Abstract
The immunomodulatory family of Siglecs recognize sialic acid-containing glycans as ‘self’, which is exploited in cancer for immune-evasion. The biochemical nature of Siglec ligands remains incompletely understood with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs (CHST1, CHST2, or CHST4) significantly enhances the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, pharmacological blockade of N- and O-glycan maturation reveals a cell type-specific pattern of importance for either class of glycan. Production of a highly homogenous CD33 (Siglec-3) fragment enabled a mass spectrometry-based binding assay to determine 10-fold and 3-fold enhanced affinity for Neu5Acα2-3(6-O-sulfo)Galβ1-4GlcNAc and Neu5Acα2-3Galβ1-4(6-O- sulfo)GlcNAc, respectively, over Neu5Acα2-3Galβ1-4GlcNAc. CD33 showed significant additivity in affinity (36-fold) for the disulfated ligand, Neu5Acα2-3(6-O-sulfo)Galβ1-4(6-O-sulfo)GlcNAc. Moreover, overexpression of both CHST1 and CHST2 in cells greatly enhanced the binding of several Siglecs, including CD33. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a modification that is a general mechanism for tuning Siglec ligands on cells, including in cancer.
Competing Interest Statement
The authors have declared no competing interest.
