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Identification of Siglec Ligands Using a Proximity Labeling Method
- Source :
- Journal of Proteome Research. 16:3929-3941
- Publication Year :
- 2017
- Publisher :
- American Chemical Society (ACS), 2017.
-
Abstract
- 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.
- Subjects :
- 0301 basic medicine
Glycan
Free Radicals
Sialic Acid Binding Ig-like Lectin 2
Biotin
Tyramine
Biology
Ligands
Sialidase
Proteomics
Biochemistry
Mice
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
Lectins
Animals
Humans
Sialic Acid Binding Immunoglobulin-like Lectins
B-Lymphocytes
Staining and Labeling
CD22
Pattern recognition receptor
SIGLEC
Lectin
Hydrogen Peroxide
General Chemistry
respiratory system
Sialic acid
RAW 264.7 Cells
030104 developmental biology
Immunoglobulin M
chemistry
Immune System
030220 oncology & carcinogenesis
biology.protein
Leukocyte Common Antigens
Subjects
Details
- ISSN :
- 15353907 and 15353893
- Volume :
- 16
- Database :
- OpenAIRE
- Journal :
- Journal of Proteome Research
- Accession number :
- edsair.doi.dedup.....8c1d2499536130cc5aaf86d00a93164f