5 years ago

Identification of Siglec Ligands Using a Proximity Labeling Method

Identification of Siglec Ligands Using a Proximity Labeling Method
Chun-Cheng Lin, Takashi Angata, Yu-Ju Chen, Yi-Hsiu Chen, Penk-Yeir Low, Chin-Ju Tang, Chan-Yo Fan, Yi-Ju Chen, Albert Ventura, Lanyi Chang
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.

Publisher URL: http://dx.doi.org/10.1021/acs.jproteome.7b00625

DOI: 10.1021/acs.jproteome.7b00625

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