A combination of three distinct trafficking signals mediates axonal targeting and presynaptic clustering of GAD65

The signals involved in axonal trafficking and presynaptic clustering are poorly defined. Here we show that targeting of the gamma-aminobutyric acid-synthesizing enzyme glutamate decarboxylase 65 (GAD65) to presynaptic clusters is mediated by its palmitoylated 60-aa NH(2)-terminal domain and that this region can target other soluble proteins and their associated partners to presynaptic termini. A Golgi localization signal in aa 1-23 followed by a membrane anchoring signal upstream of the palmitoylation motif are required for this process and mediate targeting of GAD65 to the cytosolic leaflet of Golgi membranes, an obligatory first step in axonal sorting. Palmitoylation of a third trafficking signal downstream of the membrane anchoring signal is not required for Golgi targeting. However, palmitoylation of cysteines 30 and 45 is critical for post-Golgi trafficking of GAD65 to presynaptic sites and for its relative dendritic exclusion. Reduction of cellular cholesterol levels resulted in the inhibition of presynaptic clustering of palmitoylated GAD65, suggesting that the selective targeting of the protein to presynaptic termini is dependent on sorting to cholesterol-rich membrane microdomains. The palmitoylated NH(2)-terminal region of GAD65 is the first identified protein region that can target other proteins to presynaptic clusters.     

The hydrophilic isoform of glutamate decarboxylase, GAD67, is targeted to membranes and nerve terminals independent of dimerization with the hydrophobic membrane-anchored isoform, GAD65

GAD67, the larger isoform of the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase, is a hydrophilic soluble molecule, postulated to localize at nerve terminals and membrane compartments by heterodimerization with the smaller membrane-anchored isoform GAD65. We here show that the dimerization region in GAD65 is distinct from the NH(2)-terminal membrane-anchoring region and that a membrane anchoring GAD65 subunit can indeed target a soluble subunit to membrane compartments by dimerization. However, only a fraction of membrane-bound GAD67 is engaged in a heterodimer with GAD65 in rat brain. Furthermore, in GAD65-/- mouse brain, GAD67, which no longer partitions into the Triton X-114 detergent phase, still anchors to membranes at similar levels as in wild-type mice. Similarly, in primary cultures of neurons derived from GAD65-/- mice, GAD67 is targeted to nerve terminals, where it co-localizes with the synaptic vesicle marker SV2. Thus, axonal targeting and membrane anchoring is an intrinsic property of GAD67 and does not require GAD65. The results suggest that three distinct moieties of glutamate decarboxylase localize to membrane compartments, an amphiphilic GAD65 homodimer, an amphiphilic GAD65/67 heterodimer, tethered to membranes via the GAD65 subunit, and a hydrophilic GAD67 homodimer, which associates with membranes by a distinct mechanism.     

Compartmentalization of GABA Synthesis by GAD67 Differs between Pancreatic Beta Cells and Neurons

The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.     

Two distinct mechanisms target GAD67 to vesicular pathways and presynaptic clusters

The inhibitory neurotransmitter γ-amino butyric acid (GABA) is synthesized by two isoforms of the enzyme glutamic acid decarboxylase (GAD): GAD65 and GAD67. Whereas GAD67 is constitutively active and produces >90% of GABA in the central nervous system, GAD65 is transiently activated and augments GABA levels for rapid modulation of inhibitory neurotransmission. Hydrophobic lipid modifications of the GAD65 protein target it to Golgi membranes and synaptic vesicles in neuroendocrine cells. In contrast, the GAD67 protein remains hydrophilic but has been shown to acquire membrane association by heterodimerization with GAD65. Here, we identify a second mechanism that mediates robust membrane anchoring, axonal targeting, and presynaptic clustering of GAD67 but that is independent of GAD65. This mechanism is abolished by a leucine-103 to proline mutation that changes the conformation of the N-terminal domain but does not affect the GAD65-dependent membrane anchoring of GAD67. Thus two distinct mechanisms target the constitutively active GAD67 to presynaptic clusters to facilitate accumulation of GABA for rapid delivery into synapses.     

Glutamate decarboxylase and GABA in pancreatic islets: lessons from knock-out mice.

The GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) is expressed in pancreatic beta-cells and GABA has been suggested to play a role in islet cell development and function. Mouse beta-cells predominantly express the larger isoform of the enzyme, GAD67, and very low levels of the second isoform, GAD65. Yet GAD65 has been shown to be a target of very early autoimmune T-cell responses associated with beta-cell destruction in the non-obese diabetic (NOD) mouse model of Type 1 diabetes. Mice deficient in GAD67, GAD65 or both were used to assess whether GABA is important for islet cell development, and whether GAD65 is required for initiation of insulitis and progression to Type 1 diabetes in the mouse. Lack of either GAD65 or GAD67 did not effect the development of islet cells and the general morphology of islets. When GAD65-/-(129/Sv) mice were backcrossed into the NOD strain for four generations, GAD65-deficient mice developed insulitis similar to GAD65+/+ mice. Furthermore, at the low penetrance of diabetes in this backcross, GAD65-deficient mice developed disease at the same rate and incidence as wildtype mice. The results suggest that GABA generated by either GAD65 or GAD67 is not critically involved in islet formation and that GAD65 expression is not an absolute requirement for development of autoimmune diabetes in the NOD mouse.     

Improved in Planta Expression of the Human Islet Autoantigen Glutamic Acid Decarboxylase (GAD65)

The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH₂-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD67_1-87/GAD65_88-585 molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes.     

High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase

The smaller isoform of the GABA-synthesizing enzyme, glutamic acid decarboxylase 65 (GAD65), is unusually susceptible to becoming a target of autoimmunity affecting its major sites of expression, GABA-ergic neurons and pancreatic beta-cells. In contrast, a highly homologous isoform, GAD67, is not an autoantigen. We used homolog-scanning mutagenesis to identify GAD65-specific amino acid residues which form autoreactive B-cell epitopes in this molecule. Detailed mapping of 13 conformational epitopes, recognized by human monoclonal antibodies derived from patients, together with two and three-dimensional structure prediction led to a model of the GAD65 dimer. GAD65 has structural similarities to ornithine decarboxylase in the pyridoxal-5'-phosphate-binding middle domain (residues 201-460) and to dialkylglycine decarboxylase in the C-terminal domain (residues 461-585). Six distinct conformational and one linear epitopes cluster on the hydrophilic face of three amphipathic alpha-helices in exons 14-16 in the C-terminal domain. Two of those epitopes also require amino acids in exon 4 in the N-terminal domain. Two distinct epitopes reside entirely in the N-terminal domain. In the middle domain, four distinct conformational epitopes cluster on a charged patch formed by amino acids from three alpha-helices away from the active site, and a fifth epitope resides at the back of the pyridoxal 5'-phosphate binding site and involves amino acid residues in exons 6 and 11-12. The epitopes localize to multiple hydrophilic patches, several of which also harbor DR*0401-restricted T-cell epitopes, and cover most of the surface of the protein. The results reveal a remarkable spectrum of human autoreactivity to GAD65, targeting almost the entire surface, and suggest that native folded GAD65 is the immunogen for autoreactive B-cells.     

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

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.     

The amino-terminal domain of the vacuolar proton-translocating ATPase a subunit controls targeting and in vivo dissociation, and the carboxyl-terminal domain affects coupling of proton transport and ATP hydrolysis

The 100-kDa "a" subunit of the vacuolar proton-translocating ATPase (V-ATPase) is encoded by two genes in yeast, VPH1 and STV1. The Vph1p-containing complex localizes to the vacuole, whereas the Stv1p-containing complex resides in some other intracellular compartment, suggesting that the a subunit contains information necessary for the correct targeting of the V-ATPase. We show that Stv1p localizes to a late Golgi compartment at steady state and cycles continuously via a prevacuolar endosome back to the Golgi. V-ATPase complexes containing Vph1p and Stv1p also differ in their assembly properties, coupling of proton transport to ATP hydrolysis, and dissociation in response to glucose depletion. To identify the regions of the a subunit that specify these different properties, chimeras were constructed containing the cytosolic amino-terminal domain of one isoform and the integral membrane, carboxyl-terminal domain from the other isoform. Like the Stv1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Stv1p localized to the Golgi and the complex did not dissociate in response to glucose depletion. Like the Vph1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Vph1p localized to the vacuole and the complex exhibited normal dissociation upon glucose withdrawal. Interestingly, the V-ATPase complex containing the chimera with the carboxyl-terminal domain of Vph1p exhibited a higher coupling of proton transport to ATP hydrolysis than the chimera containing the carboxyl-terminal domain of Stv1p. Our results suggest that whereas targeting and in vivo dissociation are controlled by sequences located in the amino-terminal domains of the subunit a isoforms, coupling efficiency is controlled by the carboxyl-terminal region.     

