Isolation, identification, and characterization of the human airway ligand for the eosinophil and mast cell immunoinhibitory receptor Siglec-8

Anabel Gonzalez-Gil, T. August Li, Ryan N. Porell, Steve M. Fernandes, Haley E. Tarbox, Hyun Sil Lee, Kazuhiro Aoki, Michael Tiemeyer, Jean Kim, Ronald L. Schnaar

Research output: Contribution to journalArticlepeer-review

Abstract

Background: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid–containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. Objective: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. Methods: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. Results: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. Conclusion: A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.

Original languageEnglish (US)
Pages (from-to)1442-1452
Number of pages11
JournalJournal of Allergy and Clinical Immunology
Volume147
Issue number4
DOIs
StatePublished - Apr 2021

Keywords

  • DMBT1
  • GP340
  • SALSA
  • Siglec-8
  • keratan sulfate
  • mucus layer
  • nasal lavage
  • sialic acid
  • submucosal gland
  • upper airway

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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