GlycoCAP: A Cell-Free, Bacterial Glycosylation Platform for Building Clickable Azido-Sialoglycoproteins
GlycoCAP: A Cell-Free, Bacterial Glycosylation Platform for Building Clickable Azido-Sialoglycoproteins
复制标题
GlycoCAP：用于构建可点击叠氮基唾液酸糖蛋白的无细胞细菌糖基化平台
DOI：
10.1021/acssynbio.3c00017
复制
发表时间：
2023
影响因子：
4.7
通讯作者：
Jewett, Michael C.
中科院分区：
文献类型：
--
作者：
Thames, Ariel Helms;Moons, Sam J.;Wong, Derek A.;Boltje, Thomas J.;Bochner, Bruce S.;Jewett, Michael C.
被称为凝集素的聚糖结合受体代表了一类潜在的治疗靶点。然而，靶向凝集素的治疗潜力在很大程度上仍未开发，部分原因是由于构建基于聚糖的药物的工具的限制。一类理想的结构是具有非典型聚糖的蛋白质。无细胞蛋白合成系统已经成熟，是一种很有前途的制造糖蛋白的方法，可以克服目前的限制并使新的糖蛋白药物成为可能。然而，这种方法尚未应用于非典型聚糖的蛋白质构建。为了解决这一限制，我们开发了一个无细胞糖蛋白合成平台，用于构建非典型聚糖，特别是可点击的叠氮-唾液糖蛋白（称为GlycoCAP）。glycap平台使用基于大肠杆菌的无细胞蛋白质合成系统，以高度的均匀性和效率将非典型聚糖安装到蛋白质上。作为模型，我们在尘螨过敏原（Der p 2）上构建了四种非规范聚糖：α2,3 c5 -叠氮-唾液基乳糖、α2,3 c9 -叠氮-唾液基乳糖、α2,6 c5 -叠氮-唾液基乳糖和α2,6 c9 -叠氮-唾液基乳糖。通过一系列优化，我们实现了超过60%的非标准叠氮-唾液酸的唾液化效率。然后，我们证明叠氮化物点击手柄可以使用菌株促进和铜催化的点击化学与模型荧光基团共轭。我们预计，通过提供更广泛的可能的非规范聚糖结构，glycap将促进基于聚糖的药物的开发和发现，并提供一种通过点击化学偶联实现糖蛋白功能化的方法。
Glycan-binding receptors known as lectins represent a class of potential therapeutic targets. Yet, the therapeutic potential of targeting lectins remains largely untapped due in part to limitations in tools for building glycan-based drugs. One group of desirable structures is proteins with noncanonical glycans. Cell-free protein synthesis systems have matured as a promising approach for making glycoproteins that may overcome current limitations and enable new glycoprotein medicines. Yet, this approach has not been applied to the construction of proteins with noncanonical glycans. To address this limitation, we develop a cell-free glycoprotein synthesis platform for building noncanonical glycans and, specifically, clickable azido-sialoglycoproteins (called GlycoCAP). The GlycoCAP platform uses anEscherichia coli-based cell-free protein synthesis system for the site-specific installation of noncanonical glycans onto proteins with a high degree of homogeneity and efficiency. As a model, we construct four noncanonical glycans onto a dust mite allergen (Der p 2): α2,3 C5-azido-sialyllactose, α2,3 C9-azido-sialyllactose, α2,6 C5-azido-sialyllactose, and α2,6 C9-azido-sialyllactose. Through a series of optimizations, we achieve more than 60% sialylation efficiency with a noncanonical azido-sialic acid. We then show that the azide click handle can be conjugated with a model fluorophore using both strain-promoted and copper-catalyzed click chemistry. We anticipate that GlycoCAP will facilitate the development and discovery of glycan-based drugs by granting access to a wider variety of possible noncanonical glycan structures and also provide an approach for functionalizing glycoproteins by click chemistry conjugation.