Maternal immune activation induces GAD1 and GAD2 promoter remodeling in the offspring prefrontal cortex

Maternal infection during pregnancy increases the risk of neurodevelopmental disorders in the offspring. In addition to its influence on other neuronal systems, this early-life environmental adversity has been shown to negatively affect cortical γ-aminobutyric acid (GABA) functions in adult life, including impaired prefrontal expression of enzymes required for GABA synthesis. The underlying molecular processes, however, remain largely unknown. In the present study, we explored whether epigenetic modifications represent a mechanism whereby maternal infection during pregnancy can induce such GABAergic impairments in the offspring. We used an established mouse model of prenatal immune challenge that is based on maternal treatment with the viral mimetic poly(I:C). We found that prenatal immune activation increased prefrontal levels of 5-methylated cytosines (5mC) and 5-hydroxymethylated cytosines (5hmC) in the promoter region of GAD1, which encodes the 67-kDa isoform of the GABA-synthesising enzyme glutamic acid decarboxylase (GAD67). The early-life challenge also increased 5mC levels at the promoter region of GAD2, which encodes the 65-kDa GAD isoform (GAD65). These effects were accompanied by elevated GAD1 and GAD2 promoter binding of methyl CpG-binding protein 2 (MeCP2) and by reduced GAD67 and GAD65 mRNA expression. Moreover, the epigenetic modifications at the GAD1 promoter correlated with prenatal infection-induced impairments in working memory and social interaction. Our study thus highlights that hypermethylation of GAD1 and GAD2 promoters may be an important molecular mechanism linking prenatal infection to presynaptic GABAergic impairments and associated behavioral and cognitive abnormalities in the offspring.     

Enzymatic characterization of a recombinant isoform hybrid of glutamic acid decarboxylase (rGAD67/65) expressed in yeast

BACKGROUND AND AIMS: Glutamic acid decarboxylase (GAD, EC 4.1.1.15) catalyses the conversion of glutamate to gamma-aminobutyric acid (GABA). The 65 kDa isoform, GAD65 is a potent autoantigen in type 1 diabetes, whereas GAD67 is not. A hybrid cDNA was created by fusing a human cDNA for amino acids 1-101 of GAD67 to a human cDNA for amino acids 96-585 of GAD65; the recombinant (r) protein was expressed in yeast and was shown to have equivalent immunoreactivity to mammalian brain GAD with diabetes sera. We here report on enzymatic and molecular properties of rGAD67/65. METHODS: Studies were performed on enzymatic activity of rGAD67/65 by production of 3H-GABA from 3H-glutamate, enzyme kinetics, binding to the enzyme cofactor pyridoxal phosphate (PLP), stability according to differences in pH, temperature and duration of storage, and antigenic reactivity with various GAD-specific antisera. RESULTS: The properties of rGAD67/65 were compared with published data for mammalian brain GAD (brackets). These included a specific enzyme activity of 22.7 (16.7) nKat, optimal pH for enzymatic activity 7.4 (6.8), K(m) of 1.3 (1.3) mM, efficient non-covalent binding to the cofactor PLP, and high autoantigenic potency. The stability of rGAD67/65 was optimal over 3 months at -80 degrees C, or in lyophilized form at -20 degrees C. CONCLUSIONS: Hybrid rGAD67/65 has enzymatic and other properties similar to those of the mixed isoforms of GAD in preparations from mammalian brain as described elsewhere, in addition to its previously described similar immunoreactivity.     

Evidence that GAD65 mediates increased GABA synthesis during intense neuronal activity in vivo

In this study we tested the hypothesis that the 65-kDa isoform of glutamate decarboxylase (GAD(65)) mediates activity-dependent GABA synthesis as invoked by seizures in anesthetized rats. GABA synthesis was measured following acute GABA-transaminase inhibition by gabaculine using spatially localized (1)H NMR spectroscopy before and after bicuculline-induced seizures. Experiments were conducted with animals pre-treated with vigabatrin 24 h earlier in order to reduce GAD(67) protein and also with non-treated controls. GAD isoform content was quantified by immunoblotting. GABA was higher in vigabatrin-treated rats compared to non-treated controls. In vigabatrin-treated animals, GABA synthesis was 28% lower compared to controls [p < 0.05; vigabatrin-treated, 0.043 +/- 0.011 micromol/(g min); non-treated, 0.060 +/- 0.014 micromol/(g min)] and GAD(67) was 60% lower. No difference between groups was observed for GAD(65). Seizures increased GABA synthesis in both control [174%; control, 0.060 +/- 0.014 micromol/(g min) vs. seizures, 0.105 +/- 0.043 micromol/(g min)] and vigabatrin-treated rats [214%; control, 0.043 +/- 0.011 micromol/(g min); seizures, 0.092 +/- 0.018 micromol/(g min)]. GAD(67) could account for at least half of basal GABA synthesis but only 20% of the two-fold increase observed in vigabatrin-treated rats during seizures. The seizure-induced activation of GAD(65) in control cortex occurs concomitantly with a 2.3-fold increase in inorganic phosphate, known to be a potent activator of apoGAD(65)in vitro. Our results are consistent with a major role for GAD(65) in activity-dependent GABA synthesis.     

