识别α-synuclein N端结构域的单克隆抗体鉴定及免疫应用

doi:10.3969/j.issn.1006-7795.2023.06.018

首都医科大学学报 ›› 2023, Vol. 44 ›› Issue (6): 1022-1028.doi: 10.3969/j.issn.1006-7795.2023.06.018

识别α-synuclein N端结构域的单克隆抗体鉴定及免疫应用

贾焕珍¹，焦洁²，高歌^2#*，杨慧^2#*

1.首都医科大学燕京医学院中心实验室，北京 101321；2.首都医科大学基础医学院神经生物学系北京脑重大疾病研究院帕金森病研究所北京市神经再生修复重点实验室神经变性病教育部重点实验室, 北京 100069

收稿日期:2022-12-31 出版日期:2023-12-21 发布日期:2023-12-21
通讯作者: 高歌，杨慧 E-mail:gaog@ccmu.edu.cn,huiyang@ccmu.edu.cn
基金资助:
国家自然科学基金项目(81870994)。

Identification the monoclonal antibodies recognizing the α-synuclein N-terminal domain and their application in immunotherapy

Jia Huanzhen¹，Jiao Jie²，Gao Ge^2#*，Yang Hui^2#*

1.Central lab，Yanjing Medical College，Capital Medical University，Beijing 101321，China；2.Department of Neurobiology, School of Basic Medical Sciences, Capital Medical University，Center of Parkinsons Disease Beijing Institute for Brain Disorders, Beijing Key Laboratory of Neural Regeneration and Repair, Key Laboratory for Neurodegenerative Diseases of Ministry of Education，Beijing 100069，China

Received:2022-12-31 Online:2023-12-21 Published:2023-12-21
Supported by:
This study was supported by National Natural Science Foundation of China(81870994).

摘要/Abstract

摘要： 目的分析5种识别α-突触核蛋白（α-synuclein，α-syn)N端结构域的单克隆抗体1C16、2B8、2P21、3O18和1J6的特异性，为帕金森病(Parkinson's disease, PD)的早期诊断和免疫治疗提供抗体支持。方法体外蛋白重组技术获得人源α-syn蛋白 (human-α-syn,h-α-syn)、鼠源α-syn蛋白 (mouse-α-syn,m-α-syn)、β-syn蛋白、N端人源α-syn蛋白（α-syn/N)和去N端人源α-syn蛋白（α-syn/ΔN)。斑点印迹法鉴定5种单克隆抗体的特异性识别结构域，使用蛋白质印迹技术检测其对变性后的纯蛋白和鼠脑组织的识别情况。结果抗体1C16可以识别非变性的h-α-syn纯蛋白和小鼠脑组织中的变性的h-α-syn蛋白，不识别m-α-syn和β-syn。抗体1J6仅识别非变性的h-α-syn及α-syn/N纯蛋白, 不识别变性后的全长α-syn纯蛋白和小鼠脑组织中的变性h-α-syn蛋白。结论筛选出的2种单克隆抗体具有特异性，可为本实验下一步生物标志物的酶联免疫吸附法测定检测和针对α-syn的免疫治疗提供基础。

关键词: 帕金森病, α-突触核蛋白, α-突触核蛋白 N端, 蛋白纯化, 单克隆抗体

Abstract: Objective To analyze the specificity of five monoclonal antibodies 1C16, 2B8, 2P21, 3O18 and 1J6 that recognize α-synuclein (α-syn) N-terminal domains, to provide antibody support for early diagnosis and immunotherapy of Parkinson's disease (PD). Methods We generated human α-syn(h-α-syn), mouse α-syn (m-α-syn), β-syn, N-terminus human α-syn (α-syn/N) and delete N-terminus human α-syn (α-syn/ΔN) protein using recombinant protein purification technology in vitro. The specific recognition domains of the five monoclonal antibodies were detected by dot blotting. Using Western blotting to detect the denatured α-syn from purified protein and mice brain. Results Antibody 1C16 recognized h-α-syn proteins in native form and h-α-syn protein in the mice brain in denatured form rather than m-α-syn and β-syn. Antibody 1J6 could recognize h-α-syn and α-syn/N protein in native form rather than full-length α-syn protein and h-α-syn protein in the mouse brain in denatured form. Conclusion The two selected antibodies are specific to human α-syn, which provide the basis for the detection of biomarkers by enzyme linked immunosorbent assay and can be used for immunotherapy in PD.

