title: Galactose 6-O-sulfotransferases are not required for the generation of Siglec-F ligands in leukocytes or lung tissue.
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.
authors.affiliation: From the Department of Anatomy and Program in Biomedical Sciences, University of California, San Francisco, California 94143-0452.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Patnode
authors.forename: Chu-Wen
authors.initials: CW
authors.lastname: Cheng
authors.forename: Chi-Chi
authors.initials: CC
authors.lastname: Chou
authors.forename: Mark S
authors.initials: MS
authors.lastname: Singer
authors.forename: Matilda S
authors.initials: MS
authors.lastname: Elin
authors.forename: Kenji
authors.initials: K
authors.lastname: Uchimura
authors.forename: Paul R
authors.initials: PR
authors.lastname: Crocker
authors.forename: Kay-Hooi
authors.initials: KH
authors.lastname: Khoo
authors.forename: Steven D
authors.initials: SD
authors.lastname: Rosen
mesh_terms: D000818:Animals; D015214:Antigens, Differentiation, Myelomonocytic / Q000737:chemistry* / Q000378:metabolism; D001992:Bronchoalveolar Lavage Fluid; D002462:Cell Membrane / Q000378:metabolism; D004804:Eosinophils / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005434:Flow Cytometry; D005690:Galactose / Q000737:chemistry*; D007962:Leukocytes / Q000378:metabolism*; D008024:Ligands; D008168:Lung / Q000378:metabolism* / Q000469:parasitology; D016676:Macrophages, Alveolar / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D009077:Mucins / Q000378:metabolism; D009559:Nippostrongylus; D011134:Polysaccharides / Q000032:analysis; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D017206:Strongylida Infections / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism* / Q000502:physiology; D000097811:Carbohydrate Sulfotransferases
chemical_list: D015214:Antigens, Differentiation, Myelomonocytic; D005690:Galactose; D008024:Ligands; D009077:Mucins; D011134:Polysaccharides; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases; C443130:Siglecf protein, mouse
keywords: Eosinophils; Galactose-6-O-Sulfate; Lectin; Lung; Mucins; Siglec-F; Sulfotransferase