Sialylation of Prion Protein Controls the Rate of Prion Amplification, the Cross-Species Barrier, the Ratio of PrPSc Glycoform and Prion Infectivity

The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC) into the disease-associated, transmissible form (PrPSc). PrPC is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrPC and the strain-specific structure of PrPSc. The current work highlights the previously unappreciated role of sialylation of PrPC glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrPSc glycoform ratio. The current study demonstrates that undersialylated PrPC is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrPC. As a result, PMCAb-derived PrPSc was less sialylated than brain-derived PrPSc. A decrease in PrPSc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrPC using sialidase was found to increase the rate of PrPSc amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrPC reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrPC was also found to control PrPSc glycoform ratio. A decrease in PrPC sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrPSc. 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrPC differed from that of spleen-derived PrPC. Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrPC, suggesting that Neu1 is not responsible for desialylation of PrPC. The current work highlights previously unappreciated role of PrPC sialylation in prion diseases and opens multiple new research directions, including development of new therapeutic approaches.

朊病毒疾病的核心致病事件，是细胞型朊蛋白（PrPC）发生构象转变，成为与疾病相关且具有传染性的致病型朊蛋白（PrPSc）。PrPC是一种唾液酸糖蛋白，含有两个保守的N-糖基化位点。多年来已被确定的调控朊病毒复制的关键参数包括宿主PrPC的氨基酸序列，以及PrPSc的毒株特异性结构。本研究着重阐明了此前未被重视的PrPC聚糖唾液酸化在朊病毒发病机制中的作用，包括其对朊病毒复制速率、感染性、跨物种屏障以及PrPSc糖型比例的调控作用。本研究证实，在蛋白质错误折叠循环扩增（PMCAb）的朊病毒扩增过程中，唾液酸化程度较低的PrPC会被选择性富集，而唾液酸化程度较高的PrPC则会被淘汰。由此，经PMCAb扩增得到的PrPSc的唾液酸化程度低于脑组织来源的PrPSc。PrPSc唾液酸化程度的降低，与其来源材料的感染性下降呈显著相关。不过，采用唾液酸酶对PrPC进行酶促去唾液酸化处理后，PrPSc在PMCAb中的扩增速率可提升10至10000倍，且该效应具有毒株依赖性。此外，对PrPC进行去唾液酸化处理，可降低甚至消除朊病毒在PMCAb中扩增时的物种屏障。上述结果表明，唾液酸所携带的负电荷可调控同源与异源朊病毒复制的能垒。令人意外的是，研究还发现PrPC的唾液酸化状态同样可调控PrPSc的糖型比例：PrPC唾液酸化水平的降低，会导致PrPSc中双糖基化糖型的占比升高。电荷分布二维分析显示，脑组织来源的PrPC与脾脏来源的PrPC的唾液酸化状态存在差异。敲除溶酶体唾液酸酶Neu1并不会改变脑组织来源PrPC的唾液酸化状态，这表明Neu1并不参与PrPC的去唾液酸化过程。本研究揭示了PrPC唾液酸化在朊病毒疾病中此前未被重视的作用，并为诸多新研究方向提供了思路，包括新型治疗策略的开发。