Site-directed mutagenesis of K396R of the 65 kDa glutamic acid decarboxylase active site obliterates enzyme activity but not antibody binding

The role of K396 in the enzymatic catalysis and the antigenicity of the 65 kDa isoform of glutamate decarboxylase (GAD65) was analyzed using the K396R GAD65 mutant. GAD65 is a major autoantigen in Type 1 diabetes and autoantibodies directed to GAD65 are widely used markers for this disease. We found that (1) recombinant human GAD65 is fully enzymatically active; (2) the K396R mutation abolished GAD65 activity; and (3) the K396R mutant retained full antigenicity to GAD65 autoantibodies in serum from Type 1 diabetes patients, but not to polyclonal antibodies raised to the catalytic domain.     

Anti-idiotypic antibody specific to GAD65 autoantibody prevents type 1 diabetes in the NOD mouse

Overt autoantibodies to the smaller isoform of glutamate decarboxylase (GAD65Ab) are a characteristic in patients with Type 1 diabetes (T1D). Anti-idiotypic antibodies (anti-Id) directed to GAD65Ab effectively prevent the binding of GAD65 to GAD65Ab in healthy individuals. Levels of GAD65Ab-specific anti-Id are significantly lower in patients with T1D, leading to overt GAD65Ab in these patients. To determine the possible protective role of GAD65Ab-specific anti-Id in T1D pathogenesis, we developed the monoclonal anti-Id MAb 8E6G4 specifically targeting human monoclonal GAD65Ab b96.11. MAb 8E6G4 was demonstrated as a specific anti-Id directed to the antigen binding site of b96.11. MAb 8E6G4 recognized human antibodies in sera from healthy individuals, T2D patients, and T1D patients as established by ELISA. We confirmed these MAb 8E6G4-bound human antibodies to contain GAD65Ab by testing the eluted antibodies for binding to GAD65 in radioligand binding assays. These findings confirm that GAD65Ab are present in sera of individuals, who test GAD65Ab-negative in conventional detection assays. To test our hypothesis that GAD65Ab-specific anti-Id have an immune modulatory role in T1D, we injected young Non Obese Diabetic (NOD) mice with MAb 8E6G4. The animals were carefully monitored for development of T1D for 40 weeks. Infiltration of pancreatic islets by mononuclear cells (insulitis) was determined to establish the extent of an autoimmune attack on the pancreatic islets. Administration of MAb 8E6G4 significantly reduced the cumulative incidence rate of T1D and delayed the time of onset. Insulitis was significantly less severe in animals that received MAb 8E6G4 as compared to control animals. These results support our hypothesis that anti-Id specific to GAD65Ab have a protective role in T1D.     

Analysis of GAD65 autoantibodies in Stiff-Person syndrome patients

Autoantibodies to the 65-kDa isoform of glutamate decarboxylase GAD65 (GAD65Ab) are strong candidates for a pathological role in Stiff-Person syndrome (SPS). We have analyzed the binding specificity of the GAD65Ab in serum and cerebrospinal fluid (CSF) of 12 patients with SPS by competitive displacement studies with GAD65-specific rFab-derived from a number of human and mouse mAbs specific for different determinants on the Ag. We demonstrate considerable differences in the epitope specificity when comparing paired serum and CSF samples, suggesting local stimulation of B cells in the CSF compartment of these patients. Moreover, these autoantibodies strongly inhibit the enzymatic activity of GAD65, thus blocking the formation of the neurotransmitter gamma-aminobutyric acid. The capacity of the sera to inhibit the enzymatic activity of GAD65 correlated with their binding to a conformational C-terminal Ab epitope. Investigation of the inhibitory mechanism revealed that the inhibition could not be overcome by high concentrations of glutamate or the cofactor pyridoxal phosphate, suggesting a noncompetitive inhibitory mechanism. Finally, we identified a linear epitope on amino acids residues 4-22 of GAD65 that was recognized solely by autoantibodies from patients with SPS but not by serum from type 1 diabetes patients. A mAb (N-GAD65 mAb) recognizing this N-terminal epitope was successfully humanized to enhance its potential therapeutic value by reducing its overall immunogenicity.     

