Isolation by anion-exchange of immunologically and enzymatically active human islet glutamic acid decarboxylase 65 overexpressed in Sf9 insect cells

Abstract

The enzyme L-glutamic acid decarboxylase is a major autoantigen of the beta cell. Autoantibodies against this enzyme are observed before the onset of insulin-dependent diabetes mellitus (IDDM) in man and may be of predictive value. There is evidence that this enzyme is involved in the development of autoimmune diabetes in animals. In order to facilitate the investigation of the role of L-glutamine acid decarboxylase in IDDM, we expressed the 65 kDa isoform of human islet L-glutamic acid decarboxylase in insect cells using a baculovirus-based vector. The material was expressed at high levels (up to 50 mg/l of cells). Partially purified metabolically labelled L-glutamic acid decarboxylase bound to immunoglobulins in the sera from 20 of 49 subjects with newly-diagnosed IDDM. The enzyme was isolated in high yields (up to 26 mg/l cell culture) with fully maintained enzymatic activity by either ion-exchange chromatography or immunoaffinity chromatography. Purified L-glutamic acid decarboxylase inhibited the binding of radioactive L-glutamic acid decarboxylase, prepared by in vitro translation of mRNA, to immunoglobulins in the sera of subjects with IDDM. Recombinant human islet L-glutamic acid decarboxylase, isolated from Sf9 cells, is a suitable material for the large scale investigation of the utility of this enzyme in the prediction and prevention of autoimmune diabetes.

Two amino acids in glutamic acid decarboxylase act in concert for maintainance of conformational determinants recognised by Type I diabetic autoantibodies

“…All mutant GAD 65 recombinant proteins co-migrated in SDS polyacrylamide gels with the control, wild-type GAD 65 expressed in insect cells (not shown). None of the mutants showed decarboxylase enzymatic activity; replacement of residues A, B, C and D in the mutant with those present in wild-type GAD 65 led to restoration of decarboxylase activity, which matched the control GAD 65 expressed in insect cells [37] (not shown).…”

Maturation of the humoral autoimmune response to epitopes of GAD in preclinical childhood type 1 diabetes.

“…In sera with reactivity to GAD67, the reactivity to the GAD65 portion of the chimeric constructs was verified by competition with unlabeled GAD67. Competition experiments in the study were performed by the addition of either purified baculovirus human GAD65 (1 µg), produced and purified in our laboratory in a manner similar to previously described procedures (19), or 15 µl of an in vitro translated unlabeled GAD67 reaction to 2 µl of serum for 1 h before performing the radiobinding assay. Statistical analysis.…”

Regulation of GAD expression in rat pancreatic islets and brain by γ-vinyl-GABA and glucose

“…The enzymatic activity of GAD was assayed as previously described [14,17] by measuring the conversion of L-(1-14 C)glutamic acid (Amersham Amersham, UK) to 14 CO 2 . One enzyme unit is defined as 1 mmol of product formed per minute at 37 C. The actual level of GAD enzyme activity for islets incubated in 5 mmol/l glucose was found to be 18±22 mU/islet equivalent in accordance with previous reports.…”

“…The GAD65 and GAD67 monoclonal antibodies (mAbs), named EJ65 and EJ67 respectively, were produced by immunizing RBF mice [13] with purified recombinant human islet GAD65 [14] or partially purified rat islet GAD67 (Moody et al, unpublished). Over a period of 2 months each animal received three subcutaneous injections of antigen in Freunds adjuvant (the first complete, the last two incomplete).…”