α-突触核蛋白在帕金森病患者血清中的磷酸化修饰  

Phosphorylation modification of alpha synuclein in serum of patients with Parkinson’s disease

作　　者：齐雪 李家慧 朱远峰 禹璐 王鹏[1,2] Qi Xue;Li Jiahui;Zhu Yuanfeng;Yu Lu;Wang Peng(Department of Human Anatomy,School of Basic Medical Sciences,Beihua University,Jilin 132013,Jilin Province,China;Laboratory of Neurodegenerative Diseases,School of Basic Medical Sciences,Beihua University,Jilin 132013,Jilin Province,China)

机构地区：[1]北华大学基础医学院人体解剖学教研室,吉林省吉林市132013 [2]北华大学基础医学院神经退行性疾病研究室,吉林省吉林市132013

出　　处：《中国组织工程研究》2023年第35期5577-5582,共6页Chinese Journal of Tissue Engineering Research

基　　金：吉林省自然科学基金(YDZJ202201ZYTS575),项目负责人:王鹏;吉林省卫生与健康技术创新项目(2018J083),项目负责人:王鹏;北华大学研究生创新项目(北华研创合字[2021]017),项目负责人:齐雪。

摘　　要：背景:α-突触核蛋白翻译后修饰后形成的聚集体是帕金森病主要的病理学改变。α-突触核蛋白可通过血脑屏障由中枢进入外周血分布至机体各部,这成为帕金森病病理播散的重要途径。因此研究血液中的α-突触核蛋白变化对揭示帕金森病的机制以及早期诊断尤为重要。目的:拟分析α-突触核蛋白在帕金森病患者血清中的磷酸化修饰位点及生成聚集体间的结构稳定性的差异。方法:构建重组人α-突触核蛋白原核表达系统,亲和层析法纯化蛋白,采用SDS-PAGE电泳和Western blot方法检测α-突触核蛋白单体纯度和特异性。收集北华大学附属医院神经内科住院的26例帕金森病患者血清和26例正常人血清,完成各组血清中α-突触核蛋白聚集体的制备;采用质谱SWATH方法检测帕金森病患者血清中α-突触核蛋白发生磷酸化修饰的修饰位点,并对不同位点的蛋白聚集体进行定量分析。将不同磷酸化修饰的蛋白聚集体免疫亲和层析法纯化后,Western blot方法检测帕金森病患者血清中各磷酸化修饰后聚集体的稳定性。结果与结论:①实验通过SDS电泳和Western blot方法检测显示得到纯度较高、特异性较高的α-突触核蛋白单体。②质谱SWATH分析结果显示,在帕金森病患者和正常人血清中均发生磷酸化修饰,其中帕金森病患者血清中磷酸化修饰的位点明显多于正常人,帕金森病患者血清中发生磷酸化的位点有丝氨酸(Serine,Ser)87、Ser129、酪氨酸(L-tyrosine,Tyr)125、Tyr133和Tyr136等。③帕金森病患者血清中孵育后的α-突触核蛋白中,Ser129位点磷酸化修饰占总磷酸化的53.65%,Tyr125,Tyr133,Tyr136和Ser87磷酸化各为17.21%,15.79%,15.79%,9.52%和1.03%。④Western blot检测结果显示,帕金森病患者血清中Ser129位点磷酸化修饰后形成的聚集体较Tyr125,Tyr133和Tyr136更为稳定。⑤上述数据证实,帕金森病患者血清中的α-突触核蛋白磷酸�BACKGROUND:The aggregates formed after translational modification ofα-synuclein are the main pathological changes of Parkinson’s disease.α-Synuclein can penetrate the blood-brain barrier and be delivered from the central nervous system to the peripheral blood to all parts of the body,which becomes an important pathway for the pathological dissemination of Parkinson’s disease.Therefore,it is particularly important to study the changes of blood markers in patients with Parkinson’s disease to reveal the mechanism of Parkinson‘s disease as well as for early diagnosis.OBJECTIVE:To analyze the differences in the structural stability of phosphorylation modification sites and generated aggregates ofα-synuclein in serum of patients with Parkinson’s disease.METHODS:A recombinant humanα-synuclein prokaryotic expression system was constructed,and the protein was purified by affinity chromatography.The purity and specificity ofα-synuclein monomer was detected by SDS-PAGE and western blot.Serum samples of 26 normal controls and 26 patients with Parkinson’s disease in the Department of Neurology,Affiliated Hospital of Beihua University were collected to complete the preparation of serumα-synuclein aggregates.The modified sites for phosphorylation modification ofα-synuclein protein in normal serum and serum of patients with Parkinson’s disease were identified using SWATH-mass spectrometry,and protein aggregates at different sites were quantitatively analyzed.The protein aggregates with different phosphorylation modifications were purified by immunoaffinity chromatography,and then detected for stability in the serum of patients with Parkinson’s disease by western blot.RESULTS AND CONCLUSION:The results of SDS-PAGE and western blot showed thatα-synuclein monomers with high purity and specificity were obtained.The results of the SWATH-mass spectrometry analysis showed that phosphorylation modifications occurred in both Parkinson’s disease patients and normal human serum,with significantly more phosphorylat

关 键 词：Α-突触核蛋白 帕金森病 磷酸化 血清 丝氨酸 酪氨酸 聚集体 诊断生物标志物 

分 类 号：R459.9[医药卫生—治疗学] R363[医药卫生—临床医学] R74