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, similar to40 structures were sialylated Among these, avid binding was detected to a single defined glycan, NeuAcalpha-2-3(6-O-sulfo)Galbeta1-4[fucalpha1-3]GlcNAc, also referred to in the literature as 6'-sulfo-sLe(x). Notably, neither unsulfated sLe(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc) nor an isomer with the sulfate on the 6-position of the GlcNAc residue (6-sulfo-sLe(x), NeuAcalpha2-3Galbeta1-4[Fucalpha1-3](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'-sulfosLe(x) supported significant binding with a K-d of 2.3 mum. 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-sLe(x) 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.