Preparation of magnetic polyacrylamide hydrogel with chitosan for immobilization of glutamate decarboxylase to produce γ-aminobutyric acid.

Gamma-aminobutyric acid (GABA) is an vital neurotransmitter, and the reaction to obtain GABA through biocatalysis requires coenzymes, which are therefore limited in the production of GABA. In this study, polyacrylamide hydrogels doped with chitosan and waste toner were synthesized for glutamate decarboxylase (GAD) and coenzyme co-immobilization to realize the production of GABA and the recovery of coenzymes. Enzymatic properties of immobilized GAD were discussed. The immobilized enzymes have significantly improved pH and temperature tolerance compared to free enzymes. In terms of reusability, after 10 repeated reuses of the immobilized GAD, the residual enzyme activity of immobilized GAD still retains 100% of the initial enzyme activity, and the immobilized coenzyme can also be kept at about 32%, with better stability and reusability. And under the control of no exogenous pH, immobilized GAD showed good performance in producing GABA. Therefore, in many ways, the new composite hydrogel provides another way for the utilization of waste toner and promises the possibility of industrial production of GABA.

Autoantibodies directed against glutamate decarboxylase interfere with glucose-stimulated insulin secretion in dispersed rat islets.

Islet autoantibodies, including autoantibodies directed against the 65kDa isoform of glutamate decarboxylase (GAD65Ab), are present in the majority of patients with newly diagnosed type 1 diabetes (T1D). Whereas these autoantibodies are historically viewed as an epiphenomenon of the autoimmune response with no significant pathogenic function, we consider in this study the possibility that they impact the major islet function, namely glucose-stimulated insulin secretion. Two human monoclonal GAD65Ab (GAD65 mAb) (b78 and b96.11) were investigated for uptake by live rat beta cells, subcellular localization and their effect on glucose-stimulated insulin secretion. The GAD65 mAbs were internalized by live pancreatic beta cells, where they localized to subcellular structures in an epitope-specific manner. Importantly, GAD65 mAb b78 inhibited, while GAD65 mAb b96.11 enhanced, glucose-stimulated insulin secretion (GSIS). These opposite effects on GSIS rule out non-specific effects of the antibodies and suggest that internalization of the antibody leads to epitope-specific interaction with intracellular machinery regulating insulin granule release. The most likely explanation for the alteration of GSIS by GAD65 Abs is via changes in GABA release due to inhibition or change in GAD65 enzyme activity. This is the first report indicating an active role of GAD65Ab in the pathogenesis of T1D.

Characterization and mutagenesis of a novel Mycobacterium smegmatis-derived glutamate decarboxylase active at neutral pH.

γ-aminobutyric acid (GABA) has various physiological functions and is widely used in medicine, food, and other fields. Glutamate decarboxylase (GAD) is a key enzyme that catalyzes the decarboxylation of L-glutamate to synthesize GABA. However, the industrial application of microorganism-derived GAD is limited by its rapid loss of enzymatic activity with pH approaching neutrality. In this study, a novel glutamate decarboxylase, GADMSM, from Mycobacterium smegmatis was overexpressed and purified. On the basis of homologous modeling and substrate molecular docking, several GADMSM mutants were constructed, and their enzymatic properties were analyzed. The results showed that the optimal pH of wild-type GADMSM is 5.4; at pH 6.2, 22.8% enzymatic activity was retained. The T211I replacement in GAD and C-terminal deletion mutant GADMSMΔC showed relatively high catalytic activity in a pH range of 5.0-7.0. The Vmax and Km values of GADMSMΔC were 14.69 and 5.70, respectively, at pH 5.5, and 9.87 and 6.17, respectively, at pH 7.0. Compared with the wild-type GAD, GADMSMΔC maintained higher affinity and enzymatic activity of the substrate, maintaining 78.5% of the highest enzymatic activity even at pH 7.0, which is the highest reported activity retention for GAD under neutral pH condition. Therefore, GADMSMΔC can be used for the transformation of high-yielding strains and industrial production of GABA.

