Combinatorial chemoenzymatic synthesis and high-throughput screening of sialosides.
Combinatorial chemoenzymatic synthesis and high-throughput screening of sialosides.
复制标题
组合化学酶合成和唾液酸的高通量筛选。
DOI：
10.1021/cb800127n
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发表时间：
2008-09-19
影响因子：
4
通讯作者：
Chen, Xi
中科院分区：
文献类型：
--
作者：
Chokhawala, Harshal A.;Huang, Shengshu;Lau, Kam;Yu, Hai;Cheng, Jiansong;Thon, Vireak;Hurtado-Ziola, Nancy;Guerrero, Juan A.;Varki, Ajit;Chen, Xi
虽然含有唾液酸的结构在生物分子识别中的重要作用已被充分证明，但关于唾液酸结构修饰、唾液酸键和潜在的聚糖结构如何影响唾液酸识别蛋白的结合或活性以及相关的下游生物过程的信息有限。以生物素化唾液基转移酶受体和唾液酸前体为原料，建立了一种新的组合化学酶法，用于在96孔板上高效合成含有不同唾液酸结构和不同糖基的生物素化唾液苷。通过将反应混合物转移到neutravidin包被板上，并使用识别唾液转移酶受体而不是唾液化产物的凝集素检测酶促反应的产率，生物素化的唾液苷产物可以直接用于高通量筛选，而无需纯化，以快速识别唾液酸结合蛋白的配体特异性。为了证明原理，我们采用一锅三酶体系，从4个生物素化的唾液基转移酶受体和18个唾液酸前体中合成了72个生物素化的α2,6-连接的唾液苷。在neutravidin包被的微滴板上进行的高通量筛选实验表明，尽管黑Sambucus凝集素与α2,6-连接的唾液苷具有高度混杂性，但人类Siglec-2 （CD22）对其唾液苷配体中的许多唾液酸结构和潜在聚糖具有高度选择性。
Although the vital roles of sialic acid-containing structures in biomolecular recognition are well documented, limited information is available on how sialic acid structural modifications, sialyl linkages, and the underlying glycan structures affect the binding or the activity of sialic acid-recognizing proteins and related downstream biological processes. A novel combinatorial chemoenzymatic method has been developed for the highly efficient synthesis of biotinylated sialosides containing different sialic acid structures and different underlying glycans in 96-well plates from biotinylated sialyltransferase acceptors and sialic acid precursors. By transferring the reaction mixtures to NeutrAvidin-coated plates and assaying for the yields of enzymatic reactions using lectins recognizing sialyltransferase acceptors but not the sialylated products, the biotinylated sialoside products can be directly used, without purification, for high-throughput screening to quickly identify the ligand specificity of sialic acid-binding proteins. For a proof-of-principle experiment, 72 biotinylated α2,6-linked sialosides were synthesized in 96-well plates from 4 biotinylated sialyltransferase acceptors and 18 sialic acid precursors using a one-pot three-enzyme system. High-throughput screening assays performed in NeutrAvidin-coated microtiter plates show that while Sambucus nigra Lectin binds to α2,6-linked sialosides with high promiscuity, human Siglec-2 (CD22) is highly selective for a number of sialic acid structures and the underlying glycans in its sialoside ligands.
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影响因子：
3
作者：
KELM, S;SCHAUER, R;CROCKER, PR
通讯作者：
CROCKER, PR
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