Characterization of continuous B-cell epitopes in the N-terminus of glutamate decarboxylase67 using monoclonal antibodies
Characterization of continuous B-cell epitopes in the N-terminus of glutamate decarboxylase67 using monoclonal antibodies
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DOI：
10.1002/psc.2703
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发表时间：
2014-12-01
影响因子：
2.1
通讯作者：
Trier, Nicole Hartwig
中科院分区：
文献类型：
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作者：
Agca, Selin;Houen, Gunnar;Trier, Nicole Hartwig
谷氨酸脱羧酶（GAD）是一种与自身免疫性疾病1型糖尿病（T1D）和僵硬人综合征（SPS）相关的自身抗原。该蛋白是一种参与产生抑制性神经递质-氨基丁酸的必需酶，存在于GAD67和GAD65两种亚型中。在SPS和T1D患者中，这两种亚型都可能被自身抗体靶向，尽管SPS主要与GAD67自身抗体的存在相关，而T1D主要与GAD65自身抗体的存在相关。在这项研究中，我们通过修饰肽酶联免疫吸附法描述了抗体对覆盖完整蛋白序列的重叠GAD67肽的反应性，以便使用两种单克隆抗体（mab）鉴定潜在的GAD67表位。这两种GAD67单抗均对位于GAD67 n端的线性表位具有反应性。GAD mAb 1和2的表位分别鉴定为氨基酸序列NAGADPNTTN和TETDFSNLF，分别对应于氨基酸14-23和91-99。对表位的精细定位表明，抗体的反应性与氨基酸侧链的功能有关，而不是氨基酸侧链的特异性。此外，结果表明，表位结构中的非接触氨基酸对抗体的反应性至关重要。这些氨基酸的确切作用仍有待确定，但它们被认为与主氢键或稳定表位结构有关。由于对GAD67抗原区域的了解有限，因此该研究有助于GAD67表位的表征，并可能是开发基于肽的SPS治疗方法的第一步。版权所有(c) 2014欧洲多肽协会和约翰威利父子有限公司
Glutamate decarboxylase (GAD) is an autoantigen associated with the autoimmune disorders Type-1 diabetes (T1D) and stiff-person syndrome (SPS). The protein, being an essential enzyme involved in the production of the inhibitory neurotransmitter -aminobutyric acid, exists in two isoforms, GAD67 and GAD65. Both isoforms may be targeted by autoantibodies in SPS and T1D patients, although SPS primarily is associated with the presence of GAD67 autoantibodies, whereas T1D mainly is associated with the presence of GAD65 autoantibodies. In this study, we describe antibody reactivity to overlapping GAD67 peptides covering the complete protein sequence by modified peptide enzyme-linked immunosorbent assay in order to identify potential GAD67 epitopes using two monoclonal antibodies (mAbs). Both GAD67 mAbs showed reactivity to linear epitopes located at the N-terminal end of GAD67. The epitopes of GAD mAb 1 and 2 were identified as the amino acid sequences NAGADPNTTN and TETDFSNLF, respectively, corresponding to amino acids 14-23 and 91-99. Fine mapping of the epitopes revealed that antibody reactivity was related to amino acid side-chain functionality, rather than amino acid side-chain specificity. Additionally, results suggested that non-contact amino acids in the epitope structure were essential for antibody reactivity. The exact role of these amino acids remains to be determined, but they are thought to be involved in backbone hydrogen bonds or stabilization of the epitope structure. As only limited knowledge is available in relation to antigenic regions of GAD67, this study contributes to characterization of GAD67 epitopes and may be a first step in the development of peptide-based therapeutics against SPS. Copyright (c) 2014 European Peptide Society and John Wiley & Sons, Ltd.