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

Anabel Gonzalez-Gil, Ryan N. Porell, Steve M. Fernandes, Yadong Wei, Huifeng Yu, Daniela J. Carroll, Ryan McBride, James C. Paulson, Michael Tiemeyer, Kazuhiro Aoki, Bruce S. Bochner, Ronald Lee Schnaar

Research output: Contribution to journalArticle

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 Neu5Acα2-3(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.

Original languageEnglish (US)
Pages (from-to)786-801
Number of pages16
JournalGlycobiology
Volume28
Issue number10
DOIs
StatePublished - Oct 1 2018

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Gonzalez-Gil, A., Porell, R. N., Fernandes, S. M., Wei, Y., Yu, H., Carroll, D. J., McBride, R., Paulson, J. C., Tiemeyer, M., Aoki, K., Bochner, B. S., & Schnaar, R. L. (2018). Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways. Glycobiology, 28(10), 786-801. https://doi.org/10.1093/glycob/cwy057