Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0011849 (diabetes)
 277,896 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutamic acid decarboxylase (GAD) is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). Two GAD isoforms exist, GAD65 and GAD67, which differ mostly in the first 100 amino acids of the amino terminus. IDDM sera are predominantly reactive with GAD65 but autoepitopes have been localised only to regions of GAD65 highly homologous with GAD67. In this study we investigated the contribution of the amino terminus to the IDDM epitope on GAD65, in order to test whether this region of GAD could explain the difference in reactivity between GAD65 and GAD67. A recombinant hybrid GAD molecule consisting of amino acids 1-101 of GAD67 and 96-585 of GAD65 was constructed and a truncated GAD65 was also constructed consisting of amino acids 98-585 of GAD65. The reactivity with the hybrid GAD molecule, GAD65 and GAD67, and truncated GAD65 was examined by radioimmunoprecipitation using 50 IDDM sera with known reactivity to purified porcine brain GAD. Over 90% of the IDDM sera were reactive with the hybrid GAD molecule confirming that the amino terminus of GAD65 does not contribute to the autoepitope and that the IDDM epitope is localised to the middle and carboxyl terminal domains of GAD65. Furthermore, evidence is presented that autoantibodies to GAD65 in IDDM sera react with an epitope formed on a dimeric configuration of the molecule.
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PMID:Autoantigenic reactivity of diabetes sera with a hybrid glutamic acid decarboxylase GAD67-65 molecule GAD67(1-101)/GAD65(96-585). 989 8

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.
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PMID:Glutamate decarboxylase and GABA in pancreatic islets: lessons from knock-out mice. 1042 32

Autoantibodies to GAD, an important marker of the autoimmune process in type I or insulin-dependent diabetes mellitus (IDDM), are also found in non-diabetic individuals with autoimmune polyendocrine syndrome type 1 (APS1), APS2, and stiff man syndrome (SMS). Most IDDM sera contain two distinct GAD antibody specificities, one of which targets an epitope region in the middle-third of GAD65 (IDDM-E1; amino acids 221-359) and one of which targets the carboxy-third of GAD65 (IDDM-E2; amino acids 453-569). Using 11 chimeric GAD65/GAD67 proteins to maintain conformation-dependent epitopes of GAD65, we compared the humoral repertoire of IgG antibodies from an individual with APS2-like disease (b35, b78, and b96) and MoAbs from an IDDM patient (MICA-2, MICA-3, and MICA-4). Neither the APS2 IgG antibodies nor the IDDM MoAbs bind the amino-terminal third of GAD65, but instead target the carboxy-terminal two-thirds of GAD65. Amino acids 270-359 (IDDM-E1) are targeted by one APS2 IgG antibody and MICA-4, while two other APS2 IgG antibodies, MICA-2 and MICA-3, target amino acids 443-585 (IDDM-E2). Using GAD65/67 chimera that span the IDDM-E2 region, we found that MICA-2 binds amino acids 514-528 of GAD65, but two APS2 IgG antibodies require this region and amino acids 529-570. In contrast, the binding of MICA-3 requires two discontinuous amino acid segments of GAD65 (452-513 and 528-569), but not amino acids 514-528. These results indicate that there are both similarities and differences in the humoral response to GAD65 in APS2 and IDDM.
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PMID:Comparative analysis of epitope recognition of glutamic acid decarboxylase (GAD) by autoantibodies from different autoimmune disorders. 1059 51

Two forms of glutamic-acid decarboxylase (GAD) have been identified in mammalian tissues: a 65-kDa form (GAD65) and a 67-kDa form (GAD67). Alternate splicing produces one or two smaller variants of GAD67 in the brain of embryonic mice and rats. Additionally, a short, heretofore unidentified transcript homologous to GAD67 has been detected in human testis RNA. Because GAD, the enzyme responsible for gamma-aminobutyric acid production and a key autoantigen in type I diabetes, has unclear function in non-neural tissue, it is important to understand its pattern of expression. Unlike GAD65, GAD67 is not produced in human pancreatic islets. Here, we describe a novel splice variant of GAD67 that is produced in human islets, testis, adrenal cortex, and perhaps other endocrine tissues, but not in brain. This transcript directs the synthesis of a protein without GAD enzymatic activity: GAD25. A unique peptide sequence at the carboxyl terminus of GAD25 is highly conserved between mice, rats, and humans. We conclude that humans produce a third form of GAD in non-neural tissues and that human islets, although they do not synthesize full-length GAD67, do express this shortened variant.
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PMID:Alternative splicing of GAD67 results in the synthesis of a third form of glutamic-acid decarboxylase in human islets and other non-neural tissues. 1067 65

