SIGLEC-8 LIGANDS IN HUMAN AIRWAY AND AIRWAY SECRETIONS

Siglecs – sialic acid binding Ig-like lectins – are regulatory molecules expressed on subsets of immune cells where most inhibit inflammation when engaged by complementary sialoglycan ligands on target tissues. Both eosinophils and mast cells express Siglec-8 on the cell surface, and when Siglec-8 binds to sialoglycan ligands on tissues apoptosis of eosinophils and inhibition of mediator release by mast cells is induced, limiting inflammation. Although Siglec-8 has been shown to bind a synthetic glycan 6’-sulfated sialyl N-acetyllactosamine, the endogenous Siglec-8 ligand in human airways was still unknown. This study provides evidence of endogenous high molecular weight Siglec-8 ligands in human airway and airway secretions that are sensitive to sialidase and keratanase treatment. Siglec-8 ligands were isolated and identified from normal postmortem human airways or from nasal lavage. Glycoproteins were separated by size exclusion chromatography and resolved by composite agarose-acrylamide gel electrophoresis, blotted, and probed with human Fc-tagged Siglec-8, revealing three binding species (270 kDa, 600 kDa and 1000 kDa) in tracheal extracts and one major binding specie (~900 kDa) in nasal lavage. Ligand-containing fractions were pooled, and ligands were captured by immunoprecipitation using His-tagged pentameric Siglec-8 bound to nickel-Sepharose beads. Siglec-8-precipitated ligands were subjected to mass spectrometric proteomic analysis, revealing the proteoglycan aggrecan as the predominant protein in all three-size species of Siglec-8 ligands extracted from trachea and glycoprotein-340 as the predominant protein in sample purified from nasal lavage. Anti-aggrecan antibody immunoblots of electrophoresed tracheal purified Siglec-8 ligand revealed co-migrating aggrecan immunoreactivity that was sensitive to aggrecanase treatment. Anti-GP340 antibody immunoblots of electrophoresed nasal lavage purified Siglec-8 ligand revealed co-migrating GP340 immunoreactivity. All Siglec-8 ligands were sensitive to sialidase and keratanase treatment. In conclusion, human airway Siglec-8 ligands are sialylated keratan sulfate chains presented on different proteins dependent on where they are expressed