Sialylated keratan sulfate chains are ligands for Siglec-8 in human airways

asthma;glycan;siglec;eosinophil;apoptosis;trachea;Biochemistry

Porell, RyanSchnaar, Ronald L ;

Johns Hopkins University

关键词: asthma;    glycan;    siglec;    eosinophil;    apoptosis;    trachea;    Biochemistry;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60094/PORELL-DISSERTATION-2018.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Airway inflammatory diseases are characterized by infiltration of immune cells, which are tightly regulated to limit inflammatory damage. Most members of the Siglec family of sialoglycan binding proteins are expressed on the surfaces of immune cells and are immune inhibitory when they bind their sialoglycan ligands. When Siglec-8 on activated eosinophils and mast cells binds to its sialoglycan ligands, apoptosis or inhibition of mediator release is induced. We identified human airway Siglec-8 ligands as sialylated and 6’-sulfated keratan sulfate (KS) chains carried on large proteoglycans. Siglec-8-binding proteoglycans from human airways increase eosinophil apoptosis in vitro.Given the structural complexity of intact proteoglycans, target KS chains were isolated from airway tissue and lavage. Biological samples were extensively proteolyzed, the remaining sulfated glycan chains captured and resolved by anion exchange chromatography, methanol-precipitated then chondroitin and heparan sulfates enzymatically hydrolyzed. The resulting preparation consisted of KS chains attached to a single amino acid or a short peptide. Purified KS chains were hydrolyzed with either hydrochloric acid or trifluoroacetic acid to release acidic and neutral sugars, respectively, followed by DIONEX carbohydrate analysis. To isolate Siglec-8-binding KS chains, purified KS chains from biological samples were biotinylated at the amino acid, resolved by affinity and/or size-exclusion chromatography, the resulting fractions immobilized on streptavidin microwell plates, and probed for binding of Siglec-8-Fc. Siglec-8 affinity chromatography of the tagged KS chains was performed on a nickel column derivatized with a pentameric construct of Siglec-8. Most of the KS chains flowed through the column, whereas Siglec-8 binding was retained through 150 mM NaCl and eluted with 1.5 M NaCl. Based on size separation, Siglec-8-binding KS chains are high molecular weight (>40 kD). Similarly, purified KS chains were cleaved with keratanase I, hydrazine-biotin tagged on their reducing ends, size-separated, and resulting fractions immobilized on streptavidin microwell plates. Keratanase I-cleaved KS chains showed a Siglec-8-binding peak at ~2 kD, indicating a terminal oligosaccharide epitope resistant to keratanase I that retained robust Siglec-8 binding. These data reveal a minor fraction of terminally sialylated KS chains as the Siglec-8 ligands in human airway tissues and secretions.