Biologically Derived Neoproteoglycans for Profiling Protein–Glycosaminoglycan Interactions
Biologically Derived Neoproteoglycans for Profiling Protein–Glycosaminoglycan Interactions
复制标题
用于分析蛋白质与糖胺聚糖相互作用的生物衍生新蛋白聚糖
DOI：
10.1021/acschembio.2c00205
复制
发表时间：
2022
影响因子：
4
通讯作者：
Godula, Kamil
中科院分区：
文献类型：
--
作者：
Porell, Ryan N.;Follmar, Julianna L.;Purcell, Sean C.;Timm, Bryce;Laubach, Logan K.;Kozirovskiy, David;Thacker, Bryan E.;Glass, Charles A.;Gordts, Philip L.;Godula, Kamil
糖胺聚糖（GAG）是一类高度带负电荷的膜相关和细胞外基质多糖，其参与调节无数生物功能，包括细胞粘附、迁移、信号传导和分化等。GAG通常附着于称为蛋白聚糖（PG）的核心蛋白，并且可以接合>500个结合蛋白，使它们成为感知外部刺激和转导细胞反应的重要中继。然而，赋予其结合特异性的其独特的亚结构蛋白识别结构域仍然难以捉摸。虽然聚糖阵列的出现已经快速地使得能够分析一系列聚糖结合蛋白的配体特异性，但是它们用于分析GAG结合蛋白的适应性已经相当具有挑战性。目前的GAG微阵列主要采用合成定义的寡糖，其仅捕获天然GAG多糖的一部分结构多样性。增强现有阵列平台以包括从组织中纯化或在具有工程化聚糖生物合成途径的细胞中产生的GAG结构，可以显著促进对GAG-蛋白质相互作用中的结构-活性关系的理解。在这里，我们展示了一种有效的和可调的策略，以模拟细胞蛋白聚糖的结构，通过缀合生物衍生的GAG链的蛋白质支架，定义为新蛋白聚糖（neoproteoglycans，neoPGs）。反应性荧光接头的使用使得能够实时监测缀合反应效率和调节neoPG价态。将试剂固定在96孔阵列平台上允许有效探测配体结合和酶-底物特异性，包括生长因子和人硫酸酯酶1。neoPG也可以直接用作可溶性探针，以评估细胞中的GAG依赖性生长因子信号传导。
Glycosaminoglycans (GAGs) are a class of highly negatively charged membrane-associated and extracellular matrix polysaccharides involved in the regulation of myriad biological functions, including cell adhesion, migration, signaling, and differentiation, among others. GAGs are typically attached to core proteins, termed proteoglycans (PGs), and can engage >500 binding proteins, making them prominent relays for sensing external stimuli and transducing cellular responses. However, their unique substructural protein-recognition domains that confer their binding specificity remain elusive. While the emergence of glycan arrays has rapidly enabled the profiling of ligand specificities of a range of glycan-binding proteins, their adaptation for the analysis of GAG-binding proteins has been considerably more challenging. Current GAG microarrays primarily employ synthetically defined oligosaccharides, which capture only a fraction of the structural diversity of native GAG polysaccharides. Augmenting existing array platforms to include GAG structures purified from tissues or produced in cells with engineered glycan biosynthetic pathways may significantly advance the understanding of structure–activity relationships in GAG–protein interactions. Here, we demonstrate an efficient and tunable strategy to mimic cellular proteoglycan architectures by conjugating biologically derived GAG chains to a protein scaffold, defined as neoproteoglycans (neoPGs). The use of a reactive fluorogenic linker enabled real-time monitoring of the conjugation reaction efficiency and tuning of the neoPG valency. Immobilization of the reagents on a 96-well array platform allowed for efficient probing of ligand binding and enzyme–substrate specificity, including growth factors and the human sulfatase 1. The neoPGs can also be used directly as soluble probes to evaluate GAG-dependent growth factor signaling in cells.