Glycan array screening reveals a candidate ligand for Siglec-8

Sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8) is selectively expressed on human eosinophils, basophils, and mast cells, where it regulates their function and survival. Previous studies demonstrated sialic acid-dependent binding of Siglec-8 but failed to reveal significant substructure specificity or high affinity of that binding. To test a broader range of potential ligands, a Siglec-8-Ig chimeric protein was tested for binding to 172 different glycan structures immobilized as biotinylated glycosides on a 384-well streptavidin-coated plate. Of these, ∼40 structures were sialylated. Among these, avid binding was detected to a single defined glycan, NeuAca2r-3(6-O-sulfo)Galβ1-4[Fucα1-3]GlcNAc, also referred to in the literature as 6′-sulfo-sLe^x. Notably, neither unsulfated sLe^x (NeuAcα2-3Galβ1-4[Fucα1-3]GlcNAc) nor an isomer with the sulfate on the 6-position of the GlcNAc residue (6-sulfo-sLe ^x, NeuAcα2-3Galβ1-4[Fucα1-3](6-O-sulfo)GlcNAc) supported detectable binding. Subsequent secondary screening was performed using surface plasmon resonance. Biotin glycosides immobilized on streptavidin biosensor chips were exposed to Siglec-8-Ig in solution. Whereas surfaces derivatized with sLe^x and 6-sulfo-sLe^x failed to support detectable Siglec-8 binding, 6′-sulfo-sLe^x supported significant binding with a K_d of 2.3 μM. In a separate test of binding specificity, aminopropyl glycosides were covalently immobilized at different concentrations on activated (N-hydroxysuccinimidyl) glass surfaces (Schott-Nexterion Slide H). Subsequent exposure to Siglec-8-Ig precomplexed with fluorescein isothiocyanate anti-human Fc resulted in fluorescent signals at immobilized concentrations of 6′-sulfo-sLe^x of <5 pmol/spot. In contrast, sLe^x and 6-sulfo-sLex did not support any Siglec-8 binding at the highest concentration tested (300 pmol/spot). We conclude that Siglec-8 binds preferentially to the sLe^x structure bearing an additional sulfate ester on the galactose 6-hydroxyl.