scholarly journals A signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region.

1994 ◽  
Vol 126 (2) ◽  
pp. 331-341 ◽  
Author(s):  
M Solimena ◽  
R Dirkx ◽  
M Radzynski ◽  
O Mundigl ◽  
P De Camilli
Keyword(s):  
Amino Acids ◽  
Golgi Complex ◽  
Pancreatic Beta Cells ◽  
Insulin Dependent Diabetes Mellitus ◽  
Site Directed Mutagenesis ◽  
Dependent Diabetes Mellitus ◽  
Acid Decarboxylase ◽  
Complex Region ◽  
Insulin Dependent ◽  
Targeting Signal

The mechanisms involved in the targeting of proteins to different cytosolic compartments are still largely unknown. In this study we have investigated the targeting signal of the 65-kD isoform of glutamic acid decarboxylase (GAD65), a major autoantigen in two autoimmune diseases: Stiff-Man syndrome and insulin-dependent diabetes mellitus. GAD65 is expressed in neurons and in pancreatic beta-cells, where it is concentrated in the Golgi complex region and in proximity to GABA-containing vesicles. GAD65, but not the similar isoform GAD67 which has a more diffuse cytosolic distribution, is palmitoylated within its first 100 amino acids (a.a.). We have previously demonstrated that the domain corresponding to a.a. 1-83 of GAD65 is required for the targeting of GAD65 to the Golgi complex region. Here we show that this domain is sufficient to target an unrelated protein, beta-galactosidase, to the same region. Site-directed mutagenesis of all the putative acceptor sites for thiopalmitoylation within this domain did not abolish targeting of GAD65 to the Golgi complex region. The replacement of a.a. 1-29 of GAD67 with the corresponding a.a. 1-27 of GAD65 was sufficient to target the otherwise soluble GAD67 to the Golgi complex region. Conversely, the replacement of a.a. 1-27 of GAD65 with a.a. 1-29 of GAD67 resulted in a GAD65 protein that had a diffuse cytosolic distribution and was primarily hydrophilic, suggesting that targeting to the Golgi complex region is required for palmitoylation of GAD65. We propose that the domain corresponding to a.a. 1-27 of GAD65, contains a signal required for the targeting of GAD65 to the Golgi complex region.

scholarly journals Identification of a dominant epitope of glutamic acid decarboxylase (GAD-65) recognized by autoantibodies in stiff-man syndrome.

1993 ◽  
Vol 178 (6) ◽  
pp. 2097-2106 ◽  
Author(s):  
M H Butler ◽  
M Solimena ◽  
R Dirkx ◽  
A Hayday ◽  
P De Camilli
Keyword(s):  
Diabetes Mellitus ◽  
Amino Acids ◽  
Amino Acid ◽  
Insulin Dependent Diabetes Mellitus ◽  
Dependent Diabetes Mellitus ◽  
Acid Decarboxylase ◽  
Gad Autoantibodies ◽  
Gad 65 ◽  
Insulin Dependent ◽  
Stiff Man Syndrome

Glutamic acid decarboxylase (GAD) is the enzyme that synthesizes the neurotransmitter gamma-aminobutyric acid (GABA) in neurons and in pancreatic beta cells. It is a major target of autoimmunity in Stiff-Man syndrome (SMS), a rare neurological disease, and in insulin-dependent diabetes mellitus. The two GAD isoforms, GAD-65 and GAD-67, are the products of two different genes. GAD-67 and GAD-65 are very similar to each other in amino acid sequence and differ substantially only at their NH2-terminal region. We have investigated the reactivity of autoantibodies of 30 Stiff-Man syndrome patients to GAD. All patient sera contained antibodies that recognize strongly GAD-65, but also GAD-67, when tested by immunoprecipitation on brain extracts and by immunoprecipitation or immunocytochemistry on cells transfected with either the GAD-65 or the GAD-67 gene. When tested by Western blotting, all patient sera selectively recognized GAD-65. Western blot analysis of deletion mutants of GAD-65 demonstrated that autoantibodies are directed predominantly against two regions of the GAD-65 molecule. All SMS sera strongly recognized a fragment contained between amino acid 475 and the COOH terminus (amino acid 585). Within this region, amino acids 475-484 and 571-585 were required for reactivity. The requirement of these two discontinuous segments implies that the epitope is influenced by conformation. This reactivity is similar to that displayed by the monoclonal antibody GAD 6, suggesting the presence of a single immunodominant epitope (SMS-E1) in this region of GAD-65. In addition, most SMS sera recognized at least one epitope (SMS-E2) in the NH2-terminal domain of GAD-65 (amino acids 1-95). The demonstration in SMS patients of a strikingly homogeneous humoral autoimmune response against GAD and the identification of dominant autoreactive target regions may help to elucidate the molecular mechanisms of GAD processing and presentation involved in GAD autoimmunity. Moreover, the reactivity reported here of GAD autoantibodies in SMS partially differs from the reactivity of GAD autoantibodies in insulin-dependent diabetes mellitus, suggesting a link between the pattern of humoral autoimmunity and the clinical condition.

