Glycan Array Screening Reveals a Candidate Ligand for Siglec-8*

Bruce S. Bochner,John R. White,Ronald L. Schnaar,Richard A. Alvarez,Nicolai V. Bovin,Ola Blixt,Padmaja Mehta

doi:10.1074/jbc.m412378200

Abstract

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, approximately 40 structures were sialylated. Among these, avid binding was detected to a single defined glycan, NeuAcalpha2-3(6-O-sulfo)Galbeta1-4[Fucalpha1-3]GlcNAc, also referred to in the literature as 6'-sulfo-sLex. Notably, neither unsulfated sLex (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc) nor an isomer with the sulfate on the 6-position of the GlcNAc residue (6-sulfo-sLex, 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 sLex and 6-sulfo-sLex failed to support detectable Siglec-8 binding, 6'-sulfo-sLex supported significant binding with a Kd of 2.3 microm. 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-sLex of <5 pmol/spot. In contrast, sLex 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 sLex structure bearing an additional sulfate ester on the galactose 6-hydroxyl.

Highlights

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
Additional experiments using monoclonal antibodies revealed that Siglec-8 is expressed on the surface of eosinophils and on basophils and mast cells [14], and the existence of both the Siglec-8 and Siglec-8L isoforms was verified in human eosinophils, basophils, and mast cells [18, 19]
Of additional relevance was that closely related structures to 181, such as structure 182, known as 6-sulfo-sLex NeuAc␣2–3Gal␤1– 4(Fuc␣1–3)(6-O-Su)GlcNAc␤1-O(CH2)3NHCO(CH2)5NH-biotin, which only differs from structure 181 by the location of the 6-O-Su (Fig. 2), and structure 108, known as sLex or NeuAc␣2–3Gal␤1– 4(Fuc␣1–3)GlcNAc␤1O(CH2)2NHCO(CH2)5NHCO(CH2)5NH-biotin, had minimally increased binding affinity (1.2:1 and 0.8:1 signal-to-noise binding, respectively) for Siglec-8

Summary

A Candidate Ligand for Siglec-8

Rosettes are formed with Siglec-8, and neuraminidase treatment alters rosette formation [14, 15]. Specific structures shown to bind Siglec-8 include forms of sialic acid that are linked ␣2–3 or ␣2– 6 to Gal␤1– 4GlcNAc [15]. Among several cores is the protein-carbohydrate interaction core H and carbohydrate synthesis protein expression core D These cores have developed a high throughput screening platform for identifying glycan-binding protein-ligand interactions using a streptavidin/biotin-based glycan array containing ϳ180 different glycan structures immobilized as biotinylated glycosides on a 384-well streptavidin-coated plate. This was developed as an expansion of previous efforts [29] and in conjunction with the carbohydrate synthesis core D to determine the glycan binding specificity of glycan-binding proteins. We report that these and other approaches have been used to determine that Siglec-8 is a highly specific lectin, binding preferentially to the sLex structure bearing an additional sulfate ester on the galactose 6-hydroxyl, namely NeuAc␣2– 3(6-O-sulfo)Gal␤1– 4[Fuc␣1–3]GlcNAc, referred to in the literature as 6Ј-sulfo-sLex, which is a structure closely related to 6-sulfo-sLex, a candidate ligand for L-selectin

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION