Abstract
Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.
Highlights
The cell surface lectin Siglec-F is thought to preferentially recognize ligands modified with galactose 6-O-sulfate
None of the ligands we describe are diminished in mice lacking KSGal6ST and chondroitin 6-O-sulfotransferase 1 (C6ST-1), indicating that neither of the known galactose 6-Osulfotransferases is required for ligand synthesis
We demonstrate that the cell types previously known to express Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages [15,16,17,18], correspondingly express ligands for this receptor on their surface
Summary
The cell surface lectin Siglec-F is thought to preferentially recognize ligands modified with galactose 6-O-sulfate. Endogenous ligands have yet to be biochemically defined, experiments using polyacrylamide-linked glycans have established that Siglec-F prefers ␣2,3-linked sialic acid residues [25] Consistent with this specificity, Siglec-F-Fc staining of airway epithelium and alveolar cells is blocked by Maackia amurensis agglutinin which recognizes ␣2,3-linked sialic acids, and absent in mice lacking the ␣2,3-sialyltransferase ST3Gal3 [26, 27]. An antibody that recognizes 6Ј-sulfo-sLex stains mouse airway epithelium where Siglec-F ligands have been detected [30], this antibody binds other glycan structures Based on these data, the prevailing view has been that galactose 6-O-sulfate (Gal6S) is likely to be a critical recognition element for Siglec-F in vivo. Contrary to expectations, neither KSGal6ST nor C6ST-1 is required for the generation of these ligands
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