Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.1.15 (glutamate decarboxylase)
2,169 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The autoimmune phenomena associated with destruction of the beta cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.
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PMID:Human monoclonal islet cell antibodies from a patient with insulin-dependent diabetes mellitus reveal glutamate decarboxylase as the target antigen. 138 89

Insulin-dependent (type 1) diabetes mellitus is an organ-specific autoimmune disease frequently associated with an islet-specific humoral autoimmune response. The role of islet cell autoantibodies in the disease process is unclear; in particular, it is not known whether they are a non-specific side effect of islet cell destruction or play a role in the autoimmune network leading to type 1 diabetes. Here we report the immunoglobulin gene usage and somatic mutation rates of a panel of seven human monoclonal islet cell autoantibodies (MICA 1-7) directed towards the major islet cell autoantigen glutamate decarboxylase (GAD). These autoantibodies were produced from cells from two patients with newly diagnosed type 1 diabetes. VH1, VH4 and V lambda 2 gene segments were frequently used in the MICA, but no correlation between V gene usage and epitope recognition was found. The nonrandom ratio of replacement versus silent mutations in the variable gene region, an accumulation of replacement mutations in the complementarity determining regions, which confer antigen binding, and the high relative avidity for GAD observed for MICA 1, 3, 4, and 6, suggested that the immune response to GAD is driven by the antigen. In contrast, MICA 2, 5, and 7, revealing a lower affinity for antigen, have accumulated a large number of silent mutations. These latter antibodies may, therefore, be characteristic for later stages of the chronic autoimmune disease. Our results argue in favor of an antigen-driven autoantibody response to islets in human type 1 diabetes. They suggest that GAD is an important target of autoimmunity associated with type 1 diabetes.
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PMID:Immunoglobulin variable gene analysis of human autoantibodies reveals antigen-driven immune response to glutamate decarboxylase in type 1 diabetes mellitus. 761 98

The gamma-aminobutyrate-synthesizing enzyme glutamate decarboxylase (GAD; L-glutamate 1-carboxy-lyase, EC 4.1.1.15) is a major target of autoantibodies associated with both early and late stages of pancreatic beta-cell destruction and development of type 1 diabetes. We have used five monoclonal anti-islet-cell antibodies (MICAs 1,2,3,4, and 6) derived from a newly diagnosed diabetic patient to probe the autoimmune epitopes in the enzyme. All the MICAs specifically recognized the smaller GAD protein, GAD65, and did not recognize the nonallelic GAD67 protein. A series of N-terminal, C-terminal, and internal deletion mutants, as well as protein footprinting, were used to identify the target regions in GAD65. Immunoprecipitation revealed two major native epitope areas in the GAD65 molecule. The first, defined by MICAs 1 and 3, is destroyed by deleting 41 amino acids at the C terminus but is also dependent on intact amino acids 244-295. This epitope (or epitopes) may span both middle and C-terminal domains of the protein. The second conformational epitope region, defined by MICAs 4 and 6, is dependent on intact amino acids 245-295 but is not affected by deletion of 110 amino acids at the C terminus and is therefore confined to domain(s) in the middle of the molecule. MICA 2 recognizes a linear epitope close to the C terminus. Thus, the N-terminal domain of GAD65, which differs most significantly from GAD67, does not harbor the MICA epitopes. Rather subtle amino acid differences in the middle and C-terminal domains define the GAD65-specific autoimmune epitopes. Analysis of sera from 10 type 1 diabetic patients suggests that MICAs 1, 3, 4, and 6 represent a common epitope recognition in this disease, whereas the MICA 2 epitope is rare. Furthermore, autoantibodies in some sera are restricted to the MICA 1/3 epitope, suggesting that this epitope may represent a single dominant epitope in the early phases of beta-cell autoimmunity.
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PMID:Autoreactive epitopes defined by diabetes-associated human monoclonal antibodies are localized in the middle and C-terminal domains of the smaller form of glutamate decarboxylase. 768 90