Key words: Parkinson's disease, α-synuclein, α-synuclein N terminal, protein purification, monoclonal antibody

中图分类号:

R394

贾焕珍, 焦洁, 高歌, 杨慧. 识别α-synuclein N端结构域的单克隆抗体鉴定及免疫应用[J]. 首都医科大学学报, 2023, 44(6): 1022-1028.

Jia Huanzhen, Jiao Jie, Gao Ge, Yang Hui. Identification the monoclonal antibodies recognizing the α-synuclein N-terminal domain and their application in immunotherapy[J]. Journal of Capital Medical University, 2023, 44(6): 1022-1028.

参考文献

［1］Lashuel H A, Overk C R, Oueslati A, et al. The many faces of α-synuclein: from structure and toxicity to therapeutic target［J］. Nat Rev Neurosci, 2013, 14(1): 38-48.
［2］Binolfi A, Limatola A, Verzini S, et al. Intracellular repair of oxidation-damaged α-synuclein fails to target C-terminal modification sites［J］. Nat Commun, 2016, 7(1): 10251.
［3］Kang L J, Janowska M K, Moriarty G M, et al. Mechanistic insight into the relationship between N-terminal acetylation of α-synuclein and fibril formation rates by NMR and fluorescence［J］. PLoS One, 2013, 8(9): e75018.
［4］Oueslati A, Fournier M, Lashuel H A. Role of post-translational modifications in modulating the structure, function and toxicity of α-synuclein: implications for Parkinson's disease pathogenesis and therapies［J］. Prog Brain Res, 2010, 183: 115-145.
［5］Gribaudo S, Tixador P, Bousset L, et al. Propagation of α-synuclein strains within human reconstructed neuronal network［J］. Stem Cell Reports, 2019, 12(2): 230-244.
［6］Liu W J, Zhang Q D, Xing H, et al. Characterization of a novel monoclonal antibody for serine-129 phosphorylated α-synuclein: a potential application for clinical and basic research［J］. Front Neurol, 2022, 13: 821792.
［7］Shen J M, Du T T, Wang X, et al. α-Synuclein amino terminus regulates mitochondrial membrane permeability［J］. Brain Res, 2014, 1591: 14-26.
［8］Foulds P G, Mitchell J D, Parker A, et al. Phosphorylated α-synuclein can be detected in blood plasma and is potentially a useful biomarker for Parkinson's disease［J］. FASEB J, 2011, 25(12): 4127-4137.
［9］Inglis K J, Chereau D, Brigham E F, et al. Polo-like kinase 2 (PLK2) phosphorylates α-synuclein at serine 129 in central nervous system［J］. J Biol Chem, 2009, 284(5): 2598-2602.
［10］Delenclos M, Burgess J D, Lamprokostopoulou A, et al. Cellular models of α-synuclein toxicity and aggregation［J］. J Neurochem, 2019, 150(5): 566-576.
［11］Zhang Z T, Kang S S, Liu X, et al. Asparagine endopeptidase cleaves α-synuclein and mediates pathologic activities in Parkinson's disease［J］. Nat Struct Mol Biol, 2017, 24(8): 632-642.
［12］Games D, Valera E, Spencer B, et al. Reducing C-terminal-truncated α-synuclein by immunotherapy attenuates neurodegeneration and propagation in Parkinson's disease-like models［J］. J Neurosci, 2014, 34(28): 9441-9454.
［13］Wang Z P, Gao G, Duan C L, et al. Progress of immunotherapy of anti-α-synuclein in Parkinson's disease［J］. Biomed Pharmacother, 2019, 115: 108843.
［14］Jankovic J, Goodman I, Safirstein B, et al. Safety and tolerability of multiple ascending doses of PRX002/RG7935, an anti-α-synuclein monoclonal antibody, in patients with Parkinson disease: a randomized clinical trial［J］. JAMA Neurol, 2018, 75(10): 1206-1214.
［15］Lynch S M, Zhou C, Messer A. An scFv intrabody against the nonamyloid component of α-synuclein reduces intracellular aggregation and toxicity［J］. J Mol Biol, 2008, 377(1): 136-147.

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识别α-synuclein N端结构域的单克隆抗体鉴定及免疫应用

Identification the monoclonal antibodies recognizing the α-synuclein N-terminal domain and their application in immunotherapy

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