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.
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PMID:Comparative expression and purification of human glutamic acid decarboxylase from Saccharomyces cerevisiae and Pichia pastoris. 1086 68

GAD is a major target of autoimmunity in preclinical type 1 diabetes. Here we examine the maturation of the humoral response to GAD epitopes sequentially from birth to diabetes onset or current follow-up in 29 GAD antibody (GADA)+ offspring of parents with diabetes from the BABYDIAB Study. Antibodies were measured against GAD65, GAD67, and GAD65/67 chimeras by radiobinding assay. In 28 of 29 offspring, the first GADAs contained reactivity against epitopes within GAD65 residues 96-444, suggesting that the middle GAD65 region is a primary target of GAD humoral autoimmunity. In 7 of these 28 offspring, initial antibody reactivity was against all epitope regions tested (middle GAD65, COOH-terminal GAD65 residues 445-585, NH2-terminal GAD65 residues 1-95, and GAD67); in 16 offspring, reactivity was to middle and COOH-terminal GAD65 epitopes, and in 5 offspring, reactivity was only to the middle GAD65 epitopes. The single offspring without middle GAD65 reactivity had antibodies to the NH2-terminal epitopes in the absence of all other islet autoimmunity. Subsequent GADA epitope spreading was frequent and seen in 10 of 15 offspring with informative follow-up samples. Spreading was mostly (eight cases) to NH2-terminal GAD65 epitopes. In two offspring, spreading to new epitopes was found when antibody titers to GAD65 and early epitopes were declining, suggesting determinant-specific regulation of the humoral response. None of the GADA reactivities nor any changes in reactivity over time were specifically associated with diabetes onset. The findings suggest that the humoral autoimmune response to GAD found in childhood is dynamic, is initially against epitopes within the middle portion of GAD65, and spreads to epitopes in other regions of GAD65 and GAD67.
Diabetes 2000 Feb
PMID:Maturation of the humoral autoimmune response to epitopes of GAD in preclinical childhood type 1 diabetes. 1086 36

Autoantibodies against the smaller isoform of glutamic acid decarboxylase (GAD) are markers for Type 1 diabetes. GAD65 autoantibody (GAD65Ab)-positive individuals in the general population are, however, mostly at low risk of developing Type 1 diabetes, suggesting that GAD65Ab phenotypes may be associated with different underlying pathogenic processes. The aim of this study was to test the hypothesis that Type 1 diabetes patients (n = 243; group I), GAD65Ab-positive healthy individuals (n = 28; group II), and healthy first-degree relatives of Type 1 diabetes patients (n = 41; group III) have antibody phenotypes that recognize different GAD65 epitopes. Sera from groups I-III were tested for their binding to GAD65 and GAD67, as well as six different GAD65/67 fusion proteins. Regardless of group, sera reactive to both GAD65 and GAD67 showed broader epitope reactivity than GAD65-specific sera. Furthermore, Type 1 diabetes patients showed a more restricted epitope binding than healthy individuals and first-degree relatives, demonstrating significantly less binding to the N-terminal part of GAD65 and to GAD67. Our analysis demonstrates that the N-terminal part is essential for full antibody binding to GAD65, in particular, to the middle epitope. It is suggested that Type 1 diabetes is associated with restricted GAD65Ab epitope specificity.
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PMID:Recognition of glutamic acid decarboxylase (GAD) by autoantibodies from different GAD antibody-positive phenotypes. 1113 26

