Journal
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
Volume 147, Issue 4, Pages 1442-1452Publisher
MOSBY-ELSEVIER
DOI: 10.1016/j.jaci.2020.08.001
Keywords
Siglec-8; upper airway; mucus layer; nasal lavage; DMBT1; GP340; SALSA; sialic acid; keratan sulfate; submucosal gland
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Funding
- National Institutes of Health [U19-AI136443, K12-HL141952, T32-GM008763, T32-GM080189]
- Flight Attendant Medical Research Institute
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The study identified a high molecular weight Siglec-8 ligand on the mucus layer of human airways, composed of DMBT1S8 with picomolar affinity for Siglec-8. This finding is important for understanding the pathogenesis and treatment of airway diseases.
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.
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