Monoclonal antibodies against rat brain glutamic acid decarboxylase (GAD)

Monoclonal antibodies against rat brain GAD have been produced and immunochemically characterized in comparison with a traditional anti-GAD antiserum (Oertel et al., Neuroscience6, 2689–2700, 1981). An immunopurified fraction in which GAD represented an estimated 5% of the total protein was used as immunogen. Out of 10 mice injected with this fraction, 6 appeared to be immunized: their sera immunoprecipitated quantitatively GAD activity. Three cell fusions were performed between spleen cells of the best immunized mice and SP2/OAg14 myeloma cells. Around 500 hybridoma were generated in each hybridization experiment. The culture medium of 13 hybridoma significantly trapped GAD activity. All immunoprecipitation curves established with the ascitic fluid obtained from the positive hybridoma, showed a lower titer, at least 50-fold, than the titer of the conventional antiserum. None of these ascitic fluids was able to stain directly any protein from a rat high speed supernatant after western blotting. However, the electrophoretical analysis of the proteins immunotrapped by any of the monoclonal antibodies, followed by western blotting and immunolabelling with the anti-GAD antiserum (“cross-immunoblotting”) showed the same two stained monomers. They have the same molecular weight (respectively 59 and 62 kDa ± 2 kDa) as those stained directly by the anti-GAD antiserum from a rat brain supernatant. Although all monoclonal antibodies showed a lower affinity then the conventional antiserum, which prevents them from being used directly in immunoblotting they permit to definitively establish that the two monomers immunolabelled by the conventional antiserum are constitutive subunits of the rat brain GAD.     

Development of glutamic acid decarboxylase 65 (GAD65) autoantibody assay using biotin-GAD65 fusion protein

We evaluated a biotin-glutamic acid decarboxylase 65 (GAD65)-based enzyme-linked immunosorbent assay (B-ELISA) to detect GAD65 autoantibodies (GAD65Ab) in 78 sera from individuals with newly diagnosed type 1 diabetes. The GAD65Ab index of patients with type 1 diabetes (mean value of GAD65Ab index of 1.891) was significantly higher than those in 50 sera from healthy control group (mean value of 0.068). The intra- and inter-assay coefficients of variation (CV) were calculated to be 1.042 and 10.703%, respectively. The specificity of the B-GAD65 ELISA was comparable to the standard radioimmunoassay (RIA) which is routinely used in the laboratory. We describe the optimal conditions of the binding kinetics from each assay-step for the detection of GAD65Ab using the WHO standard serum 97/550 as a model autoantibody serum. We concluded that incubation times of 15, 90, and 90 min for step 1 (pre-incubation of Biotin14-GAD65 with serum), step 2 (binding the Ab/Ag complex to NeutrAvidin plate), and step 3 (incubation with HRPO-anti-human IgG), respectively, along with human serum dilutions of 1:50, would provide an optimal assay signal within a relatively short timeframe.     

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.     

Differential presentation of glutamic acid decarboxylase 65 (GAD65) T cell epitopes among HLA-DRB1*0401-positive individuals.

Glutamic acid decarboxylase 65 (GAD65) is one of the major autoantigens in type 1 diabetes. We investigated whether there is variation in the processing of GAD65 epitopes between individuals with similar HLA backgrounds and whether the processing characteristics of certain immunogenic epitopes are different in distinct APC subpopulations. Using DR401-restricted T cell hybridomas specific for two immunogenic GAD65 epitopes (115-127 and 274-286), we demonstrate an epitope-specific presentation pattern in human B-lymphoblastoid cell lines (B-LCL). When pulsed with the GAD protein, some DRB1*0401-positive B-LCL, which presented GAD65 274-286 epitope efficiently, were unable to present the GAD65 115-127 epitope. However, all B-LCL presented synthetic peptides corresponding to either GAD epitope. In addition, when pulsed with human serum albumin, all cell lines gave equal stimulation of a DR4-restricted human serum albumin-specific T hybridoma. GAD65-transfected cell lines displayed the same presentation phenotype, showing that lack of the presentation of the 115-127 epitope was not due to inefficient uptake of the protein. Blood mononuclear adherent cells, B cells, or dendritic cells derived from the same individual displayed the same presentation pattern as observed in B cell lines, suggesting that the defect most likely is genetically determined. Therefore, individual differences in Ag processing may result in the presentation of distinct set of peptides derived from an autoantigen such as GAD65. This may be an important mechanism for the deviation of the immune response either into a regulatory pathway or into an inflammatory autoimmune reactivity.     

