Cram D S, Faulkner-Jones B, Kun J, Harrison L C
Burnet Clinical Research Unit, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
Endocrinology. 1995 Mar;136(3):1111-9. doi: 10.1210/endo.136.3.7532577.
Glutamic acid decarboxylase (GAD), a target of both autoantibodies and autoreactive T-cells in insulin-dependent diabetes (IDD), exists as two homologous forms, GAD65 and GAD67. GAD65 is preferentially expressed in human islets and recognized by autoantibodies in IDD, but which form primarily elicits GAD autoimmunity is unknown. GAD67 gene expression in human islets has been demonstrated only by the polymerase chain reaction. We, therefore, quantitatively compared the expression of each GAD gene in human islets and mapped the binding of autoantibodies to recombinant human GAD67 by enzyme-linked immunosorbent assay. In ribonuclease protection assays, both forms of GAD messenger RNA (mRNA) were detected in human islets, although GAD65 mRNA was 200 times more abundant than GAD67 mRNA. Immunoblotting of islets with GAD form-specific antisera revealed GAD65, but not GAD67. By in situ hybridization and immunohistochemistry, GAD65 mRNA and protein were localized to islets, predominantly, but not entirely, to beta-cells; GAD67 mRNA and protein were undetectable. Thus, although GAD67 protein expression was undetectable in human islets, the GAD67 gene is transcribed, albeit weakly. Antibodies that recognized multiple epitopes in recombinant GAD67 were found in 20% of sera from ICA positive "at risk" first degree relatives of IDD subjects and recent-onset IDD subjects. The majority of GAD67 epitopes were mapped within the mid- and C-terminal thirds of the protein, a region that is highly conserved in GAD65. Although GAD67 may share cross-reactive epitopes with GAD65, these findings do not exclude the possibility that autoimmunity to GAD arises as a consequence of the aberrant up-regulation of GAD67 in human islets.
谷氨酸脱羧酶（GAD）是胰岛素依赖型糖尿病（IDD）中自身抗体和自身反应性T细胞的靶标，它以两种同源形式存在，即GAD65和GAD67。GAD65在人胰岛中优先表达，并在IDD中被自身抗体识别，但主要引发GAD自身免疫的是哪种形式尚不清楚。人胰岛中GAD67基因的表达仅通过聚合酶链反应得到证实。因此，我们通过酶联免疫吸附测定法定量比较了人胰岛中每个GAD基因的表达，并绘制了自身抗体与重组人GAD67的结合图谱。在核糖核酸酶保护试验中，在人胰岛中检测到了两种形式的GAD信使核糖核酸（mRNA），尽管GAD65 mRNA的丰度比GAD67 mRNA高200倍。用GAD形式特异性抗血清对胰岛进行免疫印迹分析显示有GAD65，但没有GAD67。通过原位杂交和免疫组织化学，GAD65 mRNA和蛋白定位于胰岛，主要但并非完全定位于β细胞；未检测到GAD67 mRNA和蛋白。因此，尽管在人胰岛中未检测到GAD67蛋白表达，但GAD67基因被转录，尽管转录较弱。在IDD患者的ICA阳性“高危”一级亲属和近期发病的IDD患者的20%血清中发现了识别重组GAD67中多个表位的抗体。大多数GAD67表位定位于该蛋白的中部和C端三分之一区域，该区域在GAD65中高度保守。尽管GAD67可能与GAD65共享交叉反应性表位，但这些发现并不排除人胰岛中GAD67异常上调导致GAD自身免疫的可能性。
