Identification of Siglec Ligands Using a Proximity Labeling Method

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.