Carbohydrate sulfation as a mechanism for fine-tuning Siglec ligands

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.

Complementary Role of GlcNAc6ST2 and GlcNAc6ST3 in Synthesis of CL40-Reactive Sialylated and Sulfated Glycans in the Mouse Pleural Mesothelium

“…Notably, Siglec-9-binding to a cultured cell line was markedly enhanced by overexpression of human GlcNAc6ST2/CHST4 and GlcNAc6ST1/CHST2 [60], while the current results show a complementary action of GlcNAc6ST2 and GlcNAc6ST3 in mesothelial CL40 glycan. A previous study identified CHST4 as a mesothelium-enriched gene [61], which strongly supports the involvement of GlcNAc6ST2 in the synthesis of the CL40 epitope in the mesothelium.…”

Dissecting the Ability of Siglecs To Antagonize Fcγ Receptors

“…Strong masking of Siglec-7 was confirmed through greatly enhanced binding of Sig-7L liposome binding to cells treated with neuraminidase to remove cis ligands (Figure S17A). U937 cells lacking sialic acid, through an introduced mutation in cytidine monophospho-N-acetylneuraminic acid synthetase (CMAS), 49,68 also displayed significantly increased Sig7-L liposome binding compared to WT cells (Figure 5d). We deleted Siglec-7 in the CMAS −/− cells and then virally transduced them with empty lentiviral control or WT Siglec-7 (Figure S17B).…”

Exploiting Substrate Specificities of 6-O-Sulfotransferases to Enzymatically Synthesize Keratan Sulfate Oligosaccharides

“…31 Genetic engineering of cells has indicated that sulfation can regulate Siglec binding; however, the precise molecular mechanisms of this regulation are not well understood. 32,33 Histological studies have shown that airway tissues express high-molecular-weight ligands for Siglec-8 and 9. 34 Treatment of these tissues with keratanase-I abolished the binding of recombinant Siglec-8.…”