Glutamate decarboxylase and GABA in pancreatic islets: lessons from knock-out mice.

The GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) is expressed in pancreatic beta-cells and GABA has been suggested to play a role in islet cell development and function. Mouse beta-cells predominantly express the larger isoform of the enzyme, GAD67, and very low levels of the second isoform, GAD65. Yet GAD65 has been shown to be a target of very early autoimmune T-cell responses associated with beta-cell destruction in the non-obese diabetic (NOD) mouse model of Type 1 diabetes. Mice deficient in GAD67, GAD65 or both were used to assess whether GABA is important for islet cell development, and whether GAD65 is required for initiation of insulitis and progression to Type 1 diabetes in the mouse. Lack of either GAD65 or GAD67 did not effect the development of islet cells and the general morphology of islets. When GAD65-/-(129/Sv) mice were backcrossed into the NOD strain for four generations, GAD65-deficient mice developed insulitis similar to GAD65+/+ mice. Furthermore, at the low penetrance of diabetes in this backcross, GAD65-deficient mice developed disease at the same rate and incidence as wildtype mice. The results suggest that GABA generated by either GAD65 or GAD67 is not critically involved in islet formation and that GAD65 expression is not an absolute requirement for development of autoimmune diabetes in the NOD mouse.     

Development of glutamic acid decarboxylase 65 (GAD65) autoantibody assay using biotin-GAD65 fusion protein

We evaluated a biotin-glutamic acid decarboxylase 65 (GAD65)-based enzyme-linked immunosorbent assay (B-ELISA) to detect GAD65 autoantibodies (GAD65Ab) in 78 sera from individuals with newly diagnosed type 1 diabetes. The GAD65Ab index of patients with type 1 diabetes (mean value of GAD65Ab index of 1.891) was significantly higher than those in 50 sera from healthy control group (mean value of 0.068). The intra- and inter-assay coefficients of variation (CV) were calculated to be 1.042 and 10.703%, respectively. The specificity of the B-GAD65 ELISA was comparable to the standard radioimmunoassay (RIA) which is routinely used in the laboratory. We describe the optimal conditions of the binding kinetics from each assay-step for the detection of GAD65Ab using the WHO standard serum 97/550 as a model autoantibody serum. We concluded that incubation times of 15, 90, and 90 min for step 1 (pre-incubation of Biotin14-GAD65 with serum), step 2 (binding the Ab/Ag complex to NeutrAvidin plate), and step 3 (incubation with HRPO-anti-human IgG), respectively, along with human serum dilutions of 1:50, would provide an optimal assay signal within a relatively short timeframe.     

Comparative expression and purification of human glutamic acid decarboxylase from Saccharomyces cerevisiae and Pichia pastoris

The yeast cell factory is a potentially useful source of proteins in general. They include glutamic acid decarboxylase (GAD), which is one of the major autoantigens for Type 1 diabetes. We have created a hybrid form of GAD consisting of amino acids 1–101 of the human GAD67 protein fused to amino acids 96–585 of the human GAD65 protein, and have modified this to include a C-terminal hexa-Histidine (H6) tag sequence. This hybrid GAD67/65-H6 was expressed in two yeast hosts: constitutively under the control of the plasmid phosphoglycerate kinase promoter (PGK1) in Saccharomyces cerevisiae, and inducibly under the control of the chromosomal alcohol oxidase promoter (AOX1) in Pichia pastoris. Enzymatically active hybrid GAD was prepared from yeast lysates by purification either on an affinity column based on the GAD-1 monoclonal antibody, or by metal-affinity chromatography. The purified GAD67/65-H6 was radiolabelled with iodine-125 and tested with Type 1 diabetes sera in a radioimmunoprecipitation assay, and results were compared with those using untagged GAD67/65 and those using porcine brain GAD. The results of enzymatic and immunological assays show hybrid GAD67/65 is isolated at high specific activity and moderate yield, and the addition of the H6 tag sequences or the choice of yeast strain did not appreciably affect enzyme activity, percentage recovery of GAD, protein purification, or the utility in diagnosis of diabetes in terms of specificity and sensitivity to the various sera.     