The first human monoclonal islet cell antibodies of the IgG class (MICA 1-6) obtained from an individual with Type 1 (insulin-dependent) diabetes mellitus were cytoplasmic islet cell antibodies selected by the indirect immunofluorescence test on pancreas sections. Surprisingly, they all recognized the 64 kDa autoantigen glutamate decarboxylase. In this study we investigated which typical features of cytoplasmic islet cell antibodies are represented by these monoclonals. We show by double immunofluorescence testing that MICA 1-6 stain pancreatic beta cells which is in agreement with the beta-cell specific expression of glutamate decarboxylase. In contrast an islet-reactive IgM monoclonal antibody obtained from a pre-diabetic individual stained all islet cells but lacked the tissue specificity of MICA 1-6 and must therefore be considered as a polyreactive IgM-antibody. We further demonstrate that MICA 1-6 revealed typical features of epitope sensitivity to biochemical treatment of the target tissue which has been demonstrated for islet cell antibodies, and which has been used to argue for a lipid rather than a protein nature of target antigens. Our results provide direct evidence that the epitopes recognized by the MICA are destroyed by methanol/chloroform treatment but reveal a high stability to Pronase digestion compared to proinsulin epitopes. Conformational protein epitopes in glutamate decarboxylase therefore show a sensitivity to biochemical treatment of sections such as ganglioside epitopes. MICA 1-6 share typical features of islet cell and 64 kDa antibodies and reveal that glutamate decarboxylase-reactive islet cell antibodies represent a subgroup of islet cell antibodies present in islet cell antibody-positive sera.
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PMID:Human monoclonal islet specific autoantibodies share features of islet cell and 64 kDa antibodies. 769 67

Molecular mimicry between viral antigens and host proteins was often suggested to be involved in induction of autoimmune diseases. In type 1 diabetes where pancreatic beta cells are destroyed by autoimmune phenomena, a linear sequence homology between a major autoantigen, glutamate decarboxylase (GAD), and the 2C protein of coxsackie B4 was identified. In addition, a sequence homology between GAD and the mycobacterial heat shock protein 60 was described and the suggestions were made that molecular mimicry between GAD, coxsackievirus B4-2C protein, and/or heat shock protein 60 (hsp60) may be actively involved in an autoimmune reaction towards the pancreatic beta-cells. Our group was the first to isolate human monoclonal autoantibodies to GAD (MICA 1-6) from a patient with newly diagnosed type 1 diabetes. The MICA allowed a detailed characterization of the diabetes associated self-epitopes in GAD and represent a set of GAD autoantibodies present in sera from patients with type 1 diabetes. Using deletion mutants of GAD we demonstrated that the regions of GAD covering the homology sequences to coxsackievirus B4 and to the hsp60 were absolutely required for binding of the MICA to GAD. We now designed an antibody-based analysis to ask whether molecular mimicry between GAD and coxsackie B4-2C or hsp60 is relevant in type 1 diabetes. Since part of the MICA recognize conformational epitopes, they allow to test for conformational molecular mimicry in viruses that have been incriminated in the development of type 1 diabetes. Our data reveal no crossreactivity between the diabetes associated GAD epitopes defined by the MICA and hsp60, rubellavirus, cytomegalovirus, and coxsackie B1-B6 virus antigens. Neither coxsackie B4-specific antibodies in sera from normal individuals nor GAD-positive sera from patients with type 1 diabetes indicated a crossreactivity between coxsackie B4-2C and GAD. Although the regions in GAD homologous to coxsackie B4-2C and hsp60 represented parts of GAD indispensible for binding of diabetes associated autoantibodies they did not mediate a crossreactivity of autoantibodies between GAD and these two proteins. No evidence for molecular mimicry between GAD and a whole panel of foreign antigens was detected by autoantibodies in type 1 diabetes.
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PMID:Sequence homology of the diabetes-associated autoantigen glutamate decarboxylase with coxsackie B4-2C protein and heat shock protein 60 mediates no molecular mimicry of autoantibodies. 791 51

