially exposable to outmembrane circumstances during exocytosis of ␥-aminobutyric acid (GABA). It is tempting to speculate that circulating GAD65Ab in SPIDDM, which is accessible and is targeting to the N-terminal region of GAD65 molecules, modulate the enzyme activity of GAD in the microvesicles containing GABA and subsequently affect synthesis of GABA, an important fuel of ␤-cells (28). GAD65Ab may affect GAD65 in ␤-cells with decreased exocytosis of GABA, an important regulatory factor of intra-islet hormone homeostasis (26 –30). In SMS, circulating GAD65Ab is probably related to disorder of GABAergic neurons with subsequent characteristic clinical features, which are improved by plasmapheresis (31, 32). In addition, GAD65Ab in SMS are targeted to linear N-terminal as well as conformational Cterminal epitopes (3) that is similar to SPIDDM. Epitope mapping studies with SMS sera have localized linear epitopes to N-terminal residues 1– 8 (34) or 1–195 as well as C-terminal regions containing residues 475– 484 or 571–585 (3). GAD65Ab in SMS reduced GABA synthesis in rat cerebral extracts in vitro (35). It is possible that in SPIDDM, the GAD65Ab, which possess similar characteristics with those in SMS, contribute to disturb GABAergic systems in ␤-cells through the above mentioned mechanisms. Hampe et al. (25) reported a crucial role of the N-terminal region of GAD65 in the binding of GAD65Ab in type 1 diabetes. An inverse relationship between N-terminal binding of GAD65Ab and duration before insulin requirement may support the epitope specific in vivo action of GAD65Ab in SPIDDM. Our findings demonstrated clinical usefulness of measuring epitope-specific GAD65Ab for further prediction of ␤-cell failure in SPIDDM. Our results are not in accordance with another cross-sectional and retrospective study dealing with GADAb-positive type 2 diabetes latent autoimmune diabetes in adults (36), which emphasized the predictive usefulness of COOH-terminal epitope of GAD65Ab. The difference may be explained by different study design and different genetic background. First, N-terminal GAD65/67 chimeras that were used in our study were not examined in the other study (36). Second, our study was based on prospective study (6, 8, 16), whereas the other study mostly used samples from retrospective study. Finally, specific human leukocyte antigen (HLA) association with HLA-DQA1*0301DQB1*0401 in Japanese SPIDDM different from that in Japanese AIDDM (Table 1) may be related with characteristic epitope profiles in our study. We can distinguish two central epitope regions (244 –360 and 244 – 443 residues) and one C-terminal region (443–585 residues) of GAD65Ab in AIDDM. The locations of these epitopes on the GAD65 molecule are completely different from those in SPIDDM. The epitope region in the middle part of GAD65 (amino acids 244 – 443) in Japanese AIDDM is in agreement with IDDM-E1 by Daw et al. (13) or EP-1 by


Sohnlein et al. (37). The novel epitope (244 –360 residues) is located in a more narrow sequence than the previous epitope (IDDM-E1 or Ep-1), in which an even-more-narrow homologous sequence with Coxsackie virus protein P2-C is included. Only the sera from juvenile-onset AIDDM had the unique epitope residue (244 –360). The presence of a unique epitope region (residues 244 –360) in AIDDM and the absence of epitope spreading in this subgroup of patients with AIDDM differs from another report in a Caucasian population (38), suggesting that the mechanisms responsible for generation of GAD65Ab in AIDDM are heterogeneous and affected by many factors. These differences may be explained partly by the unique genetic background, including HLA-DQ genotypes, in Japanese patients with AIDDM as shown in the present study (Table 1). We examined the possibility that GAD65Ab recognize the pyridoxal 5⬘-phosphate-binding site (amino acids 395–398), which portion is essential for GAD65 enzyme activity. The binding of SPIDDM sera with chimeric GAD molecules, in which the pyridoxal phosphate binding site of GAD65 was replaced with GAD67 (370 – 450) (chimera B), was similar to that of GAD65. These results suggest that the epitopes of GAD65Ab in SPIDDM sera do not reside on the pyridoxal phosphate binding site of GAD65. In contrast, the binding of AIDDM sera with chimera B was lower than that with GAD65 (1–585). This may suggest the presence of an epitope of GAD65Ab in Japanese AIDDM, in accordance with previous reports (38, 39). Acknowledgments We thank Dr. B. Michelsen (Hagedorn Research Institute) for her generous gift of GAD67 cDNA inserted pcDNAI and Dr. Falorni (University of Perugia) for his generous gift of GAD65 (1– 83)/GAD67 (89 – 594) chimera molecule. We thank K. Hosaka for excellent secretarial work and T. Hughes for editorial assistance. Received October 2, 2002. Accepted July 2, 2003. Address all correspondence and requests for reprints to: Tetsuro Kobayashi, M.D., Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Yamanashi 409-3898, Japan. E-mail: [email protected]. This work was supported, in part, by a grant from the Ministry of Education, Science, Sports and Culture, Japan.

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