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 communis agglutinin 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 RCA120 or 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.

中文翻译：

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配体辅助标记探针制造的荧光“打开”凝集素传感器，用于检测蛋白质-糖蛋白相互作用。

阐明蛋白质间相互作用（PPI）通常非常具有挑战性，并且会产生复杂且不清楚的结果。由于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的固有低亲和力，因此特别难以研究凝集素与糖蛋白的相互作用。在这里，我们报告一种基于“配体定向标记探针（LLP）”的方法来制造用于阐明蛋白质-糖蛋白相互作用的蛋白质探针。LLP设计有两个可光活化基团，用于在凝集素，蓖麻毒素凝集素120（RCA120）和重组人Siglec-2-Fc的碳水化合物结合口袋附近引入炔烃手柄。在原理验证研究中，炔基化凝集素与光反应性二嗪嗪交联剂和对环境敏感的荧光团缀合，分别通过生物正交点击反应。修饰的RCA120或Siglec-2-Fc用于检测与溶液中靶糖蛋白或活HeLa细胞上内源表达糖蛋白的相互作用。我们预期这些蛋白质探针的制造将加速新型PPI的发现。