Cytoplasmic islet cell antibodies are well-established predictive markers of IDDM. Although target molecules of ICA have been suggested to be gangliosides, human monoclonal ICA of the immunoglobulin G class (MICA 1-6) produced from a patient with newly diagnosed IDDM recognized glutamate decarboxylase as a target antigen. Here we analyzed the possible heterogeneity of target antigens of ICA by subtracting the GAD-specific ICA staining from total ICA staining of sera. This was achieved 1) by preabsorption of ICA+ sera with recombinant GAD65 and/or GAD67 expressed in a baculovirus system and 2) by ICA analysis of sera on mouse pancreas, as GAD antibodies do not stain mouse islets in the immunofluorescence test. We show that 24 of 25 sera from newly diagnosed patients with IDDM recognize islet antigens besides GAD. In contrast, GAD was the only islet antigen recognized by ICA from 7 sera from patients with stiff man syndrome. Two of these sera, however, recognized antigens besides GAD in Purkinje cells. In patients with IDDM, non-GAD ICA were diverse. One group, found in 64% of the sera, stained human and mouse islets, whereas the other group of non-GAD ICA was human specific. Therefore, mouse islets distinguish two groups of non-GAD ICA and lack additional target epitopes of ICA besides GAD. Longitudinal analysis of 6 sera from nondiabetic ICA+ individuals revealed that mouse-reactive ICA may appear closer to clinical onset of IDDM in some individuals. Mouse-reactive ICAs, however, remained absent in 36% of the patients at diagnosis of IDDM.
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PMID:Cytoplasmic islet cell antibodies recognize distinct islet antigens in IDDM but not in stiff man syndrome. 840 7

Glutamate decarboxylase (GAD65) is a major autoantigen in insulin-dependent diabetes (IDDM) and the neurological disorder Stiff-Man-Syndrome (SMS). We derived a human monoclonal autoantibody (MICA 2) from peripheral blood of a patient newly diagnosed with IDDM, which reacted with GAD65 in Western blots. This indicated that a linear epitope is recognized by MICA 2. Using an epitope cDNA library we mapped the MICA 2 epitope to a contiguous stretch of 26 amino acids (506-531) in the C-terminus of GAD65. Neither blocking experiments with synthetic peptides nor analysis of overlapping decapeptides expressed as fusion proteins allowed us to further narrow down the epitope to the typical size of linear epitopes of 6-8 amino acids. We suggest that a miniconformational epitope provided by amino acids 506-531 is recognized by MICA 2, which withstands SDS gel electrophoresis without destruction or partially refolds during the Western blot procedure. A sequence homology with human heat shock protein 60 (HSP60) maps to this region of GAD65 but no cross-reactivity of MICA 2 with HSP60 occurred. Our data demonstrate that reactivity of an antibody in Western blots does not necessarily define a classic linear epitope of 6-8 amino acids and describe a new autoreactive epitope in GAD65 different from those reported for sera from patients with SMS.
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PMID:Mapping of an autoreactive epitope within glutamate decarboxylase using a diabetes-associated human monoclonal autoantibody and an epitope cDNA library. 874 89

Autoreactive islet cell Abs (ICA) accompany the pathogenic destruction of pancreatic beta cells in insulin-dependent diabetes mellitus (IDDM). Human monoclonal ICA (MICA 1-6), previously derived from a DR1/DR7-positive newly diagnosed diabetic patient, recognized the islet cell autoantigen glutamate decarboxylase 65 (GAD65) and defined two distinct conformational (MICA 1/3 and MICA 4/6) and one linear (MICA 2) autoimmune epitopes in this molecule. We have isolated 4 new ICA-reactive B cell lines, one from a DR4/DR11-positive newly diagnosed IDDM patient (MICA 7) and three from a DR3 homozygous patient with both IDDM and Graves' disease (MICA 8-10). Like MICA 1-6, MICA 7-10 are specific for GAD65, suggesting that GAD65-reactive B cells dominate the ICA response in IDDM. Comparative analysis of MICA 1-6 and MICA 7-10, using GAD65 mutants and blocking experiments, showed that MICA 7-10 define three novel conformational autoimmune epitopes in GAD65. Further structural analysis of the MICA 1-10 epitopes revealed two distinct and one overlapping region of epitope clusters. Thus, the C-terminal region, defined by amino acids 450 to 570, harbors the conformational MICA1/3 and MICA 7 epitopes as well as the linear epitope of MICA 2 (amino acids 506-531). The MICA 4/6 and MICA 10 epitopes are located in the middle region of the molecule defined by amino acids 245 to 449, whereas the N-terminal region contributes only to the MICA 8/9 epitopes (encompassed in amino acids 39-585). MICA 1-6, 7, and 8-10, derived from three IDDM patients of different HLA haplotypes, define six different epitopes in GAD65 and represent tools to determine the spectrum, possible HLA association, and temporal order of epitope recognition in IDDM.
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PMID:Immune reactivity of diabetes-associated human monoclonal autoantibodies defines multiple epitopes and detects two domain boundaries in glutamate decarboxylase. 894 34

