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, 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.
Bochner
Bruce S
BS
Division of Allergy and Clinical Immunology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA. bbochner@jhmi.edu
Alvarez
Richard A
RA
Mehta
Padmaja
P
Bovin
Nicolai V
NV
Blixt
Ola
O
White
John R
JR
Schnaar
Ronald L
RL
D000117:Acetylglucosamine / Q000737:chemistry; D015703:Antigens, CD / Q000737:chemistry*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000737:chemistry*; D015374:Biosensing Techniques; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D002240:Carbohydrate Sequence; D004305:Dose-Response Relationship, Drug; D005690:Galactose / Q000737:chemistry; D005898:Glass / Q000737:chemistry; D006027:Glycosides / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000737:chemistry*; D008024:Ligands; D008407:Mast Cells / Q000166:cytology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D011485:Protein Binding; D000080506:Sialyl Lewis X Antigen; D019809:Streptavidin / Q000737:chemistry; D013379:Substrate Specificity; D013431:Sulfates / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013997:Time Factors
D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; D006027:Glycosides; D037102:Lectins; D008024:Ligands; D009844:Oligosaccharides; D011134:Polysaccharides; C403967:SIGLEC8 protein, human; D000080506:Sialyl Lewis X Antigen; D013431:Sulfates; D001710:Biotin; D019809:Streptavidin; D000117:Acetylglucosamine; D005690:Galactose