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

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.

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Similarities Between Bacterial GAD and Human GAD65: Implications in Gut Mediated Autoimmune Type 1 Diabetes

10.1101/2021.11.29.470330 ◽

2021 ◽

Author(s):

Monica Westley ◽

Tiffany Richardson ◽

Suhana Bedi ◽

Baofeng Jia ◽

Fiona S.L. Brinkman ◽

...

Keyword(s):

Type 1 Diabetes ◽

Beta Cells ◽

Gut Microbiome ◽

Pancreatic Beta Cells ◽

Acid Decarboxylase ◽

Host Immune System ◽

Gamma Aminobutyric Acid ◽

Human Gut ◽

Autoimmune Attack

Abstract A variety of islet autoantibodies (AAbs) can predict and possibly dictate eventual type 1 diabetes (T1D) diagnosis. Upwards of 75% of those with T1D are positive for AAbs against glutamic acid decarboxylase (GAD65), a producer of gamma-aminobutyric acid (GABA) in human pancreatic beta cells. Interestingly, bacterial populations within the human gut also express GAD65 and produce GABA. Evidence suggests that dysbiosis of the microbiome may correlate with T1D pathogenesis and physiology. Therefore, autoimmune linkages between the gut microbiome and islets susceptible to autoimmune attack need to be further elucidated. Utilizing silico analyses, we show here that 25 GAD sequences from different human gut bacterial sources show sequence and motif similarities to human beta cell GAD65. Our motif analyses determined that a majority of gut GAD sequences contain the pyroxical dependent decarboxylase domain of human GAD65 which is important for its enzymatic activity. Additionally, we showed overlap with known human GAD65 T-cell receptor epitopes which may implicate the immune destruction of beta cells. Thus, we propose a physiological hypothesis in which changes in the gut microbiome in those with T1D result in a release of bacterial GAD, thus causing miseducation of the host immune system. Due to the notable similarities, we found between humans and bacterial GAD, these deputized immune cells may then go on to target human beta cells leading to the development of T1D.

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

The Journal of Cell Biology ◽

10.1083/jcb.124.6.927 ◽

1994 ◽

Vol 124 (6) ◽

pp. 927-934 ◽

Cited By ~ 55

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.

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High concentration of gamma-aminobutyric acid in pancreatic beta cells

Diabetes ◽

10.2337/diabetes.28.7.629 ◽

1979 ◽

Vol 28 (7) ◽

pp. 629-633 ◽

Cited By ~ 9

Author(s):

H. Taniguchi ◽

Y. Okada ◽

H. Seguchi ◽

C. Shimada ◽

M. Seki ◽

...

Keyword(s):

Beta Cells ◽

Pancreatic Beta Cells ◽

Aminobutyric Acid ◽

Gamma Aminobutyric Acid ◽

High Concentration

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Clinical characteristics of patients with cerebellar ataxia associated with anti-GAD antibodies

Arquivos de Neuro-Psiquiatria ◽

10.1590/0004-282x20170011 ◽

2017 ◽

Vol 75 (3) ◽

pp. 142-146 ◽

Cited By ~ 4

Author(s):

Tiago Silva Aguiar ◽

Andrea Fragoso ◽

Carolina Rouanet de Albuquerque ◽

Patrícia de Fátima Teixeira ◽

Marcus Vinícius Leitão de Souza ◽

...

Keyword(s):

Cerebellar Ataxia ◽

Pancreatic Beta Cells ◽

Clinical Characteristics ◽

Immunoglobulin Therapy ◽

Acid Decarboxylase ◽

Partial Recovery ◽

Gamma Aminobutyric Acid ◽

Intravenous Immunoglobulin Therapy ◽

Autoimmune Cerebellar Ataxia ◽

Insidious Onset

ABSTRACT The enzyme glutamic acid decarboxylase (GAD), present in GABAergic neurons and in pancreatic beta cells, catalyzes the conversion of gamma-aminobutyric acid (GABA). The cerebellum is highly susceptible to immune-mediated mechanisms, with the potentially treatable autoimmune cerebellar ataxia associated with the GAD antibody (CA-GAD-ab) being a rare, albeit increasingly detected condition. Few cases of CA-GAD-ab have been described. Methods This retrospective and descriptive study evaluated the clinical characteristics and outcomes of patients with CA-GAD-ab. Result Three patients with cerebellar ataxia, high GAD-ab titers and autoimmune endocrine disease were identified. Patients 1 and 2 had classic stiff person syndrome and insidious-onset cerebellar ataxia, while Patient 3 had pure cerebellar ataxia with subacute onset. Patients received intravenous immunoglobulin therapy with no response in Patients 1 and 3 and partial recovery in Patient 2. Conclusion CA-GAD-ab is rare and its clinical presentation may hamper diagnosis. Clinicians should be able to recognize this potentially treatable autoimmune cerebellar ataxia.

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High Concentration of Gamma-Aminobutyric Acid in Pancreatic Beta Cells

Diabetes ◽

10.2337/diab.28.7.629 ◽

1979 ◽

Vol 28 (7) ◽

pp. 629-633 ◽

Cited By ~ 36

Author(s):

H. Taniguchi ◽

Y. Okada ◽

H. Seguchi ◽

C. Shimada ◽

M. Seki ◽

...

Keyword(s):

Beta Cells ◽

Pancreatic Beta Cells ◽

Aminobutyric Acid ◽

Gamma Aminobutyric Acid ◽

High Concentration

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A gamma-aminobutyric acid transporter driven by a proton pump is present in synaptic-like microvesicles of pancreatic beta cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.11.5317 ◽

1993 ◽

Vol 90 (11) ◽

pp. 5317-5321 ◽

Cited By ~ 63

Author(s):

A. Thomas-Reetz ◽

J. W. Hell ◽

M. J. During ◽

C. Walch-Solimena ◽

R. Jahn ◽

...

Keyword(s):

Beta Cells ◽

Proton Pump ◽

Pancreatic Beta Cells ◽

Aminobutyric Acid ◽

Gamma Aminobutyric Acid ◽

Acid Transporter

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Mediators and mechanisms of pancreatic beta-cell death in type 1 diabetes

Arquivos Brasileiros de Endocrinologia & Metabologia ◽

10.1590/s0004-27302008000200003 ◽

2008 ◽

Vol 52 (2) ◽

pp. 156-165 ◽

Cited By ~ 84

Author(s):

Pierre Pirot ◽

Alessandra K. Cardozo ◽

Décio L. Eizirik

Keyword(s):

Type 1 Diabetes ◽

Beta Cell ◽

Beta Cells ◽

Pancreatic Beta Cells ◽

Map Kinases ◽

Pancreatic Beta Cell ◽

Insulin Deficiency ◽

Beta Cell Destruction ◽

Pro Inflammatory Cytokines

Type 1 diabetes mellitus (T1D) is characterized by severe insulin deficiency resulting from chronic and progressive destruction of pancreatic beta-cells by the immune system. The triggering of autoimmunity against the beta-cells is probably caused by environmental agent(s) acting in the context of a predisposing genetic background. Once activated, the immune cells invade the islets and mediate their deleterious effects on beta-cells via mechanisms such as Fas/FasL, perforin/granzyme, reactive oxygen and nitrogen species and pro-inflammatory cytokines. Binding of cytokines to their receptors on the beta-cells activates MAP-kinases and the transcription factors STAT-1 and NFkappa-B, provoking functional impairment, endoplasmic reticulum stress and ultimately apoptosis. This review discusses the potential mediators and mechanisms leading to beta-cell destruction in T1D.

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