MMDM patients are typically young at onset with low body mass index, require insulin treatment for glycemic control, have insulin resistance, and do not develop ketosis on withdrawal of insulin. WHO's revised classification in 1999, based on the etiopathogenesis of the disease, identifies only two categories: type 1 diabetes and type 2 diabetes. MMDM could be considered as type 1b diabetes. Genetic and immunological studies were done on MDDM patients (n = 72) from Cuttack and healthy controls to understand and to justify its inclusion in the category of type 1b diabetes. Antibodies (Abs) to tyrosine pyrophosphatase (IA2-Abs), glutamate decarboxylase 65 (GAD65-Abs), and other minor markers like ICA12 Abs and tissue transglutaminase Abs (TTG-Abs) were studied. HLA-DR and DQ were studied for the genetic markers. Of the MMDM patients 30% were positive for either GAD65 or IA-2 antibodies, and 14% were positive for ICA12 antibodies. All three antibody markers together accounted for 39% of PDDM patients, as some patients were positive for more than one autoantibody. TTG antibodies (specific for Celiac disease) were present in 14/71 (20%) of MMDM patients compared to 3/122 (2%) controls. All four autoantibodies accounted for 53% of PDDM patients, leaving 47% of patients free of known autoantibodies. The autoantibody-negative PDDM patients were analyzed for HLA and MICA markers, showing that DR7-DQ9 and MICA allele 9 are increased in this group compared to healthy controls, which suggests an autoimmune response to an unknown dietary autoantigen. We conclude from our data that an autoimmune mechanism is involved in the etiology of MMDM. In addition, the presence of silent celiac disease seen with MMDM patients, which has not yet been reported, is significant. It is important to note that subclinical celiac disease exists with diabetes mellitus and must be considered in the diagnosis of MMDM.
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PMID:Molecular mechanisms involved in the etiopathogenesis of malnutrition-modulated diabetes mellitus. 1202 Oct 93

Type 1 diabetes (T1D) is an autoimmune disease with a strong human leucocyte antigen (HLA) class II association. Depending on geographic locations, the disease-associated HLA class II alleles vary. We evaluated the beta cell-specific autoimmunity reflected in autoantibodies directed to the smaller isoform of glutamate decarboxylase (GAD65) in Japanese and Swedish T1D patients. GAD65Ab epitope specificities were assessed using GAD65-specific recombinant Fab. GAD65Ab epitope specificities did not differ between Swedish and Japanese patients. Only recognition of the MICA-4-defined middle epitope was significantly stronger in the Japanese T1D patient group compared to the Swedish T1D patients (P = 0.001). Binding to the b96.11-defined middle epitope was substantial in both groups and showed significant associations with high-risk HLA class II haplotypes. In the Japanese T1D group the association was with haplotype DRB1*0802-DQB1*0302 (P = 0.0008), while in the Swedish T1D patients binding to the b96.11-defined epitope as associated with the presence of high-risk HLA genotypes DR3-DQB1*0201 and/or DR4-DQB1*0302 (P = 0.02). A significant association between reduction in binding in the presence of recombinant Fab (rFab) DPD and high-risk allele DQB1*0201 was found (P = 0.008) in the Swedish T1D patients only. We hypothesize that epitope-specific autoantibodies effect the peptide presentation on HLA class II molecules by modulating antigen uptake and processing. Molecular modelling of the high-risk HLA class II molecules will be necessary to test whether these different molecules present similar peptide-binding specificities.
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PMID:Autoantibody epitopes to the smaller isoform of glutamate decarboxylase do not differ in Swedish and Japanese type 1 diabetes patients and may be associated with high-risk human leucocyte antigen class II alleles. 1795 79


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