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 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 Membrane anchoring of the autoantigen GAD65 to microvesicles in pancreatic beta-cells by palmitoylation in the NH2-terminal domain.

1992 ◽  
Vol 118 (2) ◽  
pp. 309-320 ◽  
Author(s):  
S Christgau ◽  
H J Aanstoot ◽  
H Schierbeck ◽  
K Begley ◽  
S Tullin ◽  
...  
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Immunogold Electron Microscopy ◽  
Acid Decarboxylase ◽  
Gamma Aminobutyric Acid ◽  
Beta Cell Destruction ◽  
Terminal Domain ◽  
Membrane Anchoring ◽  
Major Target ◽  
Hydrophilic Molecule

Pancreatic beta-cells and gamma-aminobutyric acid (GABA)-secreting neurons both express the enzyme glutamic acid decarboxylase (GAD) which is a major target of autoantibodies associated with beta-cell destruction and impairment of GABA-ergic neurotransmitter pathways. The predominant form of GAD in pancreatic beta-cells, GAD65, is synthesized as a soluble hydrophilic molecule, which is modified to become firmly membrane anchored. Here we show by immunogold electron microscopy that GAD65 is localized to the membrane of small vesicles which are identical in size to small synaptic-like microvesicles in pancreatic beta-cells. The NH2-terminal domain of GAD65 is the site of a two-step modification, the last of which results in a firm membrane anchoring that involves posttranslational hydroxylamine sensitive palmitoylation. GAD65 can be released from the membrane by an apparent enzyme activity in islets, suggesting that the membrane anchoring step is reversible and potentially regulated. The hydrophobic modifications and consequent membrane anchoring of GAD65 to microvesicles that store its product GABA may be of functional importance and, moreover, significant for its selective role as an autoantigen.

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 Two amino acids in glutamic acid decarboxylase act in concert for maintainance of conformational determinants recognised by Type I diabetic autoantibodies

Diabetologia ◽  
2000 ◽  
Vol 43 (7) ◽  
pp. 881-889 ◽  
Author(s):  
T.I.M. Tree ◽  
N. G. Morgenthaler ◽  
N. Duhindan ◽  
K. E. Hicks ◽  
A.-M. Madec ◽  
...  
Keyword(s):  
Amino Acids ◽  
Glutamic Acid ◽  
Glutamic Acid Decarboxylase ◽  
Acid Decarboxylase ◽  
Type I
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