Expression, purification, and characterization of glutamate decarboxylase from human gut-originated Lactococcus garvieae MJF010.

Human gut-originated lactic acid bacteria were cultivated, and high γ-aminobutyric acid (GABA)-producing Lactococcus garvieae MJF010 was identified. To date, despite the importance of GABA, no studies have investigated GABA-producing Lactococcus species, except for Lc. lactis. A recombinant glutamate decarboxylase of the strain MJF010 (rLgGad) was successfully expressed in Escherichia coli BL21(DE3) with a size of 53.9 kDa. rLgGad could produce GABA, which was verified using the silylation-derivative fragment ions of GABA. The purified rLgGad showed the highest GABA-producing activity at 35 °C and pH 5. rLgGad showed a melting temperature of 43.84 °C. At 30 °C, more than 80% of the activity was maintained even after 7 h; however, it rapidly decreased at 50 °C. The kinetic parameters, Km, Vmax, and kcat, of rLgGad were 2.94 mM, 0.023 mM/min, and 12.3 min- 1, respectively. The metal reagents of CaCl2, MgCl2, and ZnCl2 significantly had positive effects on rLgGad activity. However, most coenzymes including pyridoxal 5'-phosphate showed no significant effects on enzyme activity. In conclusion, this is the first report of Gad from Lc. garvieae species and provides important enzymatic information related to GABA biosynthesis in the Lactococcus genus.

Immobilization and enzymatic properties of glutamate decarboxylase from Enterococcus faecium by affinity adsorption on regenerated chitin.

Glutamate decarboxylase (GAD, EC 4.1.1.15) is an important enzyme in gamma-aminobutyric acid biosynthesis and DL-glutamic acid resolution. In this study, the Enterococcus faecium-derived GAD was successfully immobilized by regenerated chitin (RC) via specific adsorption of cellulose-binding domain (CBD). The optimal binding buffer was 20 mmol/L phosphate buffer saline (pH 8.0), and the RC binding capacity was 1.77 ± 0.11 mgcbd-gad/grc under this condition. The ratio of wet RC and crude enzyme solution used for immobilization was recommended to 3:50 (g/mL). To evaluate the effect of RC immobilization on GAD, properties of the immobilize GAD (RC-CBD-GAD) were investigated. Results indicated RC-CBD-GAD was relatively stable at pH 4.4-5.6 and temperature - 20-40 °C, and the optimal reaction pH value and temperature were pH 4.8 and 50 °C, respectively. When it was reacted with 5 mmol/L of follow chemical reagents respectively, the activity of RC-CBD-GAD was hardly affected by EDTA, KCl, and NaCl, and significantly inactivated by AgNO3, MnSO4, MgSO4, CuSO4, ZnSO4, FeCl2, FeCl3, AlCl3, CaCl2, and Pb(CH3COO)2. The apparent Km and Vmax were 28.35 mmol/L and 147.06 µmol/(gRC-CBD-GAD·min), respectively. The optimum time for a batch of catalytic reaction without exogenous pH control was 2 h. Under this reaction time, RC-CBD-GAD had a good reusability with a half-life of 23 cycles, indicating that it was very attractive for GABA industry. As a novel, efficient, and green CBD binding carrier, RC provides an alternative way to protein immobilization.

Engineering protonation conformation of l-aspartate-α-decarboxylase to relieve mechanism-based inactivation.