Targeting of a heterodimeric membrane protein complex to the Golgi: rubella virus E2 glycoprotein contains a transmembrane Golgi retention signal.

Rubella virus (RV) envelope glycoproteins, E2 and E1, form a heterodimeric complex that is targeted to medial/trans-Golgi cisternae. To identify the Golgi targeting signal(s) for the E2/E1 spike complex, we constructed chimeric proteins consisting of domains from RV glycoproteins and vesicular stomatitis virus (VSV) G protein. The location of the chimeric proteins in stably transfected Chinese hamster ovary cells was determined by immunofluorescence, immunoelectron microscopy, and by the extent of processing of their N-linked glycans. A trans-dominant Golgi retention signal was identified within the C-terminal region of E2. When the transmembrane (TM) and cytoplasmic (CT) domains of VSV G were replaced with those of RV E2, the hybrid protein (G-E2TMCT+) was retained in the Golgi. Transport of G-E2TMCT+ to the Golgi was rapid (t1/2 = 10-20 min). The G-E2TMCT+ protein was determined to be distal to or within the medial Golgi based on acquisition of endo H resistance but proximal to the trans-Golgi network since it lacked sialic acid. Deletion analysis revealed that only the TM domain of E2 was required for Golgi targeting. Although the cytoplasmic domain of E2 was not necessary for Golgi retention, it was required for efficient transport of VSV G-RV chimeras out of the endoplasmic reticulum. When assayed in sucrose velocity sedimentations gradients, the Golgi-retained G-E2TMCT+ protein behaved as a dimer. Unlike virtually all other Golgi targeting signals, the E2 TM domain does not contain any polar amino acids. The TM and CT domains of E1 were not required for targeting of E2 and E1 to the Golgi indicating that a heterodimer of two integral membrane proteins can be retained in the Golgi by a single retention signal.     

Structural and functional analysis of cysteine residues in human glutamate decarboxylase 65 (GAD65) and GAD67

Previously, we have shown that brain glutamate decarboxylase (GAD) is greatly inhibited by sulfhydryl reactive reagent suggesting cysteine residue(s) may play an important role in GAD function. In this report, we determined the role of cysteine residues in the recombinant human 65-kDa GAD isoform (hGAD65) and 67-kDa GAD isoform (hGAD67), using a combination of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and site-directed mutagenesis. Here, we report that cysteine 446 (C446) in hGAD65 is important for its activity and is present as free sulfhydryl group. This conclusion is based on the following observations: (i) mutation of C446 in hGAD65 to alanine reduced hGAD65 activity by more than 90%, (ii) MALDI-TOF analysis of the non-reduced, trypsin-digested GAD65 revealed that C446 is present as a free sulfhydryl group as indicated by a peak at m/z (mass/charge) 647.3446 (peptide 443-448) and, when GAD65 was treated with sulfhydryl reagent, N-ethylmaleimide (NEM), the peak is shifted to m/z 772.3702,a mass increase of 125.1 daltons (Da) as a result of modification of cysteine by NEM. Parallel studies have also been conducted with hGAD67. Cysteine 455 was found to be important for GAD67 activity.     

Multiple signals are required for alpha2,6-sialyltransferase (ST6Gal I) oligomerization and Golgi localization

A single amino acid difference in the catalytic domain of two isoforms of the alpha2,6-sialyltransferase (ST6Gal I) leads to differences in their trafficking, processing, and oligomerization. The STtyr isoform is transiently localized in the Golgi and is ultimately cleaved and secreted, whereas the STcys isoform is stably localized in the Golgi and is not cleaved and secreted. The stable localization of STcys is correlated with its enhanced ability to oligomerize. To test the hypothesis that multiple signals can mediate Golgi localization and further evaluate the role of oligomerization in the localization process, we evaluated the effects of individually and simultaneously altering the cytosolic tail and transmembrane region of the STcys isoform. We found that the localization, processing, and oligomerization of STcys were not substantially changed when either the core amino acids of the cytosolic tail were deleted or the sequence and length of the transmembrane region were altered. In contrast, when these changes were made simultaneously, the STcys isoform was converted into a form that was processed, secreted, and weakly oligomerized like STtyr. We propose that STcys oligomerization is a secondary event resulting from its concentration in the Golgi via mechanisms independently mediated by its cytosolic tail and transmembrane region.     

