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)

We report the isolation and sequencing of cDNAs encoding two human glutamate decarboxylases (GADs; L-glutamate 1-carboxy-lyase, EC 4.1.1.15), GAD65 and GAD67. Human GAD65 cDNA encodes a Mr 65,000 polypeptide, with 585 amino acid residues, whereas human GAD67 encodes a Mr 67,000 polypeptide, with 594 amino acid residues. Both cDNAs direct the synthesis of enzymatically active GADs in bacterial expression systems. Each cDNA hybridizes to a single species of brain mRNA and to a specific set of restriction fragments in human genomic DNA. In situ hybridization of fluorescently labeled GAD probes to human chromosomes localizes the human GAD65 gene to chromosome 10p11.23 and the human GAD67 gene to chromosome 2q31. We conclude that GAD65 and GAD67 each derive from a single separate gene. The cDNAs we describe should allow the bacterial production of test antigens for the diagnosis and prediction of insulin-dependent diabetes mellitus.
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PMID:Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene. 154 70

The 64-kDa pancreatic beta-cell autoantigen, which is a target of autoantibodies associated with early as well as progressive stages of beta-cell destruction, resulting in insulin-dependent diabetes (IDDM) in humans, has been identified as the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase. We have identified two autoantigenic forms of this protein in rat pancreatic beta-cells, a Mr 65,000 (GAD65) hydrophilic and soluble form of pI 6.9-7.1 and a Mr 64,000 (GAD64) component of pI 6.7. GAD64 is more abundant than GAD65 and has three distinct forms with regard to cellular compartment and hydrophobicity. A major portion of GAD64 is hydrophobic and firmly membrane-anchored and can only be released from membrane fractions by detergent. A second portion is hydrophobic but soluble or of a low membrane avidity, and a third minor portion is soluble and hydrophilic. All the GAD64 forms have identical pI and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results of pulse-chase labeling with [35S]methionine are consistent with GAD64 being synthesized as a soluble protein that is processed into a firmly membrane-anchored form in a process which involves increases in hydrophobicity but no detectable changes in size or charge. All the GAD64 forms can be resolved into two isoforms, alpha and beta, which differ by approximately 1 kDa in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but are identical with regard to all other parameters analyzed in this study. GAD65 has a shorter half-life than the GAD64 forms, remains hydrophilic and soluble, and does not resolve into isomers. Comparative analysis of the brain and beta-cell forms of GAD show that GAD65 and GAD64 in pancreatic beta-cells correspond to the larger and smaller forms of GAD in brain, respectively. The expression of different forms and the flexibility in subcellular localization of the GAD autoantigen in beta-cells may have implications for both its function and autoantigenicity.
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PMID:Pancreatic beta cells express two autoantigenic forms of glutamic acid decarboxylase, a 65-kDa hydrophilic form and a 64-kDa amphiphilic form which can be both membrane-bound and soluble. 174 45

gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.
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PMID:Two genes encode distinct glutamate decarboxylases. 206 16

Glutamic acid decarboxylase (GAD) is a major islet cell autoantigen in insulin-dependent diabetes mellitus (IDDM), and autoantibodies are found in high frequencies in patients with recent-onset IDDM, stiff-man syndrome (SMS), and autoimmune polyendocrine syndrome type I (APS I). Antigens in autoimmune disorders are often enzymes, and autoantibody binding frequently inhibit their activity. In this study, we examined the reactivity of anti-GAD-containing sera from 7 patients with IDDM, 4 patients with SMS, and 5 patients with APS I. All sera immunoprecipitated GAD from [35S]methionine-labeled rat islet lysates and the sera from patients with SMS and APS I, but none of the IDDM patients' sera, identified the GAD protein in Western blots. Two of four SMS patients' sera and 5 of 5 APS I patients' sera, in contrast to 0 of 7 IDDM patients' sera, inhibited the enzymatic activity of GAD. When the various sera were tested with the GAD65 and GAD67 isoforms, produced separately by transient expression in COS cells, the enzymatic activity of GAD65 was inhibited by sera from patients with SMS and APS I, whereas no effect on the GAD67 activity was observed. Taken together, the results demonstrate that the GAD autoantibodies in these three disorders display marked differences in epitope recognition and indicate that, during the development of the diseases, the autoantigen is being presented to the immune system through separate pathogenetic mechanisms.
Diabetes 1994 Jan
PMID:GAD autoantibodies in IDDM, stiff-man syndrome, and autoimmune polyendocrine syndrome type I recognize different epitopes. 750 44