Mechanism-based inactivation of l-aspartate-α-decarboxylase (PanD), which leads to irreversible modification of active site, is a major challenge in the efficient production of ß-alanine from L-aspartic acid. In this study, a semi-rational strategy that combined conformational dynamics and structural alignment was applied to increase the catalytic stability of Bacillus subtilis PanD (BsPanD). Using site-saturation and C-terminal deletion, the variant Q5 (BsPanDI46V/I88M/K104S/I126* ) was generated. The catalytic half-life and the total turnover number (TTN) of Q5 were 3.48-fold and 2.52-fold higher, respectively, compared with that of the parent Q0. The reasons for the differences were the prolonged distance d1 between the phenolic group of Tyr58 and pyruvoyl group of Ser25 (4.9 Å in Q0 vs. 5.5 Å in Q5), an increased difficulty for incorrect protonation to occur, and the decreased flexibility of residues in regions A, B, and C, thereby enhancing the probability of correct protonation. Variant Q5, coupled with l-aspartase (AspA) in a 15-L bioreactor, generated a linear cascade system using fumaric acid as a substrate, yielding 118.6 g/L ß-alanine with a product/catalyst (P/C) ratio of 5.9 g/g and a conversion > 99%. These results showed that reshaping the protonation conformation of PanD can efficiently relieve mechanism-based inactivation and boost catalytic stability.

Placental tissue of greenhouse muskmelon (Cucumis melo L.) contains more gamma-aminobutyric acid with antioxidant capacity than the fleshed pulp.

Our previous study revealed that gamma-aminobutyric acid (GABA) in Earl's muskmelon is more concentrated in the inner than the outer parts of the fruit. Here, the GABA and antioxidant capacity of the placental tissue of muskmelon, which is considered waste, were evaluated for possible use as a source of bioactive compounds. The concentrations of GABA and related substances in the placental tissue were significantly higher than in the fleshed pulp, whereas glutamic acid and sugar levels were significantly lower. The two sites showed no difference in GAD activity. Furthermore, the placental site showed high antioxidant capacities based on 2,2-diphenyl-1-picrylhydrazyl and oxygen radical absorbance capacity for hydrophilic compounds assays compared with the fleshed pulp, because of the higher levels of total phenolic and L-ascorbic acids. Therefore, the placental tissue of muskmelons may be useful for developing functional foods, which would also reduce the amount of residues during muskmelon processing.

Development of a dual-fluorescence reporter system for high-throughput screening of L-aspartate-α-decarboxylase.

ß-Alanine (3-aminopropionic acid) holds great potential in industrial application. It can be obtained through a chemical synthesis route, which is hazardous to the environment. It is well known that l-aspartate-α-decarboxylase (ADC) can convert l-aspartate to ß-alanine in bacteria. However, due to the low activity of ADC, industrial production of ß-alanine through the green biological route remains unclear. Thus, improving the activity of ADC is critical to reduce the cost of ß-alanine production. In this study, we established a dual-fluorescence high-throughput system for efficient ADC screening. By measuring the amount of ß-alanine and the expression level of ADC using two different fluorescence markers, we can rapidly quantify the relative activity of ADC variants. From a mutagenesis library containing 2000 ADC variants, we obtained a mutant with 33% increased activity. Further analysis revealed that mutations of K43R and P103Q in ADC significantly improved the yield of ß-alanine produced by the whole-cell biocatalysis. Compared with the previous single-fluorescence method, our system can not only quantify the amount of ß-alanine but also measure the expression level of ADC with different fluorescence, making it able to effectively screen out ADC variants with improved relative activity. The dual-fluorescence high-throughput system for rapid screening of ADC provides a good strategy for industrial production of ß-alanine via the biological conversion route in the future.

Protein Engineering of a Pyridoxal-5'-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for ß-Alanine Production.

In the present study, a pyridoxal-5'-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from Tribolium castaneum (TcPanD) was selected for protein engineering to efficiently produce ß-alanine. A mutant TcPanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (m/v), 50 °C, and 7.0, respectively. The 1mM of Na+, Ni2+, Co2+, K+, and Ca2+ stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni2+ and Na+ could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate l-aspartic acid was consumed and 170.5 g/L of ß-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.

Protein-engineered molecules carrying GAD65 epitopes and targeting CD35 selectively down-modulate disease-associated human B lymphocytes.

