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

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-突触核蛋白磷酸化修饰位点数量明显多于正常人;在诸多修饰位点中,血清α-突触核蛋白的Ser129位点经过磷酸化修饰后形成的聚集体最稳定,这可能为帕金森病早期诊断提供诊断标志物参考。

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 phosphorylated sites in Parkinson’s disease patients than in normal humans.The phosphorylation sites in the serum of patients with Parkinson’s disease were Serine(Ser)87,Ser129,L-tyrosine(Tyr)125,Tyr 133 and Tyr 136.After incubation in the serum of patients with Parkinson’s disease forα-synuclein,the phosphorylation modification at Ser129 accounted for 53.65%of the total phosphorylation,with 17.21%,15.79%,15.79%,9.52%,and 1.03%for Tyr125,Tyr133,Tyr136,and Ser87,respectively.The serum levels of Ser129 phosphorylation-modified aggregates were detected by western blot to be more stable than those of Tyr125,Tyr133 and Tyr136 in patients with Parkinson’s disease.To conclude,the number ofα-synuclein phosphorylation modification sites in the serum of patients with Parkinson’s disease was significantly higher than that of normal subjects.The aggregate structure generated by phosphorylation modification at Ser129 in the serum of patients with Parkinson’s disease was the most stable.This may provide a diagnostic marker for the early diagnosis of Parkinson’s disease.