The smaller form of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in two human diseases that affect its principal sites of expression. Thus, destruction of pancreatic beta cells, which results in insulin-dependent diabetes mellitus (IDDM), and impairment of GABA-ergic synaptic transmission in Stiff-Man syndrome (SMS) are both characterized by circulating autoantibodies to GAD65. Anti-GAD65 autoantibodies in IDDM are predominantly directed to conformational epitopes. Here we report the characterization of humoral autoimmune responses to GAD65 in 35 SMS patients, of whom 13 (37%) also had IDDM. All SMS patients immunoprecipitated native GAD65 and the main titers were orders of magnitude higher than in IDDM patients. Furthermore, in contrast to the situation in IDDM, autoantibodies in 35 of 35 (100%) of SMS patients recognized denatured GAD65 on Western blots. Two major patterns of epitope specificity were identified on Western blots. The first pattern, detected in 25 of 35 SMS patients (71%), of whom 11 had IDDM (44%), was predominantly reactive with a linear NH2-terminal epitope residing in the first eight amino acids of GAD65. Nine of nine individuals who were HLA-haplotyped in this group carried an IDDM susceptibility haplotype and HLA-DR3, DQw2 was particularly abundant. The second pattern, detected in 10 of 35 patients (29%) of whom two had IDDM (20%), included reactivity with the NH2-terminal epitope plus strong reactivity with one or more additional epitope(s) residing COOH-terminal to amino acid 101. The second epitope pattern may represent epitope spreading in the GAD65 molecule, but may also include some cases of epitope recognition associated with IDDM resistant HLA-haplotypes. The principal NH2-terminal linear epitope in GAD65 distinguishes the reactivity of SMS and IDDM autoantibodies and may be a determinant of pathogenicity for GABA-ergic neurons. The greater magnitude and distinct specificity of the humoral response to GAD65 in SMS may reflect a biased involvement of the T helper cell type 2 (Th2) subset of CD4+ T cells and antibody responses, whereas IDDM is likely mediated by the Th1 subset of CD4+ T cells and cytotoxic T cell responses.
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PMID:Higher autoantibody levels and recognition of a linear NH2-terminal epitope in the autoantigen GAD65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus. 751 42

The possible role of amino acid sequence and epitope homologies between a protein P2-C of Coxsackie virus B4 and human GAD in the development of host-specific immune response in insulin-dependent diabetes mellitus (IDDM) (molecular mimicry) was investigated. Peptide antibodies to the P2-C protein, GAD65, and GAD67 were raised to analyze their immunoreactivity by enzyme-linked immunosorbent assay and immunoblotting with GAD purified from the brain and pancreas of mice that develop hyperglycemia after the infection. Additionally, antibody reactivity to these peptide antigens was assessed in sera from the virus-infected mice and IDDM patients. All three peptide antisera reacted very strongly with homologous peptides; P2-C antiserum cross-reacted with GAD65 as efficiently as GAD65 antiserum with P2-C, but no cross-reaction was detected between P2-C and GAD67 although cross-reaction between the two GADs was quite pronounced. P2-C antiserum immunocomplexed with GAD65 from mouse brain or pancreas, whereas GAD65 and GAD67 antisera both immunocomplexed with the two GADs from these sources. Most of the sera from virus-infected mice were reactive to brain and pancreas GAD65 and also to P2-C peptide, whereas some reacted to GAD65 and a few to GAD67 peptides. A number of IDDM sera reacted with mouse GAD65 and also with P2-C and GAD65 peptides, whereas only a few reacted with GAD67 peptide. The immunoreactivity of the mouse and IDDM sera to P2-C and GAD65 peptides was blocked by pre-adsorption with mouse GAD. The results suggest that molecular mimicry may play a role in the pathogenesis of the disease.
Diabetes 1994 Oct
PMID:Antibodies to glutamic acid decarboxylase and P2-C peptides in sera from coxsackie virus B4-infected mice and IDDM patients. 752 7

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) has been implicated in autoimmune diabetes in NOD mice, but the role of the 67-kDa GAD isoform (GAD67) is less clear. We found that immunization of 4-week-old NOD mice with purified recombinant mouse GAD67 prevented or significantly delayed the onset of diabetes. To further explore this phenomenon, we characterized anti-GAD67 immune responses in naive and GAD-immunized NOD mice. Anti-GAD67 antibodies titers were relatively low in naive mice at all ages, but a single immunization with GAD67 at 4 weeks induced high titers of anti-GAD antibodies by 6 weeks of age. In both 4-week-old and diabetic NOD mice, there were significant endogenous T-cell proliferative responses against purified recombinant mouse GAD67. These T-cell proliferative responses were blocked by anti-I-ANOD and anti-CD4 antibodies. To characterize the anti-GAD T-cell responses in the NOD mice, we established T-cell lines and T-cell clones which recognized GAD67, and we used recombinant subfragments of GAD to localize the predominant T-cell epitopes in GAD67. T-cells from naive NOD mice proliferated in response to all GAD subfragments, whereas T-cells from diabetic mice responded primarily to the COOH-terminal 83 amino acids of GAD67. These results suggest that GAD67 is an autoantigen in IDDM and immunization of prediabetic NOD mice with GAD67 can prevent the onset of diabetes.
Diabetes 1994 Dec
PMID:Immunization with the larger isoform of mouse glutamic acid decarboxylase (GAD67) prevents autoimmune diabetes in NOD mice. 752 93