Type 1 diabetes mellitus is an autoimmune metabolic disorder characterized by chronic hyperglycemia, the presence of autoreactive T and B cells and autoantibodies against self-antigens. A membrane-bound enzyme on the pancreatic beta-cells, glutamic acid decarboxylase 65 (GAD65), is one of the main autoantigens in type 1 diabetes. Autoantibodies against GAD65 are potentially involved in beta-cell destruction and decline of pancreatic functions. The human complement receptor type 1 (CD35) on B and T lymphocytes has a suppressive activity on these cells. We hypothesized that it may be possible to eliminate GAD65-specific B cells from type 1 diabetes patients by using chimeric molecules, containing an anti-CD35 antibody, coupled to peptides resembling GAD65 B/T epitopes. These molecules are expected to selectively bind the anti-GAD65 specific B cells by the co-cross-linking of the immunoglobulin receptor and CD35 and to deliver a suppressive signal. Two synthetic peptides derived from GAD65 protein (GAD65 epitopes) and anti-CD35 monoclonal antibody were used for the construction of two chimeras. The immunomodulatory activity of the engineered antibodies was tested in vitro using peripheral blood mononuclear cells (PBMCs) from type 1 diabetes patients. A reduction in the number of anti-GAD65 IgG antibody-secreting plasma cells and increased percentage of apoptotic B lymphocytes was observed after treatment of these PBMCs with the engineered antibodies. The constructed chimeric molecules are able to selectively modulate the activity of GAD65-specific B lymphocytes and the production of anti-GAD65 IgG autoantibodies by co-cross-linking of the inhibitory CD35 and the B cell antigen receptor (BCR). This treatment presents a possible way to alter the autoimmune nature of these cells.

Fine Mapping of Glutamate Decarboxylase 65 Epitopes Reveals Dependency on Hydrophobic Amino Acids for Specific Interactions.

Characterization of multiple antibody epitopes has revealed the necessity of specific groups of amino acid residues for reactivity. This applies to the majority of antibody-antigen interactions, where especially charged and hydrophilic amino acids have been reported to be essential for antibody reactivity. This study describes thorough characterization of glutamic acid decarboxylase (GAD) 65 antigenic epitopes, an immunodominant autoantigen in type 1 diabetes (T1D). As linear epitopes are sparsely described for GAD65 in T1D, we aimed to identify and thoroughly characterize two GAD65 antibodies using immunoassays. A monoclonal antibody recognized an epitope in the N-terminal domain of GAD65, 8FWSFGSE14, whereas a polyclonal antibody recognized two continuous epitopes in the C-terminal domain, corresponding to amino acids 514RTLED518 and 549PLGDKVNF556. Hydrophobic amino acids were essential for antibody reactivity, which was verified by competitive inhibition assays. Moreover, the epitopes were located in flexible linker regions and turn structures. These findings confirm the versatile nature of antibody-antigen interactions and describe potential continuous epitopes related to T1D, which predominantly have been proposed to be of discontinuous nature.

Lactobacillus brevis CGMCC 1306 glutamate decarboxylase: Crystal structure and functional analysis.

Glutamate decarboxylase (GAD), which is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme, can catalyze α-decarboxylation of l-glutamate (L-Glu) to γ-aminobutyrate (GABA). The crystal structure of GAD in complex with PLP from Lactobacillus brevis CGMCC 1306 was successfully solved by molecular-replacement, and refined at 2.2 Šresolution to an Rwork factor of 18.76% (Rfree = 23.08%). The coenzyme pyridoxal 5-phosphate (PLP) forms a Schiff base with the active-site residue Lys279 by continuous electron density map, which is critical for catalysis by PLP-dependent decarboxylase. Gel filtration showed that the active (pH 4.8) and inactive (pH 7.0) forms of GAD are all dimer. The residues (Ser126, Ser127, Cys168, Ile211, Ser276, His278 and Ser321) play important roles in anchoring PLP cofactor inside the active site and supporting its catalytic reactivity. The mutant T215A around the putative substrate pocket displayed an 1.6-fold improvement in catalytic efficiency (kcat/Km) compared to the wild-type enzyme (1.227 mM-1 S-1 versus 0.777 mM-1 S-1), which was the highest activity among all variants tested. The flexible loop (Tyr308-Glu312), which is positioned near the substrate-binding site, is involved in the catalytic reaction, and the conserved residue Tyr308 plays a vital role in decarboxylation of L-Glu.