Protein phosphorylation of human brain glutamic acid decarboxylase (GAD)65 and GAD67 and its physiological implications

Previously, we reported that protein phosphorylation plays an important role in regulating soluble l-glutamic acid decarboxylase (GAD) [Bao, J. (1995) J. Biol. Chem. 270, 6464-6467] and membrane-associated GAD activity [Hsu, C. C. (1999) J. Biol. Chem. 274, 24366-24371]. Here, we report the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrate that protein kinase A (PKA) and protein kinase C isoform epsilon are the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. Direct phosphorylation of GAD65 and GAD67 was demonstrated by incorporation of [(32)P] from [gamma-(32)P]ATP into purified GAD65 and GAD67 and immunoblotting assay using anti-phosphoserine/threonine antibodies. We have identified one specific phosphorylation site, threonine 91 (T91), in hGAD67 that can be phosphorylated by PKA using MALDI-TOF. Site-directed mutation of T91 to alanine abolished PKA-mediated phosphorylation and inhibition of GAD activity. Furthermore, mutation of T91 to aspartic acid or glutamic acid mimics the effect of phosphorylation. A model depicting the effect of phosphorylation on GAD activity upon neuronal stimulation is also proposed.     

Regional distribution and relative amounts of glutamate decarboxylase isoforms in rat and mouse brain.

The levels of the two isoforms of glutamate decarboxylase (GAD) were measured in 12 regions of adult rat brain and three regions of mouse brain by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting with an antiserum that recognizes the identical C-terminal sequence in both isoforms from both species. In rat brain the amount of smaller isoform, GAD65, was greater than that of the larger isoform, GAD67, in all twelve regions. GAD65 ranged from 77-89% of total GAD in frontal cortex, hippocampus, hypothalamus, midbrain, olfactory bulb, periaqueductal gray matter, substantia nigra, striatum, thalamus and the ventral tegmental area. The proportion of GAD65 was lower in amygdala and cerebellum but still greater than half of the total. There was a strong correlation between total GAD protein and GAD activity. In the three mouse brain regions analysed (cerebellum, cerebral cortex and hippocampus) the proportion of GAD65 (35,47, and 51% of total GAD) was significantly lower than in the corresponding rat-brain regions. The amount of GAD67 was greater than the amount of GAD65 in mouse cerebellum and was approximately equal to the amount of GAD65 in mouse cerebral cortex and hippocampus.     

Site-directed mutagenesis of K396R of the 65 kDa glutamic acid decarboxylase active site obliterates enzyme activity but not antibody binding

The role of K396 in the enzymatic catalysis and the antigenicity of the 65 kDa isoform of glutamate decarboxylase (GAD65) was analyzed using the K396R GAD65 mutant. GAD65 is a major autoantigen in Type 1 diabetes and autoantibodies directed to GAD65 are widely used markers for this disease. We found that (1) recombinant human GAD65 is fully enzymatically active; (2) the K396R mutation abolished GAD65 activity; and (3) the K396R mutant retained full antigenicity to GAD65 autoantibodies in serum from Type 1 diabetes patients, but not to polyclonal antibodies raised to the catalytic domain.     

Increased expression of GAD65 and GABA in pancreatic beta-cells impairs first-phase insulin secretion

The functional role of glutamate decarboxylase (GAD) and its product GABA in pancreatic islets has remained elusive. Mouse beta-cells express the larger isoform GAD67, whereas human islets express only the smaller isoform GAD65. We have generated two lines of transgenic mice expressing human GAD65 in pancreatic beta-cells (RIP7-hGAD65, Lines 1 and 2) to study the effect that GABA generated by this isoform has on islet cell function. The ascending order of hGAD65 expression and/or activity in beta-cells was Line 1 heterozygotes < Line 2 heterozygotes < Line 1 homozygotes. Line 1 heterozygotes have normal glucose tolerance, whereas Line 1 homozygotes and Line 2 heterozygotes exhibit impaired glucose tolerance and inhibition of insulin secretion in vivo in response to glucose. In addition, fasting levels of blood glucose are elevated and insulin is decreased in Line 1 homozygotes. Pancreas perfusion experiments suggest that GABA generated by GAD65 may function as a negative regulator of first-phase insulin secretion in response to glucose by affecting a step proximal to or at the K(ATP)(+) channel.