Department of Pharmacy, Uppsala University, Uppsala, Sweden.
Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
PLoS One. 2024 Aug 29;19(8):e0308521. doi: 10.1371/journal.pone.0308521. eCollection 2024.
The aggregation of α-Synuclein (αSyn) is strongly linked to neuronal death in Parkinson's disease and other synucleinopathies. The spreading of aggregated αSyn between neurons is at least partly dependent on electrostatic interactions between positively charged stretches on αSyn fibrils and the negatively charged heparan sulphate proteoglycans on the cell surface. To date there is still no therapeutic option available that could halt the progression of Parkinson's disease and one of the major limitations is likely the relatively low proportion of αSyn aggregates accessible to drugs in the extracellular space. Here, we investigated whether a negatively charged peptide tail fused to the αSyn aggregate-specific antibodies SynO2 and 9E4 could enhance the antibodies' avidity to αSyn aggregates in order to improve their potential therapeutic effect through inhibiting cell-to-cell spreading and enhancing the clearance of extracellular aggregates. We performed ELISAs to test the avidity to αSyn aggregates of both monovalent and bivalent antibody formats with and without the peptide tail. Our results show that the addition of the negatively charged peptide tail decreased the binding strength of both antibodies to αSyn aggregates at physiological salt conditions, which can likely be explained by intermolecular repulsions between the tail and the negatively charged C-terminus of αSyn. Additionally, the tail might interact with the paratopes of the SynO2 antibody abolishing its binding to αSyn aggregates. Conclusively, our peptide tail did not fulfil the required characteristics to improve the antibodies' binding to αSyn aggregates. Fine-tuning the design of the peptide tail to avoid its interaction with the antibodies' CDR and to better mimic relevant characteristics of heparan sulphates for αSyn aggregate binding may help overcome the limitations observed in this study.
α-突触核蛋白（αSyn）的聚集与帕金森病和其他突触核蛋白病中的神经元死亡密切相关。聚集的 αSyn 在神经元之间的传播至少部分依赖于 αSyn 纤维上带正电荷的伸展部分与细胞表面带负电荷的硫酸乙酰肝素蛋白聚糖之间的静电相互作用。迄今为止，仍然没有可用的治疗方法可以阻止帕金森病的进展，其中一个主要限制因素可能是细胞外空间中可被药物接触到的 αSyn 聚集物的比例相对较低。在这里，我们研究了是否可以将带负电荷的肽尾融合到针对 αSyn 聚集物的抗体 SynO2 和 9E4 上，以提高抗体对 αSyn 聚集物的亲和力，从而通过抑制细胞间传播和增强细胞外聚集物的清除来提高其潜在的治疗效果。我们进行了 ELISA 实验来测试单价和双价抗体形式的抗体对 αSyn 聚集物的亲和力，有无肽尾。我们的结果表明，在生理盐条件下，带负电荷的肽尾的添加降低了两种抗体与 αSyn 聚集物的结合强度，这可能可以用尾部与 αSyn 的带负电荷的 C 末端之间的分子间排斥来解释。此外，尾部可能与 SynO2 抗体的互补决定区相互作用，从而使其无法与 αSyn 聚集物结合。总之，我们的肽尾没有满足改善抗体与 αSyn 聚集物结合的要求特征。微调肽尾的设计以避免其与抗体的 CDR 相互作用，并更好地模拟硫酸乙酰肝素对 αSyn 聚集物结合的相关特征，可能有助于克服本研究中观察到的限制。
