Siglecs are a family of receptor-type glycan recognition proteins (lectins) involved in self-nonself discrimination by the immune system. Identification of Siglec ligands is necessary to understand how Siglec-ligand interaction translates into biological outcomes. However, this is challenging because the interaction is weak. To facilitate identification of Siglec ligands, we adopted a proximity labeling method based on the tyramide radicalization principle. Cells that express Siglec ligands were labeled with Siglec-peroxidase complexes and incubated with biotin tyramide and hydrogen peroxide, to generate short-lived tyramide radicals that covalently label the proteins near the Siglec-peroxidase complex. A proof-of-principle experiment using CD22 (Siglec-2) probe identified its known ligands on B cells, including CD22 itself, CD45, and IgM among others, demonstrating the validity of this method. The specificity of labeling was confirmed by sialidase treatment of target cells and using glycan recognition-deficient mutant CD22 probes. Moreover, possible interactions between biotin-labeled proteins were revealed by literature-based protein-protein interaction network analysis, implying the presence of a molecular cluster comprising CD22 ligands. Further application of this method identified CD44 as a hitherto unknown Siglec-15 ligand on RAW264.7-derived osteoclasts. These results demonstrated the utility of proximity labeling for the identification of Siglec ligands, which may extend to other lectins.

中文翻译：

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使用邻近标记法鉴定Siglec配体

Siglecs是一类受体型聚糖识别蛋白（凝集素），参与免疫系统的自我非自我区分。鉴定Siglec配体是了解Siglec-配体相互作用如何转化为生物学结果的必要条件。但是，这是有挑战性的，因为交互作用很弱。为了便于识别Siglec配体，我们基于酪酰胺自由基化原理采用了邻近标记方法。用Siglec-过氧化物酶复合物标记表达Siglec配体的细胞，并与生物素酪酰胺和过氧化氢孵育，以生成短暂存在的酪酰胺基团，共价标记Siglec-过氧化物酶复合物附近的蛋白质。使用CD22（Siglec-2）探针进行的原理验证实验确定了其在B细胞上的已知配体，包括CD22本身，CD45，和IgM等，证明了这种方法的有效性。通过唾液酸酶处理靶细胞并使用聚糖识别缺陷型突变CD22探针证实了标记的特异性。此外，通过基于文献的蛋白质-蛋白质相互作用网络分析揭示了生物素标记的蛋白质之间可能的相互作用，这表明存在包含CD22配体的分子簇。该方法的进一步应用将CD44鉴定为RAW264.7来源的破骨细胞上迄今未知的Siglec-15配体。这些结果证明了邻近标记法用于鉴定Siglec配体的实用性，该配体可能会扩展到其他凝集素。通过唾液酸酶处理靶细胞并使用聚糖识别缺陷型突变CD22探针证实了标记的特异性。此外，通过基于文献的蛋白质-蛋白质相互作用网络分析揭示了生物素标记的蛋白质之间可能的相互作用，这表明存在包含CD22配体的分子簇。该方法的进一步应用将CD44鉴定为RAW264.7来源的破骨细胞上迄今未知的Siglec-15配体。这些结果证明了邻近标记法用于鉴定Siglec配体的实用性，该配体可能会扩展到其他凝集素。通过唾液酸酶处理靶细胞并使用聚糖识别缺陷型突变CD22探针证实了标记的特异性。此外，通过基于文献的蛋白质-蛋白质相互作用网络分析揭示了生物素标记的蛋白质之间可能的相互作用，这表明存在包含CD22配体的分子簇。该方法的进一步应用将CD44鉴定为RAW264.7来源的破骨细胞上迄今未知的Siglec-15配体。这些结果证明了邻近标记法用于鉴定Siglec配体的实用性，该配体可能会扩展到其他凝集素。该方法的进一步应用将CD44鉴定为RAW264.7来源的破骨细胞上迄今未知的Siglec-15配体。这些结果证明了邻近标记法用于鉴定Siglec配体的实用性，该配体可能会扩展到其他凝集素。该方法的进一步应用将CD44鉴定为RAW264.7来源的破骨细胞上迄今未知的Siglec-15配体。这些结果证明了邻近标记法用于鉴定Siglec配体的实用性，该配体可能会扩展到其他凝集素。