Song Y H, Connor E L, Muir A, She J X, Zorovich B, Derovanesian D, Maclaren N
Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Gainesville 32610.
J Clin Endocrinol Metab. 1994 May;78(5):1108-12. doi: 10.1210/jcem.78.5.7513715.
Patients with idiopathic Addison's disease are characterized by cytoplasmic adrenal autoantibodies, detectable by indirect immunofluorescence of cryocut sections of human adrenal cortex. Recently, autoantibodies that bind a 55-kilodalton protein in the microsomal fraction of adrenal gland extracts identified to be the cytochrome P450 enzyme 21-hydroxylase have been found in Addisonian patient sera. We confirm the finding and report here the autoantigenic epitopes involved in the autoantibody reactivity using recombinant DNA technology. Six cDNA fragments spanning different regions of the 21-hydroxylase gene were expressed as fusion proteins with glutathione S-transferase in Escherichia coli. Immunoblot analyses were used to evaluate the reactivity of the recombinant proteins with patients' sera to determine the autoepitopes involved. We found that a conserved region (amino acids 164-356) reacted with 25 of 30 adrenal autoantibody-positive sera tested. One serum sample reacted only with the amino portion of the 21-hydroxylase (amino acids 1-162). In addition, 4 other enzymes important to steroid hormone biosynthesis, 11 beta-hydroxylase, 17 alpha-hydroxylase, side-chain cleavage enzyme P450, and 3 beta-hydroxysteroid dehydrogenase, were expressed in E. coli, but none of them gave positive autoantibody reactions by Western blot assays, even using sera from 5 patients with type I autoimmune polyglandular syndrome. The availability of recombinant antigens has permitted structural analysis of the autoepitopes involved in the autoimmune response to 21-hydroxylase in Addison's disease. Our findings should lead to the development of a simple and specific tool for immunodiagnosis of the disease.
特发性艾迪生病患者的特征是存在细胞质肾上腺自身抗体，可通过人肾上腺皮质冷冻切片的间接免疫荧光检测到。最近，在艾迪生病患者血清中发现了能与肾上腺提取物微粒体部分中一种55千道尔顿蛋白结合的自身抗体，该蛋白被鉴定为细胞色素P450酶21-羟化酶。我们证实了这一发现，并在此报告利用重组DNA技术确定自身抗体反应所涉及的自身抗原表位。跨越21-羟化酶基因不同区域的六个cDNA片段在大肠杆菌中作为与谷胱甘肽S-转移酶的融合蛋白表达。免疫印迹分析用于评估重组蛋白与患者血清的反应性，以确定所涉及的自身表位。我们发现一个保守区域（氨基酸164 - 356）与所检测的30份肾上腺自身抗体阳性血清中的25份发生反应。一份血清样本仅与21-羟化酶的氨基部分（氨基酸1 - 162）发生反应。此外，对类固醇激素生物合成重要的其他4种酶，11β-羟化酶、17α-羟化酶、侧链裂解酶P450和3β-羟类固醇脱氢酶，在大肠杆菌中表达，但即使使用来自5例I型自身免疫性多腺体综合征患者的血清，通过蛋白质印迹分析它们也均未产生阳性自身抗体反应。重组抗原的可得性使得对艾迪生病中针对21-羟化酶的自身免疫反应所涉及的自身表位进行结构分析成为可能。我们的发现应能促成开发一种用于该疾病免疫诊断的简单且特异的工具。
