An analysis of the cross-reactivity of autoantibodies to GAD65 and GAD67 in diabetes

Abstract

Background: Autoantibodies to GAD65 (anti-GAD65) are present in the sera of 70-80% of patients with type 1 diabetes (T1D), but antibodies to the structurally similar 67 kDa isoform GAD67 are rare. Antibodies to GAD67 may represent a cross-reactive population of anti-GAD65, but this has not been formally tested.

Methodology/principal findings: In this study we examined the frequency, levels and affinity of anti-GAD67 in diabetes sera that contained anti-GAD65, and compared the specificity of GAD65 and GAD67 reactivity. Anti-GAD65 and anti-GAD67 were measured by radioimmunoprecipitation (RIP) using (125)I labeled recombinant GAD65 and GAD67. For each antibody population, the specificity of the binding was measured by incubation with 100-fold excess of unlabeled GAD in homologous and heterologous inhibition assays, and the affinity of binding with GAD65 and GAD67 was measured in selected sera. Sera were also tested for reactivity to GAD65 and GAD67 by immunoblotting. Of the 85 sera that contained antibodies to GAD65, 28 contained anti-GAD67 measured by RIP. Inhibition with unlabeled GAD65 substantially or completely reduced antibody reactivity with both (125)I GAD65 and with (125)I GAD67. In contrast, unlabeled GAD67 reduced autoantibody reactivity with (125)I GAD67 but not with (125)I GAD65. Both populations of antibodies were of high affinity (>10(10) l/mol).

Conclusions: Our findings show that autoantibodies to GAD67 represent a minor population of anti-GAD65 that are reactive with a cross-reactive epitope found also on GAD67. Experimental results confirm that GAD65 is the major autoantigen in T1D, and that GAD67 per se has very low immunogenicity. We discuss our findings in light of the known similarities between the structures of the GAD isoforms, in particular the location of a minor cross-reactive epitope that could be induced by epitope spreading.

Figure 1. Antibodies to GAD65 and GAD67 detected by RIP.

(a) Comparison of antibody reactivity to GAD65 (x-axis) and to GAD67 (y-axis), measured as the percent of the total counts of radioactivity precipitated by each serum (%Total counts) in 85 sera that contain anti-GAD65. The horizontal dotted line indicates the upper limit of normal for anti-GAD67 and the vertical dotted line indicates the upper limit of normal for anti-GAD65, based on mean+3SD of reactivity of 10 normal controls. None of the normal controls showed any reactivity beyond these levels. (b) Titration curves for anti-GAD65 and anti-GAD67 in three representative sera that contain high levels of both anti-GAD65 and anti-GAD67. , (□) and (▴) represent three different sera samples.

Figure 2. Competition assays to measure cross-reactivity of anti-GAD65 (a) and anti-GAD67 (b).

Results, shown as mean and range of duplicates, of inhibition assay for three representative sera inhibited with GAD65 and GAD67. Reactivity with ¹²⁵I-GAD65 or ¹²⁵I-GAD67 (dark grey bars) was inhibited with 100-fold excess of the same GAD (homologous inhibition, black bars). In contrast, unlabelled GAD65 substantially or completely inhibited anti-GAD67, although unlabelled GAD67 did not inhibit anti-GAD65 reactivity (heterologous inhibition, light grey bars).