scholarly journals Heterogeneity in glutamic acid decarboxylase expression among single rat pancreatic beta cells

Diabetologia ◽  
1999 ◽  
Vol 42 (9) ◽  
pp. 1086-1092 ◽  
Author(s):  
M. C. Sánchez-Soto ◽  
M. E. Larrieta ◽  
R. Vidaltamayo ◽  
M. Hiriart
Keyword(s):  
Glutamic Acid ◽  
Beta Cells ◽  
Glutamic Acid Decarboxylase ◽  
Pancreatic Beta Cells ◽  
Acid Decarboxylase

scholarly journals Amino acid residues 24-31 but not palmitoylation of cysteines 30 and 45 are required for membrane anchoring of glutamic acid decarboxylase, GAD65

1994 ◽  
Vol 124 (6) ◽  
pp. 927-934 ◽  
Author(s):  
Y Shi ◽  
B Veit ◽  
S Baekkeskov
Keyword(s):  
Amino Acids ◽  
Glutamic Acid ◽  
Beta Cells ◽  
Glutamic Acid Decarboxylase ◽  
Pancreatic Beta Cells ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Acid Decarboxylase ◽  
Post Translational Modification ◽  
Membrane Anchoring

The smaller isoform of the GABA synthesizing enzyme glutamic acid decarboxylase, GAD65, is synthesized as a soluble protein that undergoes post-translational modification(s) in the NH2-terminal region to become anchored to the membrane of small synaptic-like microvesicles in pancreatic beta cells, and synaptic vesicles in GABA-ergic neurons. A soluble hydrophilic form, a soluble hydrophobic form, and a hydrophobic firmly membrane-anchored form have been detected in beta cells. A reversible and hydroxylamine sensitive palmitoylation has been shown to distinguish the firmly membrane-anchored form from the soluble yet hydrophobic form, suggesting that palmitoylation of cysteines in the NH2-terminal region is involved in membrane anchoring. In this study we use site-directed mutagenesis to identify the first two cysteines in the NH2-terminal region, Cys 30 and Cys 45, as the sites of palmitoylation of the GAD65 molecule. Mutation of Cys 30 and Cys 45 to Ala results in a loss of palmitoylation but does not significantly alter membrane association of GAD65 in COS-7 cells. Deletion of the first 23 amino acids at the NH2 terminus of the GAD65 30/45A mutant also does not affect the hydrophobicity and membrane anchoring of the GAD65 protein. However, deletion of an additional eight amino acids at the NH2 terminus results in a protein which is hydrophilic and cytosolic. The results suggest that amino acids 24-31 are required for hydrophobic modification and/or targeting of GAD65 to membrane compartments, whereas palmitoylation of Cys 30 and Cys 45 may rather serve to orient or fold the protein at synaptic vesicle membranes.

scholarly journals Endogenous Levels of Gamma Amino-Butyric Acid Are Correlated to Glutamic-Acid Decarboxylase Antibody Levels in Type 1 Diabetes

Biomedicines ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 91
Author(s):  
Henrik Hill ◽  
Andris Elksnis ◽  
Per Lundkvist ◽  
Kumari Ubhayasekera ◽  
Jonas Bergquist ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Glutamic Acid ◽  
Beta Cells ◽  
Glutamic Acid Decarboxylase ◽  
Pancreatic Beta Cells ◽  
Drug Candidate ◽  
Acid Decarboxylase ◽  
Diabetes Research ◽  
Gad Autoantibodies

Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system (CNS) and outside of the CNS, found in the highest concentrations in immune cells and pancreatic beta-cells. GABA is gaining increasing interest in diabetes research due to its immune-modulatory and beta-cell stimulatory effects and is a highly interesting drug candidate for the treatment of type 1 diabetes (T1D). GABA is synthesized from glutamate by glutamic acid decarboxylase (GAD), one of the targets for autoantibodies linked to T1D. Using mass spectrometry, we have quantified the endogenous circulating levels of GABA in patients with new-onset and long-standing T1D and found that the levels are unaltered when compared to healthy controls, i.e., T1D patients do not have a deficit of systemic GABA levels. In T1D, GABA levels were negatively correlated with IL-1 beta, IL-12, and IL-15 15 and positively correlated to levels of IL-36 beta and IL-37. Interestingly, GABA levels were also correlated to the levels of GAD-autoantibodies. The unaltered levels of GABA in T1D patients suggest that the GABA secretion from beta-cells only has a minor impact on the circulating systemic levels. However, the local levels of GABA could be altered within pancreatic islets in the presence of GAD-autoantibodies.

scholarly journals Cytotoxic T cells specific for glutamic acid decarboxylase in autoimmune diabetes.

