Fluorescence “Turn-on” Lectin Sensors Fabricated by Ligand-Assisted Labeling Probes for Detecting Protein?Glycoprotein Interactions
Fluorescence “Turn-on” Lectin Sensors Fabricated by Ligand-Assisted Labeling Probes for Detecting Protein?Glycoprotein Interactions
复制标题
由配体辅助标记探针制造的荧光“开启”凝集素传感器用于检测蛋白质与糖蛋白相互作用
DOI：
10.1021/acs.biomac.9b01495
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发表时间：
2019
影响因子：
6.2
通讯作者：
Lin Chun-Cheng
中科院分区：
文献类型：
--
作者：
Li Pei-Jhen;Anwar Mohammad Tarigue;Fan Chen-Yo;Juang Duane S.;Lin Hsin-Yi;Chang Tsung-Che;Kawade Sachin Kisan;Chen Hsiang-Jung;Chen Yu-Ju;Tan Kui-Thong;Lin Chun-Cheng
阐明蛋白质-蛋白质相互作用（PPIs）通常是非常具有挑战性的，并且产生复杂和不明确的结果。由于凝集素-糖蛋白相互作用的非共价性质以及蛋白质与糖蛋白上的聚糖配体固有的低结合亲和力，凝集素-糖蛋白相互作用特别难以研究。在这里，我们报告了一种基于“配体定向标记探针（LLP）”的方法来制造蛋白质探针，用于阐明蛋白质-糖蛋白相互作用。LLP设计了双光活化基团，用于在凝集素（Ricinus communisagglutinin 120 （RCA120）和重组人siglece -2- fc）的碳水化合物结合袋附近引入一个烯处理。在原理验证研究中，炔基化凝集素分别通过生物正交点击反应与光反应性重氮交联剂和环境敏感荧光基团偶联。修饰后的rca120或siglece -2- fc用于检测与溶液中的靶糖蛋白或活HeLa细胞上内源性表达的糖蛋白的相互作用。我们预计这些蛋白质探针的制造将加速新型ppi的发现。
Elucidation of protein–protein interactions (PPIs) is often very challenging and yields complex and unclear results. Lectin–glycoprotein interactions are especially difficult to study due to the noncovalent nature of the interactions and inherently low binding affinities of proteins to glycan ligands on glycoproteins. Here, we report a “ligand-directed labeling probe (LLP)”-based approach to fabricate protein probes for elucidating protein–glycoprotein interactions. LLP was designed with dual photoactivatable groups for the introduction of an alkyne handle proximal to the carbohydrate-binding pocket of lectins,Ricinus communisagglutinin 120 (RCA120) and recombinant human Siglec-2-Fc. In proof-of-principle studies, alkynylated lectins were conjugated with a photoreactive diazirine cross-linker and an environment-sensitive fluorophore, respectively, by the bioorthogonal click reaction. The modified RCA120or Siglec-2-Fc was used for detecting the interaction with the target glycoprotein in the solution or endogenously expressed glycoproteins on live HeLa cells. We anticipate that the fabrication of these protein probes will accelerate the discovery of novel PPIs.