α- 突触核蛋白在帕金森病患者血清中的磷酸化修饰.

BACKGROUND: The aggregates formed after translational modification of α-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 of α-synuclein in serum of patients with Parkinson’s disease. METHODS: A recombinant human α-synuclein prokaryotic expression system was constructed, and the protein was purified by affinity chromatography. The purity and specificity of α-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 serum α-synuclein aggregates. The modified sites for phosphorylation modification of α-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 that α-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 for α-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 of α-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. [ABSTRACT FROM AUTHOR]