Identification of mutations restricting autocatalytic activation of bacterial L-aspartate α-decarboxylase.

Bacterial L-aspartate α-decarboxylase (PanD) specifically catalyzes the decarboxylation of L-aspartic acid to ß-alanine. It is translated as an inactive pro-protein, then processed by self-cleavage to form two small subunits with catalytic activity. There is a significant difference in the efficiency of this process among the reported PanDs, while the structural basis remains unclear. More PanDs with known sequences and characterized properties are needed to shed light on the molecular basis of the self-cleavage process. In this study, PanD genes from 33 selected origins were synthesized and expressed; using purified recombinant enzymes, their self-processing properties were characterized and classified. Three classes of PanDs were acquired based on their self-cleavage efficiency. Combined with the phylogenetic analysis and structure comparison, sited-directed mutagenesis was performed to investigate the effects of four mutants on self-processing. In comparison with the wild-type (96.4%), the self-cleavage efficiencies of mutants V23E, I26C, T27A, and E56S were decreased to 90.5, 83.6, 74.4 and 81.2%, respectively. The results indicated that residues of V23, I26, T27 and E56 were critical to the self-cleavage processing of PanDs. This work provided further understanding to the self-cleavage processing of PanDs, which may contribute to protein engineering of the enzyme.

Safety and efficacy of autoantigen-specific therapy with 2 doses of alum-formulated glutamate decarboxylase in children with multiple islet autoantibodies and risk for type 1 diabetes: A randomized clinical trial.

OBJECTIVE: Treatments have failed to delay or stop the autoimmune process, preceding onset of type 1 diabetes. We investigated if autoantigen-specific treatment with alum-formulated glutamate decarboxylase (GAD-Alum) was safe and affected progression to type 1 diabetes in children with islet autoimmunity. METHODS: In an investigator-initiated, double-blind, placebo-controlled clinical trial, non-diabetic children aged 4 to 17.9 years with autoantibodies to glutamate decarboxylase (GADA) and at least one of insulinoma-associated protein 2, insulin or zinc-transporter 8, were randomized, stratified by 2 or ≥3 islet autoantibodies, to 2 injections of 20 µg GAD-Alum or placebo, 30 days apart. Main outcome was safety, investigated by adverse events, hematology, chemistry, thyroid and celiac autoimmunity and titers of islet autoantibodies, and efficacy, investigated by cumulative incidence of diabetes onset over 5-year follow-up. Secondary variables: change in first-phase insulin release (FPIR) after intravenous glucose tolerance tests, fasting, 120 minutes and Area under the curve (AUC) C-peptide and p-glucose after oral glucose tolerance tests and HbA1c. RESULTS: Fifty children (median age: 5.2) were assigned 1:1 to GAD-Alum or placebo, all receiving full treatment and included in the analyses. GAD-Alum did not affect any safety parameter, while GADA titers increased (P = .001). Time to clinical diagnosis was not affected by treatment (hazard ratio, HR = 0.77, P = .574) in the full population or in the separate stratum groups. Treatment did not affect any of the secondary variables. CONCLUSIONS: GAD-Alum as a subcutaneous prime and boost injection was safe in prediabetic young children but did not affect progression to type 1 diabetes. The safety of GAD-Alum should prove useful in future prevention studies.

Study on oligomerization of glutamate decarboxylase from Lactobacillus brevis using asymmetrical flow field-flow fractionation (AF4) with light scattering techniques.

