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Endogenous airway mucins carry glycans that bind Siglec-F and induce eosinophil apoptosis.

ABSTRACT: BACKGROUND:Sialic acid-binding, immunoglobulin-like lectin (Siglec) F is a glycan-binding protein selectively expressed on mouse eosinophils. Its engagement induces apoptosis, suggesting a pathway for ameliorating eosinophilia in the setting of asthma and other eosinophil-associated diseases. Siglec-F recognizes sialylated sulfated glycans in glycan-binding assays, but the identities of endogenous sialoside ligands and their glycoprotein carriers in vivo are unknown. OBJECTIVES:To use mouse lung-derived materials to isolate, biochemically identify, and biologically characterize naturally occurring endogenous glycan ligands for Siglec-F. METHODS:Lungs from normal and mucin-deficient mice, as well as mouse tracheal epithelial cells, were investigated in vitro and in vivo for the expression of Siglec-F ligands. Western blotting and cytochemistry used Siglec-F-Fc as a probe for directed purification, followed by liquid chromatography-tandem mass spectrometry of recognized glycoproteins. Purified components were tested in mouse eosinophil-binding assays and flow cytometry-based cell death assays. RESULTS:We detected mouse lung glycoproteins that bound to Siglec-F; binding was sialic acid dependent. Proteomic analysis of Siglec-F binding material identified Muc5b and Muc4. Cross-affinity enrichment and histochemical analysis of lungs from mucin-deficient mice assigned and validated the identity of Muc5b as one glycoprotein ligand for Siglec-F. Purified mucin preparations carried sialylated and sulfated glycans, bound to eosinophils and induced their death in vitro. Mice conditionally deficient in Muc5b displayed exaggerated eosinophilic inflammation in response to intratracheal installation of IL-13. CONCLUSIONS:These data identify a previously unrecognized endogenous anti-inflammatory property of airway mucins by which their glycans can control lung eosinophilia through engagement of Siglec-F.

SUBMITTER: Kiwamoto T

PROVIDER: S-EPMC4433759 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Endogenous airway mucins carry glycans that bind Siglec-F and induce eosinophil apoptosis.

Kiwamoto Takumi T Katoh Toshihiko T Evans Christopher M CM Janssen William J WJ Brummet Mary E ME Hudson Sherry A SA Zhu Zhou Z Tiemeyer Michael M Bochner Bruce S BS

The Journal of allergy and clinical immunology 20141212 5

<h4>Background</h4>Sialic acid-binding, immunoglobulin-like lectin (Siglec) F is a glycan-binding protein selectively expressed on mouse eosinophils. Its engagement induces apoptosis, suggesting a pathway for ameliorating eosinophilia in the setting of asthma and other eosinophil-associated diseases. Siglec-F recognizes sialylated sulfated glycans in glycan-binding assays, but the identities of endogenous sialoside ligands and their glycoprotein carriers in vivo are unknown.<h4>Objectives</h4>To ...[more]

PMID: 25497369

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Project description:Mucous secretions were obtained from cat tracheas that had received [3H]glucose and [35S]sulphate to radiolabel mucus glycoproteins biosynthetically. Samples were collected under resting ('basal') conditions as well as after pilocarpine stimulation and were separated into gel and sol phases by centrifugation. Macromolecules were partially purified by using gel chromatography on Sepharose CL-4B, and the species that were eluted with the void volume were then separated into two major populations with isopycnic density-gradient centrifugation in CsCl. The major component from the gel phase of pilocarpine-induced secretions had a buoyant density typical of mucins and was observed as linear and apparently flexible chains by electron microscopy. Reduction of disulphide bonds gave subunits that could be further cleaved by trypsin digestion into components of approximately the same size as the high-Mr glycopeptides obtained from other mucins after this treatment. In contrast, the dominant species in the gel phase of the 'basal' secretion had a significantly higher buoyant density than expected for mucins and was largely unaffected by reduction, as studied by gel chromatography. The macromolecules were fragmented by trypsin, suggesting that they contain a polypeptide backbone. This more dense component also predominated in the sol phase both from the 'basal' secretions and from the pilocarpine-released secretions. Digestion with DNAase, chondroitin ABC lyase or heparan sulphate lyase had no effect, which shows that this component is not DNA, a dermatan sulphate/chondroitin sulphate or a heparan sulphate proteoglycan. In contrast, endo-beta-galactosidase and keratanase caused some fragmentation, suggesting that the molecules contain some linkages of the poly-(N-acetyl-lactosamine) type, although the degradation was not as extensive as expected for keratan sulphate. Treatment with alkaline borohydride resulted in extensive fragmentation of the high-Mr glycopeptides from both components, indicating that the glycans were oligosaccharides that were probably O-linked. The monosaccharide compositions of both components were consistent with that expected for mucins. The data are in keeping with the major component from the pilocarpine-stimulated gel secretions being a mucus glycoprotein and the more dense component being a mucin-like molecule, possibly related to the keratanase-sensitive material isolated from canine trachea by Varsano, Basbaum, Forsberg, Borson, Caughey & Nadel [(1987) Exp. Lung Res. 13, 157-184].

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Endogenous airway mucins carry glycans that bind Siglec-F and induce eosinophil apoptosis.

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Endogenous airway mucins carry glycans that bind Siglec-F and induce eosinophil apoptosis.

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