Although most individuals with insulin-dependent diabetes mellitus (IDDM) have autoantibodies to glutamic acid decarboxylase (GAD), antibodies to GAD are also present in some individuals with a low risk of developing diabetes. The GAD autoantibodies of IDDM are specific for the GAD65 isoform, do not bind denatured GAD protein, and target epitope(s) dependent on conformation of the protein. However, the IDDM epitopes have been difficult to further define because the antibodies do not bind GAD protein fragments or synthetic peptides. Since the GAD67 isoform is highly homologous to GAD65 but is usually not a target of the GAD autoantibodies in IDDM sera, we created six GAD65/GAD67 chimeric proteins to maintain the overall GAD protein conformation and used these chimeric proteins to map conformation-dependent epitopes of GAD65 targeted by IDDM sera. We find that the GAD binding present in most IDDM sera (n = 11 of 12) is composed of two distinct GAD antibody specificities that target different conformation-dependent regions of the GAD65 protein, one that is located between amino acids 240 and 435 (termed IDDM-E1) and one that is located between amino acids 451 and 570 (termed IDDM-E2). One IDDM serum (n = 1 of 12) bound only the IDDM-E1 region. Identification of epitopes targeted by IDDM sera may allow one to distinguish between GAD antibody-positive individuals at high and low risk of developing IDDM and to determine if differences in the autoimmune repertoire directed at GAD are present. The chimeric GAD65/GAD67 proteins may also be useful in designing GAD assays specific for IDDM.
Diabetes 1995 Feb
PMID:Two distinct glutamic acid decarboxylase auto-antibody specificities in IDDM target different epitopes. 753 43

Glutamic acid decarboxylase (GAD), a target of both autoantibodies and autoreactive T-cells in insulin-dependent diabetes (IDD), exists as two homologous forms, GAD65 and GAD67. GAD65 is preferentially expressed in human islets and recognized by autoantibodies in IDD, but which form primarily elicits GAD autoimmunity is unknown. GAD67 gene expression in human islets has been demonstrated only by the polymerase chain reaction. We, therefore, quantitatively compared the expression of each GAD gene in human islets and mapped the binding of autoantibodies to recombinant human GAD67 by enzyme-linked immunosorbent assay. In ribonuclease protection assays, both forms of GAD messenger RNA (mRNA) were detected in human islets, although GAD65 mRNA was 200 times more abundant than GAD67 mRNA. Immunoblotting of islets with GAD form-specific antisera revealed GAD65, but not GAD67. By in situ hybridization and immunohistochemistry, GAD65 mRNA and protein were localized to islets, predominantly, but not entirely, to beta-cells; GAD67 mRNA and protein were undetectable. Thus, although GAD67 protein expression was undetectable in human islets, the GAD67 gene is transcribed, albeit weakly. Antibodies that recognized multiple epitopes in recombinant GAD67 were found in 20% of sera from ICA positive "at risk" first degree relatives of IDD subjects and recent-onset IDD subjects. The majority of GAD67 epitopes were mapped within the mid- and C-terminal thirds of the protein, a region that is highly conserved in GAD65. Although GAD67 may share cross-reactive epitopes with GAD65, these findings do not exclude the possibility that autoimmunity to GAD arises as a consequence of the aberrant up-regulation of GAD67 in human islets.
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PMID:Glutamic acid decarboxylase-67 (GAD67): expression relative to GAD65 in human islets and mapping of autoantibody epitopes. 753 77

Two monoclonal antibodies specifically recognizing the 65 kDa isoform of the enzyme glutamic acid decarboxylase (GAD) were generated by fusion of spleen cells of a non-obese diabetic (NOD) mouse which had received a single intraperitoneal injection of 0.2 ml complete Freund's adjuvant followed three days later by one administration of a subdiabetogenic dose of streptozotocin (80 mg/kg body weight) three days before the fusion experiment was performed. Both monoclonals belong to the IgG1 isotype and were screened with an enzyme-linked immunosorbent assay using rat brain extract as a natural source of GAD and additionally with a capture assay by means of immunoglobulins of a patient with Stiff-man syndrome. The specific binding to the 65 kDa isoform of the enzyme was detected by a radioligand and an enzyme-linked immunosorbent assay using recombinant human glutamic acid decarboxylase specific for both the 67 and 65 kDa isoforms. Both monoclonal antibodies recognize the same antigenic epitope, which is located in the N-terminal region of the first 17 amino acids detected by fragments of human pancreatic 65 kDa GAD. Three out of 30 sera from Type 1 diabetic patients specifically displaced the binding of the monoclonals from 125I-labelled GAD65 measured by radio-immunoassay. A striking binding of both monoclonals M61/8F9 and M61/7E11 to the islets of cryosections of human, monkey, pig and rat pancreas but not to mouse pancreas was detectable. The antibodies failed to bind on the cell surface of viable rat islet cells. It is concluded that also in the diabetes-prone NOD mice GAD65 autoantibodies occur although GAD65 was not detectable in the mouse islets.
Diabetes Res 1994
PMID:Monoclonal antibodies specific to the glutamic acid decarboxylase 65 kDa isoform derived from a non-obese diabetic (NOD) mouse. 754 54


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