Cloning and expression of rice glutamate decarboxylase (GAD) in Escherichia coli.

Glutamate decarboxylase (GAD) converts L-glutamate to g-aminobutyric acid (GABA), which is a non-protein amino acid present in all organisms with some activities including improvement of neurve and cytoskeltal functions. Therefore, GAD is considered as a key molecule to use in molecular therapy of genetical human diseases. Accordingly, cloning of GADs from various plants is an important aim of researchers. The aim of this study was to clone rice (Oryza sativa L.) GADs in Escherichia coli (E.coli) MC 1061 bacterium.In this study, rice GADs was cloned in E.coli in both 37°C and 28°C. Two concentrations of Isopropyl-ß-D-thiogalactoside (IPTG) (0.5mM and 1mM) were investigated in TB medium. Purification was done with Ni2+-nitrilotriacetic acid (NTA) resin in various concentration of imidasol. According to SDS-PAGE analysis, rice GADs was cloned and expressed successfully in E.coli MC 1061 bacterium and the most expression was done in 37°C and 0.5mM IPTG and the best concentration of imidasol was 100mM for purification step. Based on the results, rice GADs can be expressed in E.coli MC 1061 bacterium and, hence, it is a suitable way to produce the plant enzyme in industrial scales.

Purification and characterization of glutamate decarboxylase from Enterococcus raffinosus TCCC11660.

Glutamate decarboxylase (GAD) is the sole enzyme that synthesizes γ-aminobutyric acid through the irreversible decarboxylation of L-glutamate. In this study, the purification and characterization of an unreported GAD from a novel strain of Enterococcus raffinosus TCCC11660 were investigated. The native GAD from E. raffinosus TCCC11660 was purified 32.3-fold with a recovery rate of 8.3%, using ultrafiltration and ammonium sulfate precipitation, followed by ion-exchange and size-exclusion chromatography. The apparent molecular weight of purified GAD, as determined by SDS-PAGE and size-exclusion chromatography was 55 and 110 kDa, respectively, suggesting that GAD exists as a dimer of identical subunits in solution. In the best sodium citrate buffer, metal ions of Mo6+ and Mg2+ had positive effects, while Cu2+, Fe2+, Zn2+ and Co2+ showed significant adverse effects on enzyme activity. The optimum pH and temperature of GAD were determined to be 4.6 and 45 °C, while the K m and V max values for the sole L-glutamate substrate were 5.26 and 3.45 µmol L-1 min-1, respectively.

[Simultaneous demonstration of glutamate decarboxylase and synaptophysin in paraffin sections of rat cerebellum].

The article presents highly reproducible and inexpensive protocol for simultaneous demonstration of glutamate decarboxylase (GAD67), the key enzyme of gamma-aminobutyric acid (GABA) synthesis and synaptophysin (SYP), a marker protein of synaptic vesicles using confocal laser microscopy. In the cerebellar cortex, GAD labels Purkinje cells and pinceaux in their basal parts and is unevenly distributed in the neuropil of molecular and granular layers. SYP clearly marks the contours of large dendrites of Purkinje cells in molecular layer, while in the granular layers it labels parts of cerebellar glomeruli--the terminals of the mossy fibers. GAD-immunopositive structures (GABA-ergic axons of stellate cells--Golgi cells) are often located at periphery of the glomeruli. In the peripheral zone of the glomeruli, colocalization of GAD- and SYP-immunopositive structures was observed, suggesting the presence of GABA-ergic synapses in this zone.

An archaeal glutamate decarboxylase homolog functions as an aspartate decarboxylase and is involved in ß-alanine and coenzyme A biosynthesis.

ß-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. ß-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of ß-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5'-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of ß-alanine, 4'-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the ß-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for ß-alanine biosynthesis in these organisms.

Clinical & immunological profile of newly diagnosed patients with youth onset diabetes mellitus.

