Structural basis for sulfation-dependent self-glycan recognition by the human immune-inhibitory receptor Siglec-8

Johannes M. Pröpster; Fan Yang; Said Rabbani; Beat Ernst; Frédéric H.-T. Allain; Mario Schubert

doi:10.1073/pnas.1602214113

New Research In

Edited by Carolyn R. Bertozzi, Stanford University, Stanford, CA, and approved May 20, 2016 (received for review February 10, 2016)

Significance

Siglec-8 downregulates eosinophil- and mast cell-mediated inflammatory responses upon engagement by specific self-glycans. We used solution NMR spectroscopy to determine the structure of the N-terminal lectin domain of human Siglec-8 in complex with its preferred glycan target 6′-sulfo sialyl Lewis^x. Quantitative binding studies with differently sulfated glycans and structure-based mutants demonstrate that Siglec-8 simultaneously recognizes a terminal N-acetylneuraminic acid (sialic acid) and an underlying 6-O–sulfated galactose, yielding a tight and unique specificity. We offer direct structural and mechanistic insights into how the self-glycan code is deciphered by Siglec-8, emphasize the crucial role of glycan sulfation in immunological control of inflammation, and provide a rational framework for designing Siglec-8 agonists to harness its signaling pathway in allergic and inflammatory disorders.

Abstract

Siglec-8 is a human immune-inhibitory receptor that, when engaged by specific self-glycans, triggers eosinophil apoptosis and inhibits mast cell degranulation, providing an endogenous mechanism to down-regulate immune responses of these central inflammatory effector cells. Here we used solution NMR spectroscopy to dissect the fine specificity of Siglec-8 toward different sialylated and sulfated carbohydrate ligands and determined the structure of the Siglec-8 lectin domain in complex with its prime glycan target 6′-sulfo sialyl Lewis^x. A canonical motif for sialic acid recognition, extended by a secondary motif formed by unique loop regions, recognizing 6-O–sulfated galactose dictates tight specificity distinct from other Siglec family members and any other endogenous glycan recognition receptors. Structure-guided mutagenesis revealed key contacts of both interfaces to be equally essential for binding. Our work provides critical structural and mechanistic insights into how Siglec-8 selectively recognizes its glycan target, rationalizes the functional impact of site-specific glycan sulfation in modulating this lectin–glycan interaction, and will enable the rational design of Siglec-8–targeted agonists to treat eosinophil- and mast cell-related allergic and inflammatory diseases, such as asthma.

Footnotes

↵¹To whom correspondence may be addressed. Email: mario.schubert{at}sbg.ac.at or allain{at}mol.biol.ethz.ch.
↵²Present address: Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria.

Author contributions: J.M.P., B.E., and M.S. designed research; J.M.P., F.Y., S.R., and M.S. performed research; J.M.P., F.Y., S.R., and M.S. analyzed data; and J.M.P., F.Y., B.E., F.H.-T.A., and M.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates, NMR chemical shifts, and restraints of the herein reported structures have been deposited in the Protein Data Bank (PDB), www.rscb.org, and the Biological Magnetic Resonance Data Bank (BMRB), www.bmrb.wisc.edu, with the following accession codes: ligand-free Siglec-8 (PDB, 2N7A; BMRB, 25798) and Siglec-8-6′S sLe^x complex (PDB, 2N7B; BMRB, 25799).
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