Clinical characteristics, and time course of pancreatic beta-cell function and glutamic acid decarboxylase antibodies in Thai patients with adult-onset Type 1 diabetes: distinction between patients of rapid- and slow-onset.

In order to study the clinical characteristics, time course of beta cell function and glutamic acid decarboxylase antibodies (GAD65Ab) in Thai patients with adult-onset Type 1 diabetes and to examine the distinctive features between patients with rapid-and slow-onset, 61 Thai patients with Type 1 diabetes who had age of disease onset at or after 20 years were studied. All patients were treated with insulin at the time of study and had fasting C-peptide levels +/-0.33 nmol/l. Twenty-six (42.6%) were in rapid-onset and 35 (57.4%) were in slow-onset groups. Fourty-four of 61 (70.5%) were male. About three-fourths had body mass index (BMI) < 19 kg/m2 at the time of insulin therapy. Only 7 of 61 (11.5%) patients had ketoacidosis at first presentation. Five patients had associated autoimmune thyroid disease and 10 (16.7%) patients had family history of diabetes in first-degree relatives. GAD65Ab was positive in 31 patients (50.8%); 10 (38.5%) were in rapid-onset and 21 (60.0%) were in slow-onset groups. GAD65Ab particularly of high levels were persistently elevated during 3-4 years follow-up period. The persistence of GAD65Ab were not associated with changes in fasting C-peptide levels. At the time of insulin dependency, there were no distinctive clinical features between rapid- and slow-onset patients except higher fasting C-peptide (0.08+/-0.08 vs. 0.14+/-0.10 nmol/l; p = 0.023) and GAD65Ab levels (19.6+/-17.4 vs. 46.1+/-49.7 U/ml; p = 0.036) in slow-onset patients. Fasting C-peptide levels of patients in the latter group were also demonstrated to be higher after 3-4 years of follow-up. In conclusion, most Thai patients with adult-onset Type 1 diabetes in this study were male and had significant degree of weight loss and lean BMI prior to insulin therapy. The presence of GAD65Ab did not predict clinical features or rate of beta cell loss. Patients in rapid-onset group had lower fasting C-peptide and GAD65Ab levels than those of slow-onset group which confirms the slower process of beta cell failure in the latter.     

Epitope analysis of GAD65 binding in both cord blood and at the time of clinical diagnosis of childhood type 1 diabetes.

The GAD65 epitope immunoglobulin binding pattern in cord blood of children (n=37), who later developed type 1 diabetes at 3.2-14.9 years of age, was analyzed. First, the binding at diagnosis was compared with that in the cord blood serum. The next comparison was between the cord blood serum and the mothers' serum taken at delivery. Basal GAD65 binding levels were determined in Protein A Sepharose-based radiobinding assays with (35)S-labeled human and rat GAD65, rat GAD67 and GAD65/67 fusion proteins representing N-terminal (N), middle (M) and C-terminal (C) epitopes. In the first comparison, 28/37 children had GAD65 binding above 2.44 relative units (RU) (upper three quartiles), representing a marked increase from birth in the binding to human GAD65 (p<0.0001), rat GAD65 (p<0.0001), N- (p=0.04), M- (p<0.0001), C- (p=0.001), and M + C-epitopes (p<0.0001), but not to rat GAD67. At birth, 9/37 had GAD65 binding above 1.56 RU (upper quartile) demonstrating that their binding of human (35)S-GAD65 was higher in cord blood than in the mother (p=0.008). Higher cord blood binding was also observed for the N- (p=0.02) terminal epitope but not for rat GAD65, rat GAD67, and the remaining epitopes. These data suggest that differences in the epitope GAD65 binding between mother and child at birth are limited. In contrast, the epitope pattern at diagnosis differed from that at birth, supporting the view that disease-associated epitopes develop between birth and diagnosis.     