BACKGROUND & OBJECTIVES: There has been a rise in the incidence of diabetes mellitus in the younger population of India. There are limited data available on the immunological profile of youth onset diabetes mellitus (DM) especially in type 2. Therefore, this study was undertaken to evaluate the clinical and immunological profile of youth onset DM in north India. METHODS: Fifty one consecutive patients of 8-35 yr of age with diabetes mellitus attending the Lok Nayak Hospital, Maulana Azad Medical College, New Delhi, and Hormone Care and Research Center at Ghaziabad, Uttar Pradesh, India, were included in the study. All subjects were tested for glutamic acid decarboxylase (GAD), an islet cell antigen ICA512/IA2, and insulin antibodies. GAD and ICA512/IA2 were done by ELISA and insulin autoantibodies were tested by radioimmunoassay (RIA) method. These patients were also screened for hepatitis A to E, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) as trigger factors for onset of type 1 DM. RESULTS: o0 f the total 51 patients, 38 were men and 13 were women. The mean age and BMI of the subjects was 19.7 (±7) years and 21 (± 5) kg/m [2] , respectively. Twenty patients were below the age of 18 yr and their height was more than 75 th percentile of Indian standards. All patients were symptomatic and 12 of these presented with ketoacidosis. Only 48 per cent (n=24) were positive for GAD, 14 per cent (n=7) for ICA512/IA-2, and 28% (n=14) were positive for insulin antibody. Five of these patients had evidence of hepatitis E virus infection. None of the subjects had evidence of active CMV or EBV infection. INTERPRETATION & CONCLUSIONS: About half of the youth onset diabetes mellitus patients from north India had presence of pancreatic autoimmunity in the form of GAD, ICA512/IA2, and insulin antibodies or a combination of antibodies suggestive of having type 1 DM. Further studies need to be done on a large sample size in different parts of the country.

Synthesis of ß-alanine from L-aspartate using L-aspartate-α-decarboxylase from Corynebacterium glutamicum.

ß-Alanine is mainly produced by chemical methods in current industrial processes. Here, panD from Corynebacterium glutamicum encoding L-aspartate-α-decarboxylase (ADC) was cloned and expressed in Escherichia coli BL21(DE3). ADC C.g catalyzes the α-decarboxylation of L-aspartate to ß-alanine. The purified ADC C.g was optimal at 55 °C and pH 6 with excellent stability at 16-37 °C and pH 4-7. A pH-stat directed, fed-batch feeding strategy was developed for enzymatic synthesis of ß-alanine to keep the pH value within 6-7.2 and thus attenuate substrate inhibition. A maximum conversion of 97.2 % was obtained with an initial 5 g L-aspartate/l and another three feedings of 0.5 % (w/v) L-aspartate at 8 h intervals. The final ß-alanine concentration was 12.85 g/l after 36 h. This is the first study concerning the enzymatic production of ß-alanine by using ADC.

Characterization of a glutamate decarboxylase (GAD) gene from Lactobacillus zymae.

Lactic acid bacteria (LAB) were isolated from Kimchi, a Korean traditional fermented vegetable food. LAB accumulating GABA (γ-aminobutyric acid) in the culture media were screened by TLC analysis. One isolate, GU240, produced the highest amount of GABA among the 3,000 isolates and identified as a Lactobacillus zymae strain. Glutamate decarboxylase (GAD) gene was cloned and over-expressed in E. coli BL21(DE3) using pET26b(+). The recombinant GAD was purified by using a Ni-NTA column. Its size was 53 kDa by SDS-PAGE. Maximum GAD activity was at pH 4.5 and 41 °C and the activity was dependent on pyridoxal 5'-phosphate. Km and Vmax of LzGAD were 1.7 mM and 0.01 mM/min, respectively, when glutamate was used as a substrate.

Enhancement of γ-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis.

γ-Aminobutyric acid (GABA), a non-protein amino acid, is a bioactive component in the food, feed and pharmaceutical fields. To establish an effective single-step production system for GABA, a recombinant Corynebacterium glutamicum strain co-expressing two glutamate decarboxylase (GAD) genes (gadB1 and gadB2) derived from Lactobacillus brevis Lb85 was constructed. Compared with the GABA production of the gadB1 or gadB2 single-expressing strains, GABA production by the gadB1-gadB2 co-expressing strain increased more than twofold. By optimising urea supplementation, the total production of L-glutamate and GABA increased from 22.57 ± 1.24 to 30.18 ± 1.33 g L⁻¹, and GABA production increased from 4.02 ± 0.95 to 18.66 ± 2.11 g L⁻¹ after 84-h cultivation. Under optimal urea supplementation, L-glutamate continued to be consumed, GABA continued to accumulate after 36 h of fermentation, and the pH level fluctuated. GABA production increased to a maximum level of 27.13 ± 0.54 g L⁻¹ after 120-h flask cultivation and 26.32 g L⁻¹ after 60-h fed-batch fermentation. The conversion ratio of L-glutamate to GABA reached 0.60-0.74 mol mol⁻¹. By co-expressing gadB1 and gadB2 and optimising the urea addition method, C. glutamicum was genetically improved for de novo biosynthesis of GABA from its own accumulated L-glutamate.

An examination of aspartate decarboxylase and glutamate decarboxylase activity in mosquitoes.