Transgenic plants expressing human glutamic acid decarboxylase (GAD65), a major autoantigen in insulin-dependent diabetes mellitus

Parenteral and oral administration of autoantigens can induce immune tolerance in autoimmune diseases. Prophylactic therapy based on oral administration of human autoantigens is not, however, feasible when sufficient quantities of candidate autoantigens are not available. Transgenic plants that express high levels of recombinant proteins would allow large quantities of autoantigens to be produced at relatively low costs. In addition, transgenic food would provide a simple and direct method of delivering autoantigens. The production and the characterization of transgenic tobacco and carrot plants expressing human GAD65, a major autoantigen in human insulin-dependent diabetes mellitus (IDDM), is reported. Immunogold labeling and electron microscopy of transgenic tobacco tissue shows the selective targeting of human GAD65 to chloroplast tylacoids and mitochondria. In planta expressed GAD65 has a correct immunoreactivity with IDDM-associated autoantibodies and retains enzymatic activity, a finding that suggests a correct protein folding. In transgenic tobacco and carrot the expression levels of human GAD65 varies between 0.01% and 0.04% of total soluble proteins. Transgenic edible plant organs are now available to study the feasibility of inducing immune tolerance in IDDM animals by oral administration of GAD65.     

Type 1 diabetes-predisposing MHC alleles influence the selection of glutamic acid decarboxylase (GAD) 65-specific T cells in a transgenic model

The genetic factors that contribute to the etiology of type 1 diabetes are still largely uncharacterized. However, the genes of the MHC (HLA in humans) have been consistently associated with susceptibility to disease. We have used several transgenic mice generated in our laboratory, bearing susceptible or resistant HLA alleles, in the absence of endogenous MHC class II (Abetao), to study immune responses to the autoantigen glutamic acid decarboxylase (GAD) 65 and its relevance in determining the association between autoreactivity and disease pathogenesis. Mice bearing diabetes-susceptible haplotypes, HLA DR3 (DRB1*0301) or DQ8 (DQB1*0302), singly or in combination showed spontaneous T cell reactivity to rat GAD 65, which is highly homologous to the self Ag, mouse GAD 65. The presence of diabetes-resistant or neutral alleles, such as HLA DQ6 (DQB1*0602) and DR2 (DRB1*1502) prevented the generation of any self-reactive responses to rat GAD. In addition, unmanipulated Abetao/DR3, Abetao/DQ8, and Abetao/DR3/DQ8 mice recognized specific peptides, mainly from the N-terminal region of the GAD 65 molecule. Most of these regions are conserved between human, mouse, and rat GAD 65. Further analysis revealed that the reactivity was mediated primarily by CD4(+) T cells. Stimulation of these T cells by rat GAD 65 resulted in the generation of a mixed Th1/Th2 cytokine profile in the Abetao/DR3/DQ8, Abetao/DR3, and Abetao/DQ8 mice. Thus, the presence of diabetes-associated genes determines whether immune tolerance is maintained to islet autoantigens, but autoreactivity in itself is not sufficient to induce diabetes.     

Assignment of the rat genes coding for DOPA decarboxylase (DDC) and glutamic acid decarboxylases (GAD1 and GAD2)

By use of rat cDNA probes and a panel of cell hybrids segregating rat chromosomes, the genes encoding three pyridoxal 5-phosphate (PLP)-dependent decarboxylases—namely, DOPA-decarboxylase (Ddc), glutamic acid decarboxylase 1 and 2 (Gad1 and Gad2)—were assigned to rat Chromosomes (Chrs) 14, 3, and 17, respectively. If one takes into account chromosome localizations in the human and the mouse, the present results (i) show that a synteny group is retained on rat Chr 14, human Chr 7, and mouse Chr 11 (Ddc); (ii) strengthen the homology relation known between rat Chr 3 and human and mouse Chrs 2 (Gad1); (iii) suggest that rat Chr 17 has no extensive homology to any human chromosome; and (iv) suggest the order (Prl, Fdp)-Tpl2-Gad2 on the rat Chr 17.     

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.     

Molecular engineering of biotin-glutamic acid decarboxylase 65 fusion protein (Biotin-GAD65) for non-radioactive GAD65 antibody assay

We constructed biotinylated fusion proteins that linked to three detection tags to GAD65 at the N-terminus, and expressed them in an E. coli expression system. The Biotin14-GAD65 protein exhibited the strongest binding to both the GAD65 antibody and the streptavidin among the three constructs. We describe the optimal conditions using a Biotin14-GAD65-based immunoassay for the detection of GAD65 antibody.     

