Arafat Yasir, Fenalti Gustavo, Whisstock James C, Mackay Ian R, Garcia de la Banda Maria, Rowley Merrill J, Buckle Ashley M
The Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, VIC 3800, Australia.
Mol Immunol. 2009 Dec;47(2-3):493-505. doi: 10.1016/j.molimm.2009.08.022. Epub 2009 Sep 23.
Our aim was to ascertain structural determinants of autoantigenicity based on the model of the diabetes autoantigen glutamic acid decarboxylase 65 kDa isoform (GAD65) in comparison with that of the non-autoantigenic isoform GAD67. This difference exists despite the two isoforms having the same fold and high sequence identity. Autoantibodies to GAD65 precede the development of type 1 diabetes and are clinical markers of this and certain neural autoimmune diseases. To date, epitope mapping has been based on particular amino acid differences between the two isoforms, and there is no explanation as to why autoantibodies that react with GAD65 only infrequently cross-react with GAD67. To characterize each isoform of the enzyme and gain insights into their contrasting autoantigenic properties, we have used the recently determined crystal structures of GAD65 and GAD67 to compare their structure, hydrophobicity, electrostatics, flexibility and physiochemical properties. The results revealed striking differences which appear almost exclusively at the C-terminal domain of the isoforms. Whereas GAD65 displayed a highly charged and flexible C-terminal domain containing numerous patches of high electrostatic and solvation energies, these characteristics were absent in the GAD67 molecule. Additionally, analysis indicated potential N-terminal and PLP domain binding sites surrounding the C-terminal domain of GAD65, a major region of autoantigenic activity, but not of GAD67. These features agree with good accuracy with published epitope-mapping data. Our analysis suggests that the high flexibility and charge of GAD65 in the C-terminal domain is coupled with the mobility of its catalytic loop, a property that is absolutely required for its enzymatic function. Thus, the structural features that distinguish GAD65 from GAD67 as a B cell autoantigen are related to functional requirements for its enzymatic mechanism. This could well apply to the various other enzyme autoantigens and, if so, these features could be used as the basis of future predictive strategies.
我们的目标是基于糖尿病自身抗原谷氨酸脱羧酶65 kDa亚型（GAD65）的模型，与非自身抗原性亚型GAD67相比较，确定自身抗原性的结构决定因素。尽管这两种亚型具有相同的折叠结构和高度的序列同一性，但这种差异仍然存在。针对GAD65的自身抗体在1型糖尿病发病之前就已出现，并且是这种疾病以及某些神经自身免疫性疾病的临床标志物。迄今为止，表位作图一直基于两种亚型之间特定的氨基酸差异，并且对于为什么与GAD65反应的自身抗体很少与GAD67发生交叉反应尚无解释。为了表征该酶的每种亚型并深入了解它们截然不同的自身抗原特性，我们利用最近确定的GAD65和GAD67的晶体结构来比较它们的结构、疏水性、静电学、柔韧性和物理化学性质。结果揭示了显著差异，这些差异几乎完全出现在亚型的C末端结构域。GAD65显示出一个高度带电且灵活的C末端结构域，其中包含许多具有高静电能和溶剂化能的区域，而这些特征在GAD67分子中并不存在。此外，分析表明在GAD65的C末端结构域周围存在潜在的N末端和PLP结构域结合位点，这是自身抗原活性的主要区域，但GAD67没有。这些特征与已发表的表位作图数据具有良好的准确性。我们的分析表明，GAD65在C末端结构域的高柔韧性和电荷与它催化环的流动性相关联，这是其酶功能绝对必需的特性。因此，将GAD65与GAD67区分开来作为B细胞自身抗原的结构特征与其酶促机制的功能要求有关。这很可能适用于各种其他酶自身抗原，如果是这样，这些特征可以用作未来预测策略的基础。
