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

JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY

卷:147

Article

Gonzalez-Gil, Anabel¹  Li, T. August¹  Porell, Ryan N.¹  Fernandes, Steve M.¹  Tarbox, Haley E.¹  Lee, Hyun Sil²  Aoki, Kazuhiro⁴  Tiemeyer, Michael⁴  Kim, Jean^2,3  Schnaar, Ronald L.¹ 

[1] Johns Hopkins Univ, Sch Med, Dept Pharmacol & Mol Sci, 725 N Wolfe St, Baltimore, MD 21205 USA

[2] Johns Hopkins Univ, Sch Med, Dept Med, Div Allergy & Clin Immunol, Baltimore, MD 21205 USA

[3] Johns Hopkins Univ, Sch Med, Dept Otolaryngol Head & Neck Surg, Baltimore, MD 21205 USA

[4] Univ Georgia, Complex Carbohydrate Res Ctr, 220 Riverbend Rd, Athens, GA 30602 USA

关键词: Siglec-8;    upper airway;    mucus layer;    nasal lavage;    DMBT1;    GP340;    SALSA;    sialic acid;    keratan sulfate;    submucosal gland;   

DOI  :  10.1016/j.jaci.2020.08.001

来源: Elsevier

【 摘 要 】

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.

【 授权许可】

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