Characterization of continuous B‐cell epitopes in the N‐terminus of glutamate decarboxylase67 using monoclonal antibodies

Glutamate decarboxylase (GAD) is an autoantigen associated with the autoimmune disorders Type‐1 diabetes (T1D) and stiff‐person syndrome (SPS). The protein, being an essential enzyme involved in the production of the inhibitory neurotransmitter γ‐aminobutyric acid, exists in two isoforms, GAD67 and GAD65. Both isoforms may be targeted by autoantibodies in SPS and T1D patients, although SPS primarily is associated with the presence of GAD67 autoantibodies, whereas T1D mainly is associated with the presence of GAD65 autoantibodies. In this study, we describe antibody reactivity to overlapping GAD67 peptides covering the complete protein sequence by modified peptide enzyme‐linked immunosorbent assay in order to identify potential GAD67 epitopes using two monoclonal antibodies (mAbs). Both GAD67 mAbs showed reactivity to linear epitopes located at the N‐terminal end of GAD67. The epitopes of GAD mAb 1 and 2 were identified as the amino acid sequences NAGADPNTTN and TETDFSNLF, respectively, corresponding to amino acids 14–23 and 91–99. Fine mapping of the epitopes revealed that antibody reactivity was related to amino acid side‐chain functionality, rather than amino acid side‐chain specificity. Additionally, results suggested that non‐contact amino acids in the epitope structure were essential for antibody reactivity. The exact role of these amino acids remains to be determined, but they are thought to be involved in backbone hydrogen bonds or stabilization of the epitope structure. As only limited knowledge is available in relation to antigenic regions of GAD67, this study contributes to characterization of GAD67 epitopes and may be a first step in the development of peptide‐based therapeutics against SPS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

An analysis of the cross-reactivity of autoantibodies to GAD65 and GAD67 in diabetes

Background

Autoantibodies to GAD65 (anti-GAD65) are present in the sera of 70–80% of patients with type 1 diabetes (T1D), but antibodies to the structurally similar 67 kDa isoform GAD67 are rare. Antibodies to GAD67 may represent a cross-reactive population of anti-GAD65, but this has not been formally tested.

Methodology/Principal Findings

In this study we examined the frequency, levels and affinity of anti-GAD67 in diabetes sera that contained anti-GAD65, and compared the specificity of GAD65 and GAD67 reactivity. Anti-GAD65 and anti-GAD67 were measured by radioimmunoprecipitation (RIP) using ¹²⁵I labeled recombinant GAD65 and GAD67. For each antibody population, the specificity of the binding was measured by incubation with 100-fold excess of unlabeled GAD in homologous and heterologous inhibition assays, and the affinity of binding with GAD65 and GAD67 was measured in selected sera. Sera were also tested for reactivity to GAD65 and GAD67 by immunoblotting. Of the 85 sera that contained antibodies to GAD65, 28 contained anti–GAD67 measured by RIP. Inhibition with unlabeled GAD65 substantially or completely reduced antibody reactivity with both ¹²⁵I GAD65 and with ¹²⁵I GAD67. In contrast, unlabeled GAD67 reduced autoantibody reactivity with ¹²⁵I GAD67 but not with ¹²⁵I GAD65. Both populations of antibodies were of high affinity (>10¹⁰ l/mol).

