GAD Autoantibodies in IDDM, Stiff-Man Syndrome, and Autoimmune Polyendocrine Syndrome Type I Recognize Different Epitopes

Abstract

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.

Influence of Sex and Age at Onset on Autoantibodies against Insulin, GAD₆₅ and IA2 in Recent Onset Type 1 Diabetic Patients

“…Neither GAD 65 nor IA2 is restricted to the pancreatic β cell (in contrast to insulin production) and an increased autoimmune response against more ubiquitous antigens could reflect a more generalized autoimmune process occurring in women. In fact, GADA is also found in patients with other endocrine disorders such as Graves’ disease or autoimmune polyendocrine syndrome, both more prevalent in women than in men; also, prevalence of GADA is higher in diabetic patients with other forms of autoimmunity [16, 17, 18, 19]. Moreover, the difference in frequency of GADA between women and men is most evident in the 11- to 20-year-old group, suggesting a possible implication of female hormones in postpuberal autoimmune processes.…”

Stiff-person syndrome (SPS) and anti-GAD-related CNS degenerations: Protean additions to the autoimmune central neuropathies

“…In SPS levels of anti-GAD in serum are usually highly raised, are reactive with GAD65 but almost equally so with GAD67 [7,37], can be detected by immunoblotting using purified GAD from rat brain [38], are inhibitory for the enzymatic activity of GAD [7], and react by immunofluorescence (IFL) with cells in pancreatic islets, whether or not there is coexisting diabetes, but also on sections from different regions of mammalian brain [39,40] (Fig. 2).…”

“…In a provocative study, Viannelo et al [41] used IFL on cultured hippocampal neurons, on which GABAergic synapses are well represented, and staining patterns differed according to different neurological disorders, SPS, ataxia or epilepsy; the tentative suggestion was that differences in GAD epitope recognition differentiated clinical phenotypes. In T1D on the other hand anti-GAD are usually at low to moderate levels and mostly specific for GAD65, react predominantly with highly conformational epitopes and so are rarely detected by immunoblotting [39,40,42], seldom inhibitory for the enzymatic activity of GAD [43] and, although reactive with pancreatic islets, do not react by IFL with brain [38]. However since this latter statement is based on early (1990s) reports and on [22,23] an unpublished communication to us from a neuroimmunology diagnostic laboratory, a systematic study is needed of the reactivity with brain of anti-GAD in classical T1D.…”

Autoantibodies in Diabetes

“…Titers of GADA are considerably higher in stiff-person syndrome than in type 1 diabetes [78]. Furthermore, there are three regions of the GAD65 molecule that are preferentially targeted by autoantibodies in stiff-person syndrome but rarely in type 1 diabetes [78][79][80]. These include a linear epitope between amino acids 1 and 16 at the amino terminus of the molecule and two conformational epitopes between residues 188 and 442 and 442 and 563.…”