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Glycobiology. 2018 Oct 1;28(10):786-801. doi: 10.1093/glycob/cwy057.

Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways.

Author information

1
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
2
Department of Medicine, Division of Allergy and Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
3
Departments of Chemical Physiology, Cell and Molecular Biology, and Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA.
4
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.

Abstract

Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells-primarily eosinophils and mast cells-where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose-acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5AcĪ±2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

PMID:
29924315
PMCID:
PMC6142871
[Available on 2019-07-17]
DOI:
10.1093/glycob/cwy057

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