Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
PLoS One. 2011 Apr 8;6(4):e18411. doi: 10.1371/journal.pone.0018411.
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).
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.
谷氨酸脱羧酶 65（anti-GAD65）自身抗体存在于 70-80%的 1 型糖尿病（T1D）患者血清中，但结构相似的 67 kDa 同工型 GAD67 抗体则很少见。GAD67 抗体可能代表了抗 GAD65 的交叉反应群体，但这尚未经过正式测试。
方法/主要发现：在这项研究中，我们检测了含有抗 GAD65 的糖尿病患者血清中抗 GAD67 的频率、水平和亲和力，并比较了 GAD65 和 GAD67 反应的特异性。使用（125）I 标记的重组 GAD65 和 GAD67 通过放射免疫沉淀（RIP）测量抗 GAD65 和抗 GAD67。对于每种抗体群体，通过在同源和异源抑制测定中用 100 倍过量的未标记 GAD 孵育来测量结合的特异性，并在选定的血清中测量与 GAD65 和 GAD67 的结合亲和力。还通过免疫印迹法测试了血清对 GAD65 和 GAD67 的反应性。在含有抗 GAD65 的 85 份血清中，有 28 份通过 RIP 检测到抗 GAD67。用未标记的 GAD65 抑制可显著或完全降低与（125）I GAD65 和（125）I GAD67 的抗体反应性。相比之下，未标记的 GAD67 降低了与（125）I GAD67 的自身抗体反应性，但不降低与（125）I GAD65 的反应性。这两种抗体群体的亲和力都很高（>10(10) l/mol）。
我们的研究结果表明，抗 GAD67 自身抗体代表了抗 GAD65 的一个小群体，该群体与在 GAD67 上也发现的一个交叉反应表位反应。实验结果证实 GAD65 是 T1D 的主要自身抗原，而 GAD67 本身的免疫原性非常低。我们根据 GAD 同工型结构之间的已知相似性，特别是可由表位扩展诱导的一个次要交叉反应表位的位置，讨论了我们的发现。