Conclusions

Our findings show that autoantibodies to GAD67 represent a minor population of anti-GAD65 that are reactive with a cross-reactive epitope found also on GAD67. Experimental results confirm that GAD65 is the major autoantigen in T1D, and that GAD67 per se has very low immunogenicity. We discuss our findings in light of the known similarities between the structures of the GAD isoforms, in particular the location of a minor cross-reactive epitope that could be induced by epitope spreading.     

Type 1 diabetes-predisposing MHC alleles influence the selection of glutamic acid decarboxylase (GAD) 65-specific T cells in a transgenic model

The genetic factors that contribute to the etiology of type 1 diabetes are still largely uncharacterized. However, the genes of the MHC (HLA in humans) have been consistently associated with susceptibility to disease. We have used several transgenic mice generated in our laboratory, bearing susceptible or resistant HLA alleles, in the absence of endogenous MHC class II (Abetao), to study immune responses to the autoantigen glutamic acid decarboxylase (GAD) 65 and its relevance in determining the association between autoreactivity and disease pathogenesis. Mice bearing diabetes-susceptible haplotypes, HLA DR3 (DRB1*0301) or DQ8 (DQB1*0302), singly or in combination showed spontaneous T cell reactivity to rat GAD 65, which is highly homologous to the self Ag, mouse GAD 65. The presence of diabetes-resistant or neutral alleles, such as HLA DQ6 (DQB1*0602) and DR2 (DRB1*1502) prevented the generation of any self-reactive responses to rat GAD. In addition, unmanipulated Abetao/DR3, Abetao/DQ8, and Abetao/DR3/DQ8 mice recognized specific peptides, mainly from the N-terminal region of the GAD 65 molecule. Most of these regions are conserved between human, mouse, and rat GAD 65. Further analysis revealed that the reactivity was mediated primarily by CD4(+) T cells. Stimulation of these T cells by rat GAD 65 resulted in the generation of a mixed Th1/Th2 cytokine profile in the Abetao/DR3/DQ8, Abetao/DR3, and Abetao/DQ8 mice. Thus, the presence of diabetes-associated genes determines whether immune tolerance is maintained to islet autoantigens, but autoreactivity in itself is not sufficient to induce diabetes.     

Transgenic plants expressing human glutamic acid decarboxylase (GAD65), a major autoantigen in insulin-dependent diabetes mellitus

Parenteral and oral administration of autoantigens can induce immune tolerance in autoimmune diseases. Prophylactic therapy based on oral administration of human autoantigens is not, however, feasible when sufficient quantities of candidate autoantigens are not available. Transgenic plants that express high levels of recombinant proteins would allow large quantities of autoantigens to be produced at relatively low costs. In addition, transgenic food would provide a simple and direct method of delivering autoantigens. The production and the characterization of transgenic tobacco and carrot plants expressing human GAD65, a major autoantigen in human insulin-dependent diabetes mellitus (IDDM), is reported. Immunogold labeling and electron microscopy of transgenic tobacco tissue shows the selective targeting of human GAD65 to chloroplast tylacoids and mitochondria. In planta expressed GAD65 has a correct immunoreactivity with IDDM-associated autoantibodies and retains enzymatic activity, a finding that suggests a correct protein folding. In transgenic tobacco and carrot the expression levels of human GAD65 varies between 0.01% and 0.04% of total soluble proteins. Transgenic edible plant organs are now available to study the feasibility of inducing immune tolerance in IDDM animals by oral administration of GAD65.     

