Analysis of CD22 cis-ligands using a synthetic sialoside binding to CD22 with ultra-high affinity

CD22 (also known as Siglec-2), an inhibitory receptor expressed in B lymphocytes (cells), recognizes the glycan structure α2,6-sialylated N-acetyllactosamine, and constitutively associates with various α2,6-sialylated membrane proteins expressed on the same cell (cis-ligands) although CD22 can bind to α2,6-sialylated molecules expressed in other cells (trans-ligands). The inhibitory activity of CD22 is regulated by the cis-ligands, and CD22 regulates B cells through the cis-ligands. Because of fast dissociation kinetics of the binding of CD22 with the ligands, systematic identification of CD22 ligands is not possible by conventional methods such as immunoprecipitation. Previously, we showed that proximity labeling using tyramide efficiently labels CD22-associated molecules. However, whether this association depends on the recognition of sialic acid by CD22 needs to be demonstrated to identify the cis-ligands. Here we develop the synthetic sialoside GSC-932 in which modification of the C5 position of the sialic acid core with a guanidyl group improves the affinity to mouse CD22 by 50 folds probably by generating the interaction of the guanidyl group with several amino acid residues in mouse CD22 such as E136 and R137, thereby efficiently inhibiting the binding of mouse CD22 with the α2,6-sialylated ligands. Application of GSC-932 to the proximity labeling clearly distinguishes sialic acid-dependent association with CD22 and shows that CD22 associates with a variety of cis-ligands. GSC-932 may be a useful tool to study ligand interaction of CD22.