A major pathway of beta-alanine synthesis in insects is through the alpha-decarboxylation of aspartate, but the enzyme involved in the decarboxylation of aspartate has not been clearly defined in mosquitoes and characterized in any insect species. In this study, we expressed two putative mosquito glutamate decarboxylase-like enzymes of mosquitoes and critically analyzed their substrate specificity and biochemical properties. Our results provide clear biochemical evidence establishing that one of them is an aspartate decarboxylase and the other is a glutamate decarboxylase. The mosquito aspartate decarboxylase functions exclusively on the production of beta-alanine with no activity with glutamate. Likewise the mosquito glutamate decarboxylase is highly specific to glutamate with essentially no activity with aspartate. Although insect aspartate decarboxylase shares high sequence identity with glutamate decarboxylase, we are able to closely predict aspartate decarboxylase from glutamate decarboxylase based on the difference of their active site residues.

Isolation and characterization of a Glutamate decarboxylase (GAD) gene and their differential expression in response to abiotic stresses from Panax ginseng C. A. Meyer.

Glutamate decarboxylase (GAD) catalyzes the conversion of L-glutamate to γ-aminobutyric acid (GABA). A full-length cDNA encoding GAD (designated as PgGAD) was isolated and characterized from the root of Panax ginseng C. A. Meyer. The length cDNA of PgGAD was 1881 bp and contained a 1491 bp open reading frame (ORF) encoding a glutamate decarboxylase protein of 496 amino acids, possessing a Ser-X-X-Lys active site, which belongs to the GAD group. The deduced amino acid sequence of the PgGAD was classified in the plant GAD family and has 76-85% high similarity with other plants as like petunia, Arabidopsis, tomato. Secondary structure of PgGAD was predicted by using SOPMA software program. Southern blot analysis of genomic DNA suggests that, there is more than one copy of the PgGAD gene. The organ specific gene expression pattern also studied in P. ginseng seedlings, in which the stem showed elevated expression than root, leaf, bud and rhizomes. Along with this, we also confirmed the gene expression of PgGAD under various abiotic stresses like temperature stress, osmotic stress, anoxia, oxidative stress, and mechanical damage. Temporal analysis of gene expression except exposure of oxidative stress revealed an enhanced expression after each stresses. The enzyme activity of PgGAD was stimulated to 2-fold under cold stress.

Purification and biochemical characterisation of a novel glutamate decarboxylase from rice bran.

BACKGROUND: Glutamate decarboxylase (GAD) is a useful enzyme whose main function is to catalyse the irreversible alpha-decarboxylation of L-glutamate to produce gamma-aminobutyric acid. The cheap and abundant rice-processing by-product rice bran contains a high amount of GAD, the purification and characterisation of which have not yet been reported. In this study, research on rice bran GAD was initiated. RESULTS: Rice bran GAD was purified to homogeneity via a combined purification protocol of ammonium sulfate fractionation, ion exchange chromatography and two gel filtrations, with a purification fold of 128.6 and an activity recovery of 21.3%. The enzyme was active at pH 5.5 and 40 degrees C and retained 80% of its original activity in the pH range 5-9 and the temperature range 30-50 degrees C. GAD activity was significantly enhanced in the presence of Ca2+ but strongly inhibited by Ag+, Hg2+, sodium dodecyl sulfate and CH3COOH. Kinetic determination of the apparent K(m) for L-glutamate and pyridoxal 5'-phosphate gave values of 27.4 mmol L(-1) and 1.16 micromol L(-1) respectively. CONCLUSION: Considering that rice bran is cheap and commercially available and that rice bran GAD is relatively stable, the development of cost-effective rice bran GAD-related functional foods would seem to be feasible.

Purification and characterization of the first archaeal glutamate decarboxylase from Pyrococcus horikoshii.

Glutamate decarboxylase (GAD) from the archaeon Pyrococcus horikoshii was successfully expressed and purified, with the aim of developing a hyperthermostable GAD for industrial applications. Its biochemical properties were different from those reported for other GADs. The enzyme had broad substrate specificity, and its optimum pH and temperature were pH 8.0 and > 97 degrees C.

Purification of L-glutamate decarboxylase from monkey brain.

