Diagnostic sensitivity of immunodominant epitopes of glutamic acid decarboxylase (GAD65) autoantibodies in childhood IDDM

Abstract

The prevalence and titre of epitope-specific autoantibodies to glutamic acid decarboxylase (GAD65) in 155 insulin-dependent diabetic (IDDM) and 9 GAD65 antibody (Ab)-positive healthy children were determined using four GAD65/67 chimaeric molecules which discriminate among the N-terminal (N), middle (M) and C-terminal (C) epitopes of GAD65. Radioligand binding assays for IgGAb used immunoprecipitation of in vitro translated 35S-GAD. We found autoantibodies to GAD65 in 116 of 155 (75%), to GAD67 in 19 of 155 (12%) (p < 0.0001) and to the GAD65-N-67 chimaera in 25 of 155 (16%) (p < 0.0001) IDDM sera. GAD67Ab were found almost exclusively (17 of 19, 89%) in GAD65Ab-positive sera and the levels of GAD67Ab correlated with those of GAD65Ab (r2 = 0.5913; p = 0.009). GAD65Ab directed to GAD65-M were found in 104 of 155 (67%), to GAD65-C in 104 of 155 (67%) and to GAD65-M + C in 116 of 155 (75%) of IDDM sera, and indicated reactivity to at least two distinct epitopes. Among the nine GAD65Ab-positive healthy children, two (22%) were also positive with GAD67, nine (100%) with GAD65-M + C, seven (78%) with GAD65-M, eight (89%) with GAD65-C and two (22%) with GAD65-N-67. Titres of GAD65Ab (p = 0.007), GAD65-C-Ab (p = 0.002) and GAD65-C + M-Ab (p = 0.003), but not of GAD65-M-Ab (p = 0.101) were significantly higher in IDDM than in healthy children. We conclude that GAD65Ab in IDDM and healthy children are directed to middle and C-terminal epitopes, and propose that levels of antibodies specifically directed to the carboxy-terminal end of GAD65 may distinguish IDDM from healthy children.

Antigen presentation of detergent-free glutamate decarboxylase (GAD65) is affected by human serum albumin as carrier protein

“…Our observation that the N-terminal specific T35 (115-127) cells showed the lowest response to this antigen preparation, while the middle region T33.1 (274-286) cells showed the highest response supports our speculation that the processing and presentation of the N- terminus of GAD65 may be impaired by HSA. It is noteworthy that in T1D GAD65Ab to the N-terminal region are particularly rare [31][32][33], which may be due to its binding with hydrophobic carrier proteins in circulation.…”

“…Our observation that the N-terminal specific T35 (115-127) cells showed the lowest response to this antigen preparation, while the middle region T33.1 (274-286) cells showed the highest response supports our speculation that the processing and presentation of the N- terminus of GAD65 may be impaired by HSA. It is noteworthy that in T1D GAD65Ab to the N-terminal region are particularly rare [31][32][33], which may be due to its binding with hydrophobic carrier proteins in circulation.In conclusion, our study of GAD65 complexed with HSA suggests that antigen processing and presentation by Priess cells may differ between preparations that vary in HSA concentration and the subsequent response by epitope-specific T cell clones.Possible implications of these findings on antigen processing and presentation for in vitro studies and for the T1D-associated pathogenesis need to be determined. …”

Conformation-dependent GAD65 autoantibodies in diabetes

“…Several approaches have been used to identify GAD65Ab disease-specific epitopes, including deletion constructs [14], chimeric GAD65/67 molecules [15,16] and naturally occurring GAD65 isoforms [17]. The majority of data suggest that conformation-dependent epitopes are found at the middle and at the C-terminal end of GAD65 [12,15,16,18]. We previously demonstrated that GAD65Ab that bind to the C-terminal end of GAD65 distinguished children with Type 1 diabetes from healthy children [18].…”

“…The majority of data suggest that conformation-dependent epitopes are found at the middle and at the C-terminal end of GAD65 [12,15,16,18]. We previously demonstrated that GAD65Ab that bind to the C-terminal end of GAD65 distinguished children with Type 1 diabetes from healthy children [18]. Similarly, the presence of GAD65Ab that bind to C-terminal epitopes in patients with latent autoimmune diabetes in adults (LADA) identified a subgroup of individuals with low BMI, low basal C-peptide values and an earlier requirement for insulin therapy [16].…”

“…We studied sera from six groups of GAD65Ab-positive individuals. Sera that reacted with both GAD65 and GAD67 were not included, since they represent less than 15 to 20% of sera from newly diagnosed Type 1 diabetes patients [18]. The groups included newly diagnosed Type 1 diabetes patients (n=85) and patients with LADA (n=24) who were registered in the Diabetes Incidence Study in Sweden [24,25].…”