Enzymatic characterization of a recombinant isoform hybrid of glutamic acid decarboxylase (rGAD67/65) expressed in yeast

BACKGROUND AND AIMS: Glutamic acid decarboxylase (GAD, EC 4.1.1.15) catalyses the conversion of glutamate to gamma-aminobutyric acid (GABA). The 65 kDa isoform, GAD65 is a potent autoantigen in type 1 diabetes, whereas GAD67 is not. A hybrid cDNA was created by fusing a human cDNA for amino acids 1-101 of GAD67 to a human cDNA for amino acids 96-585 of GAD65; the recombinant (r) protein was expressed in yeast and was shown to have equivalent immunoreactivity to mammalian brain GAD with diabetes sera. We here report on enzymatic and molecular properties of rGAD67/65. METHODS: Studies were performed on enzymatic activity of rGAD67/65 by production of 3H-GABA from 3H-glutamate, enzyme kinetics, binding to the enzyme cofactor pyridoxal phosphate (PLP), stability according to differences in pH, temperature and duration of storage, and antigenic reactivity with various GAD-specific antisera. RESULTS: The properties of rGAD67/65 were compared with published data for mammalian brain GAD (brackets). These included a specific enzyme activity of 22.7 (16.7) nKat, optimal pH for enzymatic activity 7.4 (6.8), K(m) of 1.3 (1.3) mM, efficient non-covalent binding to the cofactor PLP, and high autoantigenic potency. The stability of rGAD67/65 was optimal over 3 months at -80 degrees C, or in lyophilized form at -20 degrees C. CONCLUSIONS: Hybrid rGAD67/65 has enzymatic and other properties similar to those of the mixed isoforms of GAD in preparations from mammalian brain as described elsewhere, in addition to its previously described similar immunoreactivity.     

Genetic markers for glutamic acid decarboxylase do not predict insulin-dependent diabetes mellitus in pairs of affected siblings

Reliable genetic and immunological markers are important in the prediction of insulin-dependent diabetes mellitus (IDDM). Since glutamic acid decarboxylase (GAD) is a candidate primary autoantigen, we examined the possible linkage between IDDM and the genes encoding GAD₆₅ (GAD2, 10p11–12) and GAD₆₇ (GAD1, 2q31) in 58 Danish IDDM affected sib pairs. The allelic inheritance of 10 polymorphic dinucleotide repeat sequences spanning the chromosomal regions of the two GAD genes, were examined by affected sib pair analysis (ASP). In addition a restriction fragment length polymorphism (RFLP) was identified in the gene encoding GAD₆₅ using the restriction enzyme PvuII. The GAD gene markers were analyzed in relation to the presence of specific HLA types and GAD autoantibodies. No evidence of linkage was found between IDDM and either of the genes encoding GAD. This was also the case when subgroups carrying specific HLA susceptibility alleles were analyzed. Nor did we observe any association between these GAD genetic markers and the presence of GAD autoantibodies. Considering the high prevalence of GAD autoantibodies in IDDM, a putative genetic association between GAD and IDDM would be expected to affect most diabetic individuals. Therefore, our data indicate that the association between GAD and IDDM is not genetically determined, and that microsatellites used in this study do not contribute to the prediction of IDDM. Received: 1 July 1996 / Revised: 21 August 1996     

Improved in Planta Expression of the Human Islet Autoantigen Glutamic Acid Decarboxylase (GAD65)

The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH₂-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD67_1-87/GAD65_88-585 molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes.     

Distinctive interactions in the holoenzyme formation for two isoforms of glutamate decarboxylase

The interactions between glutamate decarboxylase (GAD) and its cofactor pyridoxal phosphate (PLP) play a key role in the regulation of GAD activity. The enzyme has two isoforms, GAD65 and GAD67. A comparison of binding constants, rate constants, and kinetic profiles for the formation of holoenzyme (holoGAD65 and holoGAD67) revealed that the two isoforms interact distinctively with the cofactor. GAD67 exhibits a higher binding constant for PLP binding, making it more difficult to dissociate PLP from holoGAD67 than holoGAD65. Meanwhile, PLP binding occurs at a much slower rate for GAD67 than GAD65, as evidenced by lower rate constants and a slower initial rate of the holoenzyme formation. Job's plots revealed a stoichiometry of 1:1 for PLP binding to GAD65 before and after the saturation level of PLP, while 1:2 for PLP binding to GAD67 prior to the saturation of PLP and 1:1 at the saturation level of PLP. These results suggested that the two binding sites of GAD65 exhibit similar affinities for PLP. In contrast, one binding site of GAD67 exhibits a significantly higher affinity for PLP than the other binding site. Based on these findings, it was proposed that a slower PLP binding to GAD67 than GAD65 and a less ease to dissociate PLP from holoGAD67 than holoGAD65 are important underlying factors. This attributes to GAD67 being more highly saturated by PLP and GAD65 being less saturated by PLP. A larger conformation change constant for GAD67 than GAD65 supported a significant conformational change induced by the initial PLP binding to GAD67, which affects the other binding site affinity of GAD67. The present studies provided valuable insights into distinctive properties between the two isoforms of GAD.     

Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modeling

The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing the PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive with MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.     

Population based study of prevalence of islet cell autoantibodies in monozygotic and dizygotic Danish twin pairs with insulin dependent diabetes mellitus.

OBJECTIVE: To study the comparative importance of environment and genes in the development of islet cell autoimmunity associated with insulin dependent diabetes mellitus. DESIGN: Population based study of diabetic twins. SETTING: Danish population. SUBJECTS: 18 monozygotic and 36 dizygotic twin pairs with one or both partners having insulin dependent diabetes. MAIN OUTCOME MEASURES: Presence of islet cell antibodies, insulin autoantibodies, and autoantibodies to glutamic acid decarboxylase (GAD65) in serum samples from twin pairs 10 years (range 0-30 years) and 9.5 years (2-30 years) after onset of disease. RESULTS: In those with diabetes the prevalence of islet cell antibodies, insulin autoantibodies, and autoantibodies to glutamic acid decarboxylase in the 26 monozygotic twins was 38%, 85%, and 92%, respectively, and in the dizygotic twins was 57%, 70%, and 57%, respectively. In those without diabetes the proportions were 20%, 50%, and 40% in the 10 monozygotic twins and 26%, 49%, and 40% in the 35 dizygotic twins. CONCLUSION: There is no difference between the prevalence of islet cell autoantibodies in dizygotic and monozygotic twins without diabetes, suggesting that islet cell autoimmunity is environmentally rather than genetically determined. Furthermore, the prevalence of islet cell antibodies was higher in the non-diabetic twins than in other first degree relatives of patients with insulin dependent diabetes. This implies that the prenatal or early postnatal period during which twins are exposed to the same environment, in contrast with that experienced by first degree relatives, is of aetiological importance.     

Decreased GAD(65) -specific Th1/Tc1 phenotype in children with Type 1 diabetes treated with GAD-alum

Diabet. Med. 29, 1272-1278 (2012) ABSTRACT: Aim The balance between T helper cell subsets is an important regulator of the immune system and is often examined after immune therapies. We aimed to study the immunomodulatory effect of glutamic acid decarboxylase (GAD) 65 formulated with aluminium hydroxide (GAD-alum) in children with Type?1 diabetes, focusing on chemokines and their receptors. Methods Blood samples were collected from 70 children with Type?1 diabetes included in a phase?II clinical trial with GAD-alum. Expression of CC chemokine receptor?5 (CCR5) and CCR4 was analysed on CD4+ and CD8+ lymphocytes after in vitro stimulation with GAD(65) using flow cytometry, and secretion of the chemokines CCL2, CCL3 and CCL4 was detected in peripheral blood mononuclear cell supernatants with Luminex. Results Expression of Th1-associated CCR5 was down-regulated following antigen challenge, together with an increased CCR4/CCR5 ratio and CCL2 secretion in GAD-alum-treated patients, but not in the placebo group. Conclusion Our results suggest that GAD-alum treatment has induced a favourable immune modulation associated with decreased Th1/Tc1 phenotypes upon antigen re-challenge, which may be of importance for regulating GAD(65) immunity.