Comprehensive analysis of O-glycosylation of amyloid precursor protein (APP) using targeted and multi-fragmentation MS strategy
Comprehensive analysis of O-glycosylation of amyloid precursor protein (APP) using targeted and multi-fragmentation MS strategy
复制标题
使用靶向多片段 MS 策略全面分析淀粉样前体蛋白 (APP) 的 O-糖基化。
DOI：
10.1016/j.bbagen.2021.129954
复制
发表时间：
2021-07-12
影响因子：
3
通讯作者：
Zhang, Yan
中科院分区：
文献类型：
--
作者：
Shi, Jingjing;Ku, Xin;Zhang, Yan
背景：淀粉样前体蛋白（APP）在大脑中异常蛋白水解加工成淀粉样β肽（Aβ）是阿尔茨海默病（AD）发病机制中的关键步骤。作为一种O-糖基化蛋白，APP的O-糖基化被认为与Aβ的生成有关。因此，全面分析 APP O-糖基化对于理解其功能非常重要。方法：我们开发了一种采用多片段化技术（TMMF 策略）的靶向 MS 方法，并成功表征了 HEK-293 T 细胞中表达的 APP695 的 O-糖基化谱。我们计算了具有各种O-糖位点和O-聚糖的糖肽的相对丰度，并进一步研究了TNF-α处理后APP O-糖基化的变化。结果：在APP的三个糖肽上总共鉴定了14个O-糖基化位点，以及至少四个O-聚糖，包括GalNAc（Tn抗原）、核心1和 单/二唾液酸化核心 1 聚糖在 Thr576 和 Thr577 残基处起决定性作用。我们在 E2 结构域起点附近的区域发现了密集的截短 O-聚糖簇，并且在靠近 β 切割位点的区域发现了高丰度的唾液酸化 O-聚糖。此外，我们还观察到TNF-α可以上调HEK-293 T细胞中APP和APP上截短的O-聚糖的表达。 结论：我们的研究建立了完整的O-糖肽MS分析策略，用于APP O-糖肽的鉴定，具有增强的片段化效率和检测灵敏度。这些结果提供了 HEK-293 T 细胞中表达的 APP 的全面 O-糖基化图谱。一般意义：APP 上准确的 O-糖基化位点和 O-聚糖结构可能有助于更好地了解 O-糖基化在 APP 的加工和功能中所起的作用。
Background: The aberrant proteolytic processing of amyloid precursor protein (APP) into amyloid beta peptide (A beta) in brain is a critical step in the pathogenesis of Alzheimer's disease (AD). As an O-glycosylated protein, O-glycosylation of APP is considered to be related to A beta generation. Therefore, comprehensive analysis of APP O-glycosylation is important for understanding its functions.Methods: We developed a Targeted MS approach with Multi-Fragmentation techniques (TMMF strategy), and successfully characterized O-glycosylation profiling of APP695 expressed in HEK-293 T cells. We calculated relative abundance of glycopeptides with various O-glycosites and O-glycans, and further investigated the alteration of APP O-glycosylation upon TNF-alpha treatment.Results: A total of 14 O-glycosites were identified on three glycopeptides of APP, and at least four O-glycans including GalNAc (Tn antigen), core 1, and mono-/di-sialylated core 1 glycans were determinant at the residues of Thr576 and Thr577. We found a dense cluster of truncated O-glycans on the region nearby beginning of E2 domain and high abundance of sialylated O-glycans on the region close to beta-cleavage site. Moreover, we also observed that TNF-alpha could upregulate the expression of APP and the truncated O-glycans on APP in HEK-293 T cell.Conclusion: Our study established an intact O-glycopeptide MS analysis strategy for APP O-glycopeptide identification with enhanced fragmentation efficiency and detection sensitivity. These results provide a comprehensive O-glycosylation map of APP expressed in HEK-293 T cell.General significance: The accurate O-glycosites and O-glycan structures on APP may lead to a better understanding of the roles O-glycosylation plays in the processing and functions of APP.