Studies on epitopes of glutamic acid decarboxylase 65 (GAD65) antibodies in type 1 diabetes

应用7种谷氨酸脱羧酶（GAD）65/67嵌合分子，研究了GAD 65自身抗体（GAD 65 Ab）在慢进型（胰岛素依赖型）糖尿病（SPIDDM）和急性发作型（胰岛素依赖型）糖尿病（AIDDM）中的疾病特异性表位分布。采用免疫沉淀、ELISA和Western印迹法检测日本SPIDDM患者（n = 17）和AIDDM患者（n = 46）血清中GAD 65抗体，结果表明，SPIDDM患者血清中GAD 65抗体与GAD 65 N端17-51位氨基酸的线性表位和C端443-585位氨基酸的构象表位发生特异性反应。GAD 65 Ab与N端83个残基的结合与SPIDDM患者不需要胰岛素的时间呈负相关。无论GAD 65 Ab的滴度如何，AIDDM中的GAD 65 Ab均不与位于氨基酸1-83之间的N端表位反应。17%（8/46）的AIDDM患者的GAD 65 Ab位于氨基酸244-360之间，说明SPIDDM患者GADAb具有独特的N端线性表位，位于GAD 65分子的锚定结构域。提示靶向该区域的GAD 65 Ab与SPIDDM中缓慢进行性β细胞衰竭之间存在关联。

Disease-specific epitope profiles of glutamic acid decarboxylase (GAD) 65 autoantibodies (GAD65Ab) were studied in slowly progressive type 1 (insulin-dependent) diabetes mellitus (SPIDDM) and acute onset type 1 (insulin-dependent) diabetes mellitus (AIDDM) using seven kinds of GAD65/67 chimeric molecules. Sera obtained from Japanese SPIDDM (n = 17) and AIDDM (n = 46) patients followed prospectively were analyzed by immunoprecipitation, ELISA and western blotting.GAD65Ab in all SPIDDM samples reacted specifically with an N-terminal linear epitope located on the membrane anchoring domain between amino acids 17-51 and C-terminal conformational epitope between amino acids 443-585 of GAD65. The binding of GAD65Ab with N-terminal 83 residues in SPIDDM inversely correlated with the period in which insulin was not required. GAD65Ab in AIDDM did not react with N-terminal epitope located between amino acids 1-83 irrespective of the titer of GAD65Ab. A novel epitope of GAD65Ab in AIDDM resided between amino acids 244-360 was identified in 17% (8/46) of patients whose age of onset was younger than other AIDDM patients.In conclusion, GADAb in SPIDDM has unique N-terminal linear epitopes that are located on the anchoring domain of GAD65 molecules. Association is suggested between GAD65Ab targeted to this region and slowly progressive β-cell failure in SPIDDM.