Li Qiongyu, Xie Yixuan, Xu Gege, Lebrilla Carlito B
Department of Chemistry , University of California, Davis , Davis , California , USA . Email:
Department of Biochemistry , University of California, Davis , Davis , California , USA.
Chem Sci. 2019 May 14;10(24):6199-6209. doi: 10.1039/c9sc01360a. eCollection 2019 Jun 28.
The cell membrane contains a highly interactive glycan surface on a scaffold of proteins and lipids. Sialic acids are negatively charged monosaccharides, and the proteins that bind to sialic acids play an important role in maintaining the integrity and collective functions of this interactive space. Sialic acid binding proteins are not readily identified and have nearly all been discovered empirically. In this research, we developed a proximity labeling method to characterize proteins with oxidation by localized radicals produced . The sites of oxidation were identified and quantified using a standard proteomic workflow. In this method, a clickable probe was synthesized and attached to modified sialic acids on the cell membrane, which functioned as a catalyst for the localized formation of radicals from hydrogen peroxide. The proteins in the sialic acid environment were labeled through amino acid oxidation, and were categorized into three groups including sialylated proteins, non-sialylated proteins with transmembrane domains, and proteins that are associated with the membrane with neither sialylated nor transmembrane domains. The analysis of the last group of proteins showed that they were associated with binding functions including carbohydrate binding, anion binding, and cation binding, thereby revealing the nature of the sialic acid-protein interaction. This new tool identified potential sialic acid-binding proteins in the extracellular space and proteins that were organized around sialylated glycans in cells.
细胞膜在蛋白质和脂质支架上含有高度相互作用的聚糖表面。唾液酸是带负电荷的单糖，与唾液酸结合的蛋白质在维持这个相互作用空间的完整性和集体功能方面发挥着重要作用。唾液酸结合蛋白不容易被识别，几乎都是通过经验发现的。在这项研究中，我们开发了一种邻近标记方法，以通过产生的局部自由基氧化来表征蛋白质。使用标准的蛋白质组学工作流程对氧化位点进行识别和定量。在该方法中，合成了一种可点击的探针并将其连接到细胞膜上修饰的唾液酸上，该唾液酸作为过氧化氢局部形成自由基的催化剂。唾液酸环境中的蛋白质通过氨基酸氧化进行标记，并分为三组，包括唾液酸化蛋白质、具有跨膜结构域的非唾液酸化蛋白质以及既不唾液酸化也不具有跨膜结构域但与膜相关的蛋白质。对最后一组蛋白质的分析表明，它们与包括碳水化合物结合、阴离子结合和阳离子结合在内的结合功能相关，从而揭示了唾液酸 - 蛋白质相互作用的本质。这个新工具识别出了细胞外空间中潜在的唾液酸结合蛋白以及围绕细胞中唾液酸化聚糖组织的蛋白质。
