Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways

Abstract

Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells-primarily eosinophils and mast cells-where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose-acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

Keratan sulfate (KS)‐proteoglycans and neuronal regulation in health and disease: the importance ofKS‐glycodynamics and interactive capability with neuroregulatory ligands

“…MAb BKS-1(+) was raised to a keratanase-generated [N-acetylglucosamine-6-sulfate (GlcNAc-6-S)] stub neoepitope at the non-reducing terminus of corneal and skeletal KS (Akhtar et al 2008). Two pattern recognition lectin receptors, sialic acid binding Ig-like lectins (Gonzalez-Gil et al 2018), and dendritic cell Langerin identify sialylated KS and the galactose-6 sulfate epitope on KS.…”

Structural insight into the binding of human galectins to corneal keratan sulfate, its desulfated form and related saccharides

“…Nonetheless, KS indeed apparently binds to Gal-7 as with the other galectins. On the glycosaminoglycan side, versatility to act as a molecular glue is further achieved by an α2,3-sialylated branch that is a counter-receptor for Gal-8N (aside from Gal-3) 22 and for human siglec-8 69 . The 6′-O-sulfation of Gal serves as a secondary motif for the binding of human siglec 70 (although this cannot be extrapolated among species), because murine siglec-F ligands do not require 6′-O-sulfation by any of the two galactose 6-O-sulfotransferases (KSGal6ST, C6ST-1) 71 .…”

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

“…Testing of a second saline installation from 1 patient (from donor 64 [Fig 2, B]) revealed that the initial nasal lavage efficiently removed essentially all of the Siglec-8 ligand from the airway, which is consistent with its absence from the epithelium of washed tissue sections. Because we had previously found that all Siglec-8 ligands revealed by airway tissue section overlay on both cartilage and submucosal glands were carried on KS glycan chains, 14 nasal lavage samples were resolved and blotted in duplicate and tested for the presence of Siglec-8 ligands and KS (with the latter tested by using an antibody to highly sulfated KS). The major KS proteoglycans in human lavage samples overlapped but were not congruent with Siglec-8 ligand (Fig 2, B), which is consistent with the conclusion that not all KS proteoglycans are Siglec-8 ligands.…”