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Expression of recombinant glutamic acid decarboxylase in insect larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus.

ABSTRACT: Autoimmune Diabetes Mellitus (DM) is a chronic disease caused by the selective destruction of insulin producing beta cells in human pancreas. DM is characterized by the presence of autoantibodies that bind a variety of islet-cell antigens. The 65?kDa isoform of glutamate decarboxylase (GAD65) is a major autoantigen recognized by these autoantibodies. Autoantibodies to GAD65 (GADA) are considered predictive markers of the disease when tested in combination with other specific autoantibodies. In order to produce reliable immunochemical tests for large scale screening of autoimmune DM, large amounts of properly folded GAD65 are needed. Herein, we report the production of human GAD65 using the baculovirus expression system in two species of larvae, Rachiplusia nu and Spodoptera frugiperda. GAD65 was identified at the expected molecular weight, properly expressed with high yield and purity in both larvae species and presenting appropriate enzymatic activity. The immunochemical ability of recombinant GAD65 obtained from both larvae to compete with [35S]GAD65 was assessed qualitatively by incubating GADA-positive patients' sera in the presence of 1??M of the recombinant enzyme. All sera tested became virtually negative after incubation with antigen excess. Besides, radiometric quantitative competition assays with GADA-positive patients' sera were performed by adding recombinant GAD65 (0.62 nM-1.4?µM). All dose response curves showed immunochemical identity between proteins. In addition, a bridge-ELISA for the detection of GADA was developed using S. frugiperda-GAD65. This assay proved to have 77.3% sensitivity and 98.2% of specificity. GAD65 could be expressed in insect larvae, being S. frugiperda the best choice due to its high yield and purity. The development of a cost effective immunoassay for the detection of GADA was also afforded.

SUBMITTER: Trabucchi A

PROVIDER: S-EPMC6351654 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Expression of recombinant glutamic acid decarboxylase in insect larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus.

Trabucchi Aldana A Bombicino Silvina S SS Targovnik Alexandra M AM Marfía Juan I JI Sabljic Adriana V AV Faccinetti Natalia I NI Guerra Luciano L LL Iacono Ruben F RF Miranda María V MV Valdez Silvina N SN

Scientific reports 20190129 1

Autoimmune Diabetes Mellitus (DM) is a chronic disease caused by the selective destruction of insulin producing beta cells in human pancreas. DM is characterized by the presence of autoantibodies that bind a variety of islet-cell antigens. The 65 kDa isoform of glutamate decarboxylase (GAD65) is a major autoantigen recognized by these autoantibodies. Autoantibodies to GAD65 (GADA) are considered predictive markers of the disease when tested in combination with other specific autoantibodies. In o ...[more]

PMID: 30696851

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Project description:ImportanceLittle is known of glutamic acid decarboxylase antibodies (GAD-abs) in the paraneoplastic context. Clinical recognition of such cases will lead to prompt tumor diagnosis and appropriate treatment.ObjectiveTo report the clinical and immunological features of patients with paraneoplastic neurological syndromes (PNS) and GAD-abs.Design, setting, and participantsRetrospective case series study and immunological investigations conducted in February 2014 in a center for autoimmune neurological disorders. Fifteen cases with GAD65-abs evaluated between 1995 and 2013 who fulfilled criteria of definite or possible PNS without concomitant onconeural antibodies were included in this study.Main outcomes and measuresAnalysis of the clinical records of 15 patients and review of 19 previously reported cases. Indirect immunofluorescence with rat hippocampal neuronal cultures and cell-based assays with known neuronal cell-surface antigens were used. One hundred six patients with GAD65-abs and no cancer served as control individuals.ResultsEight of the 15 patients with cancer presented as classic paraneoplastic syndromes (5 limbic encephalitis, 1 paraneoplastic encephalomyelitis, 1 paraneoplastic cerebellar degeneration, and 1 opsoclonus-myoclonus syndrome). When compared with the 106 non-PNS cases, those with PNS were older (median age, 60 years vs 48 years; P = .03), more frequently male (60% vs 13%; P < .001), and had more often coexisting neuronal cell-surface antibodies, mainly against γ-aminobutyric acid receptors (53% vs 11%; P < .001). The tumors more frequently involved were lung (n = 6) and thymic neoplasms (n = 4). The risk for an underlying tumor was higher if the presentation was a classic PNS, if it was different from stiff-person syndrome or cerebellar ataxia (odds ratio, 10.5; 95% CI, 3.2-34.5), or if the patient had coexisting neuronal cell-surface antibodies (odds ratio, 6.8; 95% CI, 1.1-40.5). Compared with the current series, the 19 previously reported cases had more frequent stiff-person syndrome (74% vs 13%; P = .001) and better responses to treatment (79% vs 27%; P = .005). Predictors of improvement in the 34 patients (current and previously reported) included presentation with stiff-person syndrome and the presence of a thymic tumor.Conclusions and relevancePatients with GAD-abs must be screened for an underlying cancer if they have clinical presentations different from those typically associated with this autoimmunity or develop classic PNS. The risk for cancer increases with age, male sex, and the presence of coexisting neuronal cell-surface antibodies.

Project description:γ-Aminobutyric acid (GABA) is a kind of non-proteinogenic amino acid which is highly soluble in water and widely used in the food and pharmaceutical industries. Enzymatic conversion is an efficient method to produce GABA, whereby glutamic acid decarboxylase (GAD) is the key enzyme that catalyzes the process. The activity of wild-type GAD is usually limited by temperature, pH or biotin concentration, and hence directional modification is applied to improve its catalytic properties and practical application. GABA was produced using whole cell transformation of the recombinant strains Escherichia coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively. The results showed that Gad B-T62S and Gad B-Q309A are two effective mutation sites. These findings may contribute to ideas for constructing potent recombinant strains for GABA production. Practical Application : Enzymatic properties of the GAD from Escherichia coli and GAD site-specific mutants were examined by analyzing their conserved sequences, substrate contacts, contact between GAD amino acid residues and mutation energy (ΔΔG) of the GAD mutants. The enzyme activity and stability of Gad B-T62S and Gad B-Q309A mutants were improved compared to Gad B. The kinetic parameters Km and Vmax of Gad B, Gad B-T62S, and Gad B-Q309A mutants were 11.3 ± 2.1 mM and 32.1 ± 2.4 U/mg, 7.3 ± 2.5 mM and 76.1 ± 3.1 U/mg, and 7.2 ± 3.8 mM and 87.3 ± 1.1 U/mg, respectively. GABA was produced using whole cell transformation of the recombinant strains E. coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S, and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively.

Dataset Information

Expression of recombinant glutamic acid decarboxylase in insect larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus.

Publications

Expression of recombinant glutamic acid decarboxylase in insect larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus.

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