The expression of pro(insulin) in the thymus may lead to the negative selection of pro(insulin) autoreactive T cells and peripheral tolerance to this autoantigen in type 1 diabetes (T1D). We investigated whether proinsulin is expressed in the thymus of young nonobese diabetic (NOD) mice, whether T cells from naive NOD female mice at weaning are reactive to mouse proinsulin, and the role of proinsulin as a pathogenic autoantigen in T1D. Proinsulin II mRNA transcripts were detected in the thymus of 2-wk-old NOD mice at similar levels to other control strains. Despite this expression, proinsulin autoreactive T cells were detected in the periphery of 2- to 3-wk-old naive NOD mice. Peripheral T cells reactive to the insulin, glutamic acid decarboxylase 65 (GAD65), GAD67, and islet cell Ag p69 autoantigens were also detected in these mice, indicating that NOD mice are not tolerant to any of these islet autoantigens at this young age. T cell reactivities to proinsulin and islet cell Ag p69 exceeded those to GAD67, and T cell reactivity to proinsulin in the spleen and pancreatic lymph nodes was directed mainly against a p24-33 epitope that spans the B chain/C peptide junction. Intraperitoneal immunization with proinsulin perinatally beginning at 18 days of age delayed the onset and reduced the incidence of T1D. However, s.c. immunization with proinsulin initiated at 5 wk of age accelerated diabetes in female NOD mice. Our findings support the notion that proinsulin p24-33 may be a primary autoantigen epitope in the pathogenesis of T1D in NOD mice.
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PMID:Evidence that a peptide spanning the B-C junction of proinsulin is an early Autoantigen epitope in the pathogenesis of type 1 diabetes. 1167 98

Molecular mimicry between the 65-kDa isoform of glutamic acid decarboxylase (GAD65) and the protein 2C (P2C) of Coxsackie B4 virus (CBV) may initiate human type 1 diabetes. GAD65 contains a motif that has a 6-amino acid identity with CBV-P2C (PEVKEK), whereas the weakly autoantigenic isoform, GAD67, contains PEVKTK. A human-derived monoclonal antibody (mAb) MICA3 reacts with a surface loop of GAD65 that includes PEVKEK, and mutagenic deletion of this loop was shown to reduce reactivity of GAD with the mAb by 70%. To establish that the PEVKEK motif on GAD65 contains a major epitope for diabetes sera and to identify the amino acids involved, mutants of nucleotides of GAD65 and GAD67 at sites in the PEVKEK motif were created and the expressed proteins used for radioimmunoprecipitation (RIP) tests with sera from patients with type 1 diabetes. A potent mouse mAb (GAD6) to GAD65, and a rabbit polyclonal antibody (AB108) to GAD67, were used to standardize the reactivity of the diabetes sera with the mutant molecules. Of 45 type 1 diabetes sera tested, 30 (67%) had an 80% or greater reduction of reactivity to GAD65(delta258-270) vs. intact GAD65. Various single-surface amino acids in the PEVKEK epitope region of GAD65 were mutated, but most molecules carrying these mutations reacted similarly to the parent molecule. However after point mutation of the equivalent motif of GAD67 (PEVKTK to PEVKEK), there was an increase in the reactivity of 12 of 49 (24%) type 1 diabetes sera tested; 7 of 8 sera reactive with GAD67 showed increased reactivity with GAD67(T273E), and 5 previously negative sera gained reactivity with GAD67(T273E). Thus, the PEVKEK motif on GAD65 contributes to serologic reactivity of type 1 diabetes sera. This favors the hypothesis that CBV infection causes type 1 diabetes by the process of viral mimicry with cross-reactivity to a critical epitope of GAD65.
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PMID:The PEVKEK region of the pyridoxal phosphate binding domain of GAD65 expresses a dominant B cell epitope for type 1 diabetes sera. 1202 Nov 3

gamma-Aminobutyric acid (GABA) is stored in microvesicles in pancreatic islet cells. Because GAD65 and GAD67, which catalyze the formation of GABA, are cytoplasmic, the existence of an islet vesicular GABA transporter has been postulated. Here, we test the hypothesis that the putative transporter is the vesicular inhibitory amino acid transporter (VIAAT), a neuronal transmembrane transporter of GABA and glycine. We sequenced the human VIAAT gene and determined that the human and rat proteins share over 98% sequence identity. In vitro expression of VIAAT and immunoblotting of brain and islet lysates revealed two forms of the protein: an approximately 52-kDa and an approximately 57-kDa form. By immunoblotting and immunohistochemistry, we detected VIAAT in rat but not human islets. Immunohistochemical staining showed that in rat islets, the distribution of VIAAT expression parallels that of GAD67, with increased expression in the mantle. GABA, too, was found to be present in islet non-beta-cells. We conclude that VIAAT is expressed in rat islets and is more abundant in the mantle and that expression in human islets is very low or nil. The rat islet mantle differs from rat and human beta-cells in that it contains only GAD67 and relatively increased levels of VIAAT. Cells that express only GAD67 may require higher levels of VIAAT expression.
Diabetes 2002 Jun
PMID:Expression of the vesicular inhibitory amino acid transporter in pancreatic islet cells: distribution of the transporter within rat islets. 1203 63


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