Glutamic acid decarboxylase (GAD65) immunoreactivity in brains of aggressive, adolescent anabolic steroid-treated hamsters

Chronic anabolic-androgenic steroid (AAS) treatment during adolescence facilitates offensive aggression in male Syrian hamsters (Mesocricetus auratus). The current study assessed whether adolescent AAS exposure influenced the immunohistochemical localization of glutamic acid decarboxylase (GAD65), the rate-limiting enzyme in the synthesis of gamma-aminobutyric acid (GABA), in areas of hamster brain implicated in aggressive behavior. Hamsters were administered high dose AAS throughout adolescence, scored for offensive aggression, and then examined for differences in GAD65 puncta to regions of the hamster brain important for aggression. When compared with control animals, aggressive AAS-treated hamsters showed significant increases in the area covered by GAD65 immunoreactive puncta in several of these aggression regions, including the anterior hypothalamus, ventrolateral hypothalamus, and medial amygdala. Conversely, aggressive AAS-treated hamsters showed a significant decrease in GAD65-ir puncta in the lateral septum when compared with oil-treated controls. However, no differences in GAD65 puncta were found in other aggression areas, such as the bed nucleus of the stria terminalis and central amygdala. Together, these results support a role for altered GAD65 synthesis and function in adolescent AAS-facilitated offensive aggression.     

Identification and functional analysis of truncated human glutamic acid decarboxylase 65

Human brain glutamate decarboxylase 65 (hGAD65) was found to exist as full-length and truncated forms when the glutathione S-transferase-tagged hGAD65 fusion protein was subjected to factor Xa cleavage. The truncated form is produced by cleavage at arginine 69 based on N-terminal amino acid sequence analysis, and has a molecular weight of 58 kD. It is resistant to further factor Xa cleavage or mild trypsin treatment and is more active and more stable than the full-length form. Both the full-length and truncated forms of GAD are also observed in brain preparations in the presence of protease inhibitors. Furthermore, full-length GAD could be converted to the truncated form by endogenous proteases, suggesting that the conversion of full-length to truncated GAD mediated by endogenous protease may represent an important mechanism in the regulation of GABA biosynthesis in the brain.     

Active tolerance induction and prevention of autoimmune diabetes by immunogene therapy using recombinant adenoassociated virus expressing glutamic acid decarboxylase 65 peptide GAD(500-585)

Tolerance induction of autoreactive T cells against pancreatic beta cell-specific autoantigens such as glutamic acid decarboxylase 65 (GAD65) and insulin has been attempted as a method to prevent autoimmune diabetes. In this study, we investigate whether adenoassociated virus (AAV) gene delivery of multiple immunodominant epitopes expressing GAD(500-585) could induce potent immune tolerance and persistently suppress autoimmune diabetes in NOD mice. A single muscle injection of 7-wk-old female NOD mice with rAAV/GAD(500-585) (3 x 10(11) IU/mouse) quantitatively reduced pancreatic insulitis and efficiently prevented the development of overt type I diabetes. This prevention was marked by the inactivation of GAD(500-585)-responsive T lymphocytes, the enhanced GAD(500-585)-specific Th2 response (characterized by increased IL-4, IL-10 production, and decreased IFN-gamma production; especially elevated anti-GAD(500-585) IgG1 titer; and relatively unchanged anti-GAD(500-585) IgG2b titer), the increased secretion of TGF-beta, and the production of protective regulatory cells. Our studies also revealed that peptides 509-528, 570-585, and 554-546 in the region of GAD(500-585) played important roles in rAAV/GAD(500-585) immunization-induced immune tolerance. These data indicate that using AAV, a vector with advantage for therapeutic gene delivery, to transfer autoantigen peptide GAD(500-585), can induce immunological tolerance through active suppression of effector T cells and prevent type I diabetes in NOD mice.     

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.     

A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65)

Background  

Human glutamic acid decarboxylase 65 (hGAD65) is a key autoantigen in type 1 diabetes, having much potential as an important marker for the prediction and diagnosis of type 1 diabetes, and for the development of novel antigen-specific therapies for the treatment of type 1 diabetes. However, recombinant production of hGAD65 using conventional bacterial or mammalian cell culture-based expression systems or nuclear transformed plants is limited by low yield and low efficiency. Chloroplast transformation of the unicellular eukaryotic alga Chlamydomonas reinhardtii may offer a potential solution.     

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.