1995 ◽  
Vol 181 (5) ◽  
pp. 1923-1927 ◽  
Author(s):  
P Panina-Bordignon ◽  
R Lang ◽  
P M van Endert ◽  
E Benazzi ◽  
A M Felix ◽  
...  
Keyword(s):  
T Cells ◽  
Glutamic Acid ◽  
Beta Cells ◽  
Glutamic Acid Decarboxylase ◽  
Cytotoxic T Cells ◽  
Antigen Presenting Cells ◽  
Acid Decarboxylase ◽  
Diabetic Patients ◽  
Recent Onset ◽  
Antigen Presenting

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that results in the destruction of the pancreatic islet beta cells. Glutamic acid decarboxylase (GAD) has been recently indicated as a key autoantigen in the induction of IDDM in nonobese diabetic mice. In human diabetes, the mechanism by which the beta cells are destroyed is still unknown. Here we report the first evidence for the presence of GAD-specific cytotoxic T cells in asymptomatic and recent diabetic patients. GAD65 peptides displaying the human histocompatibility leukocyte antigen (HLA)-A*0201 binding motif have been synthesized. One of these peptides, GAD114-123, binds to HLA-A*0201 molecules in an HLA assembly assay. Peripheral blood mononuclear cells from individuals with preclinical IDDM, recent-onset IDDM, and from healthy controls were stimulated in vitro with the selected peptide in the presence of autologous antigen-presenting cells. In three cases (one preclinical IDDM and two recent-onset IDDM), we detected specific killing of autologous antigen-presenting cells when incubated with GAD114-123 peptide or when infected with a recombinant vaccinia virus expressing GAD65. These patients were the only three carrying the HLA-A*0201 allele among the subjects studied. Our finding suggests that GAD-specific cytotoxic T lymphocytes may play a critical role in the initial events of IDDM.

scholarly journals Differential detection of rat islet and brain glutamic acid decarboxylase (GAD) isoforms with sequence-specific peptide antibodies.

1995 ◽  
Vol 43 (1) ◽  
pp. 53-59 ◽  
Author(s):  
L Li ◽  
J Jiang ◽  
W A Hagopian ◽  
A E Karlsen ◽  
M Skelly ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Beta Cells ◽  
Glutamic Acid Decarboxylase ◽  
Autoimmune Diabetes ◽  
Polyclonal Antibodies ◽  
Acid Decarboxylase ◽  
Alpha Cells ◽  
Specific Antibodies ◽  
Pancreatic Islet Beta Cells ◽  
Irrelevant Peptide

We studied the distribution of the M(r) 65,000 and M(r) 67,000 isoforms of glutamic acid decarboxylase, GAD65 and GAD67, in rat islets and brain by immunocytochemistry. Synthetic peptides representing selected GAD65 or GAD67 sequences were used to produce sequence-specific antibodies, allowing differential immunocytochemical detection of the two isoforms. GAD-specific reactivity of each peptide antiserum was confirmed by ELISA, immunoblotting, and immunoprecipitation. Immunostaining specificity was verified by displacement with either immunizing or irrelevant peptide. Dual immunostaining with GAD isoform-specific antibodies and polyclonal antibodies to glucagon showed that GAD65 was primarily detected in rat pancreatic islet beta-cells, whereas alpha-cells had weak GAD65 staining. In contrast, GAD67 was detected primarily in alpha-cells. In rat brain, GAD65 and GAD67 were present in neuron cell bodies and processes. These data demonstrate that antibodies raised against the N-terminus of GAD allow differential immunocytochemical identification of GAD67 and GAD65. Differential expression of GAD isoforms within islet alpha- and beta-cells supports the role of GAD65 in autoimmune diabetes and stiff-man syndrome.

Autoantibodies in IDDM primarily recognize the 65,000-M(r) rather than the 67,000-M(r) isoform of glutamic acid decarboxylase

Diabetes ◽  
1993 ◽  
Vol 42 (4) ◽  
pp. 631-636 ◽  
Author(s):  
W. A. Hagopian ◽  
B. Michelsen ◽  
A. E. Karlsen ◽  
F. Larsen ◽  
A. Moody ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Glutamic Acid Decarboxylase ◽  
Acid Decarboxylase
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