In this work, asymmetrical flow field-flow fractionation (AF4) coupled with UV/Vis, multi-angle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI) was employed for analysis of glutamate decarboxylase (LbGadB) from Lactobacillus brevis (L. brevis). AF4 provided molecular weight (MW) (or size)-based separation of dimer, hexamer, and aggregates of LbGadB. The effect of pH on oligomerization of LbGadB was investigated, and then AF4 results were compared to those from molecular modeling. The MWs measured by AF4-UV-MALS-dRI for dimeric and hexameric forms of LbGadB were 110 and 350 kDa, respectively, which are in good agreements with those theoretically calculated (110 and 330 kDa). The molecular sizes determined by AF4-UV-MALS-dRI were also in good agreement with those obtained from molecular modeling (6 and 10 nm, respectively, for dimeric and hexameric from AF4-UV-MALS-dRI and 6.4 × 7.6 and 7.6 × 13.1 nm from molecular modeling). The effects of temperature, salt type, and salt concentration on oligomerization of LbGadB were also investigated using dynamic light scattering (DLS). It was found that the hexameric form of LbGadB was most stable at pH 6 and in presence of NaCl or KCl. The results indicate that AF4, in combination of various online detectors mentioned above, provides an effective tool for monitoring of oligomerization of LbGadB under different conditions, such as temperature, pH, type of salts, and salt concentrations.

Expression and characterization of glutamate decarboxylase from Lactobacillus brevis HYE1 isolated from kimchi.

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni-NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including Km and Vmax values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.

The Mechanism of Regulation of Pantothenate Biosynthesis by the PanD-PanZ·AcCoA Complex Reveals an Additional Mode of Action for the Antimetabolite N-Pentyl Pantothenamide (N5-Pan).

The antimetabolite pentyl pantothenamide has broad spectrum antibiotic activity but exhibits enhanced activity against Escherichia coli. The PanDZ complex has been proposed to regulate the pantothenate biosynthetic pathway in E. coli by limiting the supply of ß-alanine in response to coenzyme A concentration. We show that formation of such a complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrate that both PanZ overexpression-linked ß-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. This both demonstrates that the PanDZ complex regulates pantothenate biosynthesis in a cellular context and validates the complex as a target for antibiotic development.

Effect of simultaneous vaccination with H1N1 and GAD-alum on GAD65-induced immune response.

AIMS/HYPOTHESIS: A European Phase III trial of GAD formulated with aluminium hydroxide (GAD-alum) failed to reach its primary endpoint (preservation of stimulated C-peptide secretion from baseline to 15 months in type 1 diabetes patients), but subgroup analysis showed a clinical effect when participants from Nordic countries were excluded, raising concern as to whether the mass vaccination of the Swedish and Finnish populations with the Pandemrix influenza vaccine could have influenced the study outcomes. In the current study, we aimed to assess whether Pandemrix vaccination affects the specific immune responses induced by GAD-alum and the C-peptide response. METHODS: In this secondary analysis, we analysed data acquired from the Swedish participants in the Phase III GAD-alum trial who received subcutaneous GAD-alum vaccination (two doses, n = 43; four doses, n = 46) or placebo (n = 48). GAD autoantibodies (GADA) and H1N1 autoantibodies, GAD65-induced cytokine secretion and change in fasting and stimulated C-peptide levels from baseline to 15 months were analysed with respect to the relative time between H1N1 vaccination and the first injection of GAD-alum. RESULTS: GADA levels at 15 months were associated with the relative time between GAD-alum and Pandemrix administration in participants who received two doses of the GAD-alum vaccine (p = 0.015, r = 0.4). Both in participants treated with two doses and four doses of GAD-alum, GADA levels were higher when the relative time between vaccines was ≥210 days (p < 0.05). In the group that received two doses of GAD-alum, levels of several GAD65-induced cytokines were higher in participants who received the H1N1 vaccination and the first GAD-alum injection at least 150 days apart, and the change in fasting and stimulated C-peptide at 15 months was associated with the relative time between vaccines. Neither of these effects were observed in individuals who received four doses of GAD-alum. CONCLUSIONS/INTERPRETATION: In individuals who received two doses of GAD-alum, receiving the Pandemrix vaccine closer to the first GAD-alum injection, i.e. <150 days, seemed to affect both GAD65-induced immune response and C-peptide preservation. TRIAL REGISTRATION: ClinicalTrials.gov NCT00723411.