Type I Insulin-Dependent Diabetes Mellitus: a Model for Autoimmune Polygenic Disorders

1996 ◽  
Vol 10 (1) ◽  
pp. 81-87 ◽  
Author(s):  
W.E. Winter
Keyword(s):  
Diabetes Mellitus ◽  
Pancreatic Beta Cells ◽  
Insulin Treatment ◽  
Premature Mortality ◽  
Insulin Dependent Diabetes Mellitus ◽  
Insulin Dependent Diabetes ◽  
Dependent Diabetes Mellitus ◽  
Acid Decarboxylase ◽  
Type I ◽  
Insulin Dependent

Insulin-dependent diabetes mellitus (IDD) affects between one in 250 and one in 500 Americans. The inheritance of IDD is non-Mendelian and polygenic. Genetic susceptibility predominantly results from specific alleles in the HLA complex and insulin gene region. Autoimmune destruction of pancreatic beta cells leads to profound insulinopenia (Atkinson and Maclaren, 1990). Without insulin treatment, fatal diabetic ketoacidosis results. Even with insulin treatment, after 10 to 20 years of IDD, devastating micro- and macrovascular complications lead to significant morbidity and premature mortality (Rosenbloom, 1983). In order to prevent this catastrophic disease and its consequences, we must understand the pathogenesis and immunogenetics of insulitis (the histologic lesion characteristic of IDD), whose ultimate expression is beta cell necrosis (Cudworth, 1978; Cahill and McDevitt, 1981; Eisenbarth, 1986). Current advances include recognition of new IDD-autoantigens (e.g., glutamic acid decarboxylase), susceptibility genes (e.g., HLA-DQB 1 *0201 and 0302), and the development of trials to prevent IDD through tolerization to IDD-autoantigens (e.g., insulin) (Maclaren, 1993; Winter et al., 1993). Unlocking the genetic mechanisms responsible for IDD can also reveal more general mechanisms involved in other organ-specific autoimmune diseases.

scholarly journals Fusion Proteins for Combined Analysis of Autoantibodies to the 65-kDa Isoform of Glutamic Acid Decarboxylase and Islet Antigen-2 in Insulin-dependent Diabetes Mellitus

2001 ◽  
Vol 47 (5) ◽  
pp. 926-934 ◽  
Author(s):  
Mathias Rickert ◽  
Jochen Seissler ◽  
Werner Dangel ◽  
Helga Lorenz ◽  
Wiltrud Richter
Keyword(s):  
Diabetes Mellitus ◽  
Fusion Protein ◽  
Cost Effective ◽  
Insulin Dependent Diabetes Mellitus ◽  
Insulin Dependent Diabetes ◽  
Dependent Diabetes Mellitus ◽  
Acid Decarboxylase ◽  
Immune Reactivity ◽  
Insulin Dependent ◽  
Islet Antigen

Abstract Background: Prediction, risk assessment, and diagnosis of autoimmune diseases often rely on detection of autoantibodies directed to multiple target antigens, such as the 65-kDa isoform of glutamic acid decarboxylase (GAD65-abs) and the tyrosine phosphatase-like protein islet antigen-2 (IA2-abs), the two major subspecificities of islet cell antibodies (ICAs) associated with insulin-dependent diabetes mellitus. We hypothesized that a combination of autoantigens in a fusion protein unifying the important immunodominant epitopes could provide an efficient target for cost-effective, one-step screening of sera. Methods: Chimeric proteins composed of GAD65 and IA2 residues were constructed, analyzed for their immune reactivity with monoclonal antibodies and sera, and used in a diagnostic assay with 35S-labeled protein as antigen. Results: Length and order of GAD65 and IA2 sequences were critical for conservation of the conformational epitopes in the fusion protein. Among four chimera tested, only IA2(606–979)/GAD65(1–585) retained wild-type-like folding of GAD65 and IA2 domains and yielded a stable protein after baculovirus expression. Reactivity of GAD65 antibody- and IA2 antibody-positive sera from patients newly diagnosed with insulin-dependent diabetes mellitus, from ICA-positive prediabetics, and from ICA-positive first-degree relatives demonstrated conservation of the relevant autoreactive epitopes. The assay based on the in vitro translated fusion antigen had a sensitivity and specificity identical to those for detection of GAD65- and IA2-abs based on the two separate GAD65 and IA2 proteins. Conclusions: Autoantigens such as GAD65 and IA2 can be combined successfully in a fusion protein of similar immune reactivity. This allows simultaneous detection of GAD65- and IA2-abs in a one-step screening assay and cost-effective identification of positive individuals at risk of diabetes or at onset of disease.

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