Carbohydrate Sulfation As a Mechanism for Fine-Tuning Siglec Ligands

Abstract

The immunomodulatory family of Siglecs recognizes 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 enhance 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, a 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 homogeneous Siglec-3 (CD33) fragment enabled a mass-spectrometry-based binding assay to determine ≥8-fold and ≥2-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 shows significant additivity in affinity (≥28-fold) for the disulfated ligand, Neu5Acα2–3­(6-O-sulfo)­Galβ1–4­(6-O-sulfo)­GlcNAc. Moreover, joint overexpression of CHST1 with CHST2 in cells greatly enhanced the binding of CD33 and several other Siglecs. 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 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.…”

Recent Progress in the Methodologies to Identify Physiological Ligands of Siglecs

“…A recent publication from this group revealed the details of glycotopes recognized by Siglecs and glycosyltransferases involved in their synthesis. They also described the importance of galactose sulfation for the generation of glycotope recognized by several Siglecs (34), which was independently confirmed by another group (35). For several Siglecs, mucin-like glycoproteins appear to be effective counter-receptors.…”

Exposure of Keratinocytes to Candida Albicans in the Context of Atopic Milieu Induces Changes in the Surface Glycosylation Pattern of Small Extracellular Vesicles to Enhance Their Propensity to Interact With Inhibitory Siglec Receptors

“…Hence, given the role of O-glycosylation for Siglec-7 recognition, it seems that the changes in the expression of this enzyme that we noted upon analysis of data from both the epidermal samples and cytokine-stimulated keratinocytes (normal (35) and FLG insufficient) may also critically contribute to the observed effect. It is unclear, however, if the direct product(s) of the enzyme (Tantigen) may be detected by Siglec-7; further glycan modifications by sequential enzymatic action of additional capping enzymes are likely required for the recognition; this is where ST6GAL1 may execute its role in our system as moiety exposed as a part of complex Siglec-7-recognized epitopes; additional role of sulfation has to be also considered given that a group of carbohydrate sulfotransferases (CHSTs) has been implicated in the binding affinity for both Siglecs we found important for sEV-cell interactions (61,63). Some moieties may be of course less involved, e.g.…”