Glutamate decarboxylase (GAD) is an enzyme that synthesizes gamma-aminobutyrate (GABA), a major inhibitory neurotransmitter in the central nervous system. Post-translational modification of GAD, such as N-terminal blockage, phosphorylation-dephosphorylation, and palmitoylation, is an important factor in the biological activity of GAD. In order to address the significance of post-translational events on GAD, we thought it crucial to obtain a non-recombinant form of GAD. In this study, we attempted to isolate GAD protein from the monkey brain, a model animal close to the human that has not been studied. Monkey brain was homogenized, fractionated with ammonium sulphate, and applied to a series of chromatographic steps, including hydrophobic, ion-exchange, and gel filtration. Purified GAD showed a single band on SDS-PAGE, and the enzyme was found to have a molecular weight of 61,000 and exhibited 1,100 nmol/min/mg of specific activity. It had an optimal pH of 7 and optimal thermal stability at 40 degrees C.

Characterization of glutamate decarboxylase from a high gamma-aminobutyric acid (GABA)-producer, Lactobacillus paracasei.

Gamma-aminobutyric acid (GABA) has several physiological functions in humans. We have reported that Lactobacillus paracasei NFRI 7415 produces high levels of GABA. To gain insight into the higher GABA-producing ability of this strain, we analyzed glutamate decarboxylase (GAD), which catalyzes the decarboxylation of L-glutamate to GABA. The molecular weight of the purified GAD was estimated to be 57 kDa by SDS-PAGE and 110 kDa by gel filtration, suggesting that GAD forms the dimer under native conditions. GAD activity was optimal at pH 5.0 at 50 degrees C. The Km value for the catalysis of glutamate was 5.0 mM, and the maximum rate of catalysis was 7.5 micromol min(-1) mg(-1). The N-terminal amino acid sequence of GAD was determined, and the gene encoding GAD from genomic DNA was cloned. The findings suggest that the ability of Lb. paracasei to produce high levels of GABA results from two characteristics of GAD, viz., a low Km value and activity at low pH.

Glutamate decarboxylase from Lactobacillus brevis: activation by ammonium sulfate.

In this study, the glutamate decarboxylase (GAD) gene from Lactobacillus brevis IFO12005 (Biosci. Biotechnol. Biochem., 61, 1168-1171 (1997)), was cloned and expressed. The deduced amino acid sequence showed 99.6% and 53.1% identity with GAD of L. brevis ATCC367 and L. lactis respectively. The His-tagged recombinant GAD showed an optimum pH of 4.5-5.0, and 54 kDa on SDS-PAGE. The GAD activity and stability was significantly dependent on the ammonium sulfate concentration, as observed in authentic GAD. Gel filtration showed that the inactive form of the GAD was a dimer. In contrast, the ammonium sulfate-activated form was a tetramer. CD spectral analyses at pH 5.5 revealed that the structures of the tetramer and the dimer were similar. Treatment of the GAD with high concentrations of ammonium sulfate and subsequent dilution with sodium glutamate was essential for tetramer formation and its activation. Thus the biochemical properties of the GAD from L. brevis IFO12005 were significantly different from those from other sources.

Expression of recombinant goldfish glutamic acid decarboxylase 65 and evidence for differential pH and PLP responsiveness compared to the human enzyme.

Glutamic acid decarboxylase (GAD) catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA) that acts as an important inhibitory neurotransmitter in the vertebrate brain, as well as in the regulation of neuroendocrine function. GAD65 and GAD67 are the two main isoforms that exist in vertebrates. The biochemical properties of recombinant forms of goldfish and human GAD65 were examined. The recombinant goldfish GAD65 (gfGAD65) was expressed at high levels using a maltose binding protein fusion system for biochemical characterization. The human GAD65 (hGAD65) was expressed as a GST fusion and was also purified. The recombinant goldfish GAD65 protein has properties that are different from the human counterpart. In particular, the gfGAD65 is less active at acidic pH compared to hGAD65, which is moderately active over a wider range of acidic and basic pH. Interestingly, however, gfGAD65 is less dependent on a cofactor pyridoxal-5'-L-phosphate (PLP) for activity. In the absence of added PLP, cleaved recombinant gfGAD65 showed approximately 20% of maximal activity whereas hGAD65 showed no detectable activity. The physiological and evolutionary significance of these findings is discussed in light of the conserved function of GAD in two vertebrate species that are separated in evolutionary time by more than 200 million years.

Enhanced GAD65 production in plants using the MagnICON transient expression system: Optimization of upstream production and downstream processing.