Biochemical and biophysical characterization of a plant calmodulin: Role of the N- and C-lobes in calcium binding, conformational change, and target interaction.

In plants, transient elevation of intracellular Ca(2+) concentration in response to abiotic stress is responsible for glutamate decarboxylase (GAD) activation via association with calmodulin (CaM), an EF-hand protein consisting of two homologous domains (N and C). An unusual 1:2 binding mode of CaM to CaM-binding domains of GAD has long been known, however the contribution of the two CaM domains in target recognition and activation remains to be clarified. Here, we explored the coupling between physicochemical properties of Arabidopsis CaM1 (AtCaM1) and Arabidopsis GAD1 activation, focusing on each AtCaM1 lobe. We found that the four EF-loops of AtCaM1 differently contribute to the ~20 µM apparent affinity for Ca(2+) and the C-lobe shows a ~6-fold higher affinity than N-lobe (Kd(app) 5.6 µM and 32 µM for C- and N-lobes, respectively). AtCaM1 responds structurally to Ca(2+) in a manner similar to vertebrate CaM based on comparison of Ca(2+)-induced changes in hydrophobicity exposure, secondary structure, and hydrodynamic behavior. Molecular dynamics simulations of AtCaM1 apo and Ca(2+)-bound reveal that the latter state is significantly less flexible, although regions of the N-lobe remain quite flexible; this suggests the importance of N-lobe for completing the transition to the extended structure of holoprotein, consistent with data from ANS fluorescence, CD spectroscopy, and SEC analysis. Moreover, enzymatic analysis reveal that mutations in the two lobes affect GAD1 activation in similar ways and only intact AtCaM1 can fully activate GAD1. Taken together, our data provide new insights into the CaM lobes role in interactions between CaM and plant GAD.

DPD epitope-specific glutamic acid decarboxylase (GAD)65 autoantibodies in children with Type 1 diabetes.

AIM: To study whether DPD epitope-specific glutamate decarboxylase autoantibodies are found more frequently in children with milder forms of Type 1 diabetes. METHODS: We prospectively evaluated 75 children with new-onset autoimmune Type 1 diabetes, in whom we collected demographic, anthropometric and clinical data and measured islet autoantibodies. Glutamate decarboxylase 65 autoantibody-positive samples were analysed for epitope specificities using recombinant Fab against the DPD-defined epitope of glutamate decarboxylase 65. RESULTS: After adjustment for age, positive DPD epitope recognition was significantly associated with higher C-peptide levels at onset (P = 0.02, r2 =0.21, n = 35), and high DPD recognition in the highest quartile tended to be associated with HbA1c ≤ 53 mmol/mol (7%) at the last follow-up [mean (sd) follow-up 1.3 (0.4) years; P = 0.07; for the model, P = 0.044, n = 30)]. Age- and sex-adjusted BMI percentile was significantly correlated with recognition of the DPD-defined epitope (P < 0.03, r2 =0.14, n = 34), but this correlation was driven by the older age group (age ≥ 10 years; P = 0.016, r2 =0.27, n = 21) and was not significant in younger children (P = 0.93, n = 13). There were no independent associations with sex, race/ethnicity, diabetic ketoacidosis, HbA1c , HLA DR3-DQ2/DR4-DQ8 or autoantibody number. CONCLUSIONS: Our findings suggest that recognition of the DPD-defined glutamate decarboxylase 65 autoantibody epitope at Type 1 diabetes onset is directly associated with ß-cell function, BMI and age, which supports the hypothesis that immunological factors contribute to the clinical heterogeneity of Type 1 diabetes. Larger studies relating epitope-specific glutamate decarboxylase 65 autoantibody to clinical phenotype in children with Type 1 diabetes are warranted.