Plants have emerged as competitive production platforms for pharmaceutical proteins that are required in large quantities. One example is the 65-kDa isoform of human glutamic acid decarboxylase (GAD65), a major autoimmune diabetes autoantigen that has been developed as a vaccine candidate for the primary prevention of diabetes. The expression of GAD65 in plants has been optimized but large-scale purification is hampered by its tendency to associate with membranes. We investigated the potential for large-scale downstream processing by evaluating different combinations of plant-based expression systems and engineered forms of GAD65 in terms of yield, subcellular localization and solubility in detergent-free buffer. We found that a modified version of GAD65 lacking the first 87 amino acids accumulates to high levels in the cytosol and can be extracted in detergent-free buffer. The highest yields of this variant protein were achieved using the MagnICON transient expression system. This combination of truncated GAD65 and the MagnICON system dramatically boosts the production of the recombinant protein and helps to optimize downstream processing for the establishment of a sustainable plant-based production platform for an autoimmune diabetes vaccine candidate.

Purification of L-glutamate decarboxylase by affinity chromatography.

L-Glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from rat brain synaptosomal extract was partially purified by affinity chromatography. On further purification by DEAE-Sephadex A 50 and Sephadex G-200, L-glutamate decarboxylase was purified to greater extent. It was found that a single affinity chromatography by appropriate elution gave a highly purified protein giving a single band of high specific activity on polyacrylamide gradient gel slab electrophoresis with minimal contamination. Substrate specificity of the purified enzyme was modified in the presence of 6-azauracil or phenylalanine resulting in decreased specificity to L-glutamate and increased specificity to L-aspartate.

Associations of anti-GAD antibodies with islet cell antibodies and insulin autoantibodies in first-degree relatives of type I diabetic patients.

Sera from 114 first-degree relatives of insulin-dependent diabetes mellitus (type I diabetes) patients and 81 healthy individuals living in Germany were analyzed for antibodies to rat brain glutamic acid decarboxylase (GAD-ab) using an immunoprecipitation assay. The determination of GAD-ab in the 81 islet cell antibody (ICA) and insulin autoantibody (IAA) negative healthy individuals established a normal range (mean +/- 2 SD); 2 healthy individuals (2.5%) possessed GAD-ab levels above this range, but became negative on follow-up. None of 86 ICA-/IAA- first-degree relatives had GAD-ab; whereas, 42.9% of 28 ICA+ and/or IAA+ relatives were positive for GAD-ab. Presence of GAD-ab was negatively correlated with IAA (P = 0.02) and positively with ICA (P = 0.0006). Follow-up samples were obtained from 25 of 28 ICA+ and/or IAA+ relatives with a mean (+/- SD) follow-up period of 20.6 +/- 12.1 months. In these 25 relatives, GAD-ab were positive in 70% ICA+/IAA-, 0% ICA-/IAA+, and 57.1% ICA+/IAA+ relatives in the first sample and in 57.1% ICA+/IAA-, 0% ICA-/IAA+, and 70% ICA+/IAA+ relatives in the last sample. GAD-ab, once detected, persisted in 9 of 11 GAD-ab+ relatives. Of the relatives, 2 converted to GAD-positivity, concomitant with the appearance of ICA, and 2 others lost GAD-ab during follow-up. Of the 28 ICA+ and/or IAA+ relatives, 6 progressed to overt type I diabetes on follow-up, and GAD-ab were detectable in 4 of these relatives.(ABSTRACT TRUNCATED AT 250 WORDS)

GAD autoantibodies in IDDM, stiff-man syndrome, and autoimmune polyendocrine syndrome type I recognize different epitopes.

Glutamic acid decarboxylase (GAD) is a major islet cell autoantigen in insulin-dependent diabetes mellitus (IDDM), and autoantibodies are found in high frequencies in patients with recent-onset IDDM, stiff-man syndrome (SMS), and autoimmune polyendocrine syndrome type I (APS I). Antigens in autoimmune disorders are often enzymes, and autoantibody binding frequently inhibit their activity. In this study, we examined the reactivity of anti-GAD-containing sera from 7 patients with IDDM, 4 patients with SMS, and 5 patients with APS I. All sera immunoprecipitated GAD from [35S]methionine-labeled rat islet lysates and the sera from patients with SMS and APS I, but none of the IDDM patients' sera, identified the GAD protein in Western blots. Two of four SMS patients' sera and 5 of 5 APS I patients' sera, in contrast to 0 of 7 IDDM patients' sera, inhibited the enzymatic activity of GAD. When the various sera were tested with the GAD65 and GAD67 isoforms, produced separately by transient expression in COS cells, the enzymatic activity of GAD65 was inhibited by sera from patients with SMS and APS I, whereas no effect on the GAD67 activity was observed. Taken together, the results demonstrate that the GAD autoantibodies in these three disorders display marked differences in epitope recognition and indicate that, during the development of the diseases, the autoantigen is being presented to the immune system through separate pathogenetic mechanisms.