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Link to original content: https://pubmed.ncbi.nlm.nih.gov/23880769
Galactose 6-O-sulfotransferases are not required for the generation of Siglec-F ligands in leukocytes or lung tissue - PubMed Skip to main page content
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. 2013 Sep 13;288(37):26533-45.
doi: 10.1074/jbc.M113.485409. Epub 2013 Jul 23.

Galactose 6-O-sulfotransferases are not required for the generation of Siglec-F ligands in leukocytes or lung tissue

Michael L Patnode  1 , Chu-Wen Cheng, Chi-Chi Chou, Mark S Singer, Matilda S Elin, Kenji Uchimura, Paul R Crocker, Kay-Hooi Khoo, Steven D Rosen
Affiliations

Galactose 6-O-sulfotransferases are not required for the generation of Siglec-F ligands in leukocytes or lung tissue

Michael L Patnode et al. J Biol Chem. .

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.

Keywords: Eosinophils; Galactose-6-O-Sulfate; Lectin; Lung; Mucins; Siglec-F; Sulfotransferase.

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Figures

FIGURE 1.
FIGURE 1.
Siglec-F ligand expression in peripheral blood leukocytes. A, flow cytometry analysis of leukocyte subsets stained with Siglec-F-Fc, Siglec-E-Fc, and CD22-Fc (red histograms). Staining after sialidase treatment (black histograms) and staining with human IgG (gray histograms) are shown. Results are representative of four independent experiments. B, flow cytometry analysis of Siglec-F expression on leukocytes from wild type (black histograms) or Siglec-F KO mice (gray histograms). Results are representative of two independent experiments. Eos, eosinophils; Neut, neutrophils; Baso, basophils; Mono, classical monocytes; NK, natural killer cells; T, T cells; B, B cells; Alv Mac, alveolar macrophages.
FIGURE 2.
FIGURE 2.
Siglec-F ligand expression in resident lung cells. A, cryostat-cut sections of lungs from WT mice stained with Siglec-F-Fc (green), anti-sialoadhesin (red), anti-proSP-C (blue), and DAPI (white). Arrowheads mark alveolar macrophages. Arrows mark type II alveolar epithelial cells. The asterisk marks airway epithelium. B, a high power view of the same section shown in A. Siglec-F-Fc staining after sialidase treatment, Siglec-E-Fc staining, and CD22-Fc staining are shown. The inset depicts a type II AEC from a different field (scale bar 10 μm). Results are representative of four independent experiments. C, flow cytometry analysis of Siglec-F-Fc staining of leukocytes from wild type (red histograms) or Siglec-F KO (blue histograms) mice. Sialidase treatment eliminated staining on cells from both Siglec-F KO (gray histograms) and wild type mice (not shown). Results are representative of two independent experiments. Eos, eosinophils; Neut, neutrophils; Alv Mac, alveolar macrophages. Scale bars represent 50 μm unless otherwise stated.
FIGURE 3.
FIGURE 3.
Siglec-F ligands in BAL fluid. A, fractions of BAL fluid assayed by ELISA using Siglec-F-Fc (filled circles), or anti-MUC5B (filled squares). Total protein was determined by measuring absorbance at 280 nm (dotted line). No signal was observed when wells were reacted with CD22-Fc (open squares), human IgG (open triangles), or treated with sialidase before incubation with Siglec-F-Fc (open circles). Results are representative of two independent experiments.
FIGURE 4.
FIGURE 4.
Siglec-F ligand expression during N. brasiliensis infection. A, cryostat-cut serial sections of lungs from N. brasiliensis infected or uninfected mice. Sections were treated with sialidase or buffer alone, then stained with Siglec-F-Fc (green), anti-CD11b (red), and DAPI (blue). B, high power images of the same lung sections from A, treated with sialidase or buffer alone, then stained with Siglec-F-Fc, Siglec-E-Fc, or CD22-Fc (green); anti-eMBP (red); anti-proSP-C (blue); and DAPI (white). Scale bar represents 50 μm. Results are representative of two independent experiments.
FIGURE 5.
FIGURE 5.
MS analysis of sulfated glycans in eosinophils. A, flow cytometry analysis of IL-5 transgenic eosinophils stained with Siglec-F-Fc (red histogram). Staining after sialidase treatment (black histogram) and staining with human IgG (gray histogram) is shown. Results are representative of three independent experiments. B, extracted ion chromatograms of the major sulfated O-glycans from IL-5 transgenic eosinophils as detected by nanoLC-MS/MS analysis. The m/z values for the [M − H]− molecular ions afforded by the mono-sulfated permethylated O-glycans were annotated along with the assigned structures based on interpretation of the HCD and CID MS/MS data. The relative peak heights are indicative of the relative abundance of each of the sulfated, sialylated core 1 and core 2 O-glycans. C, low mass regions of the negative ion mode nanoESI HCD and CID MS/MS spectra of mono-sulfated di-sialylated (left) and mono-sulfated mono-sialylated (right) structures. Assignment of the major peaks for all spectra is annotated using the standard schematic symbols. Eos, eosinophils.
FIGURE 6.
FIGURE 6.
Analysis of sulfated glycans in BAL fluid and lung KS. A, representative negative ion mode nanoESI HCD MS/MS spectra of mono-sulfated mono-sialylated (top) and mono-sulfated non-sialylated (bottom) O-glycan structures found in BAL fluid from wild type mice. Identification of terminal Gal3S and internal GlcNAc6S was based on previously established diagnostic ions. A weak signal at m/z 167 (bold) may indicate the presence of Gal6S in the non-sialylated structures. Additional clusters of fragment ions around m/z 500–800 are assigned as shown, which are mostly fragment ions resulting from cleavage along the GalNAcitol and consistent with the sulfated LacNAc being extended from the 6-arm. B, reversed-phase ion-pair chromatography analysis of KS from lungs of wild type mice. Standard substances were eluted at the peak positions indicated by arrows. The fragment (6S)Galβ1→4(6S)GlcNAc was not detected in the sample.
FIGURE 7.
FIGURE 7.
Siglec-F ligand expression in lungs from KSGal6ST/C6ST-1 DKO mice. A, cryostat-cut sections of lungs from WT or DKO mice stained with Siglec-F-Fc (green), anti-sialoadhesin (red), anti-proSP-C (blue), and DAPI (white). Scale bar represents 50 μm. Results are representative of two independent experiments. B, Siglec-F-Fc reactivity (left) in BAL fluid fractions from WT (filled circles) or DKO mice (filled squares), assayed by ELISA. Results are representative of two independent experiments. The signal was eliminated by sialidase treatment (open circles, open squares). Anti-MUC5B reactivity (right) in BAL fluid fractions from WT (filled circles) and DKO mice (filled squares) was assayed by ELISA. Isotype control signal was minimal (open circles, open squares). Total protein was determined by measuring absorbance at 280 nm for wild type (dotted line) and DKO mice (dashed line). WT, wild type; DKO, KSGal6ST/C6ST-1 double knock-out.
FIGURE 8.
FIGURE 8.
Siglec-F ligand expression in leukocytes from KSGal6ST/C6ST-1 DKO mice. A, flow cytometry analysis of leukocyte subsets stained with Siglec-F-Fc (red histograms). Staining after sialidase treatment (black histograms) and staining with human IgG (gray histograms) are shown. Scale bars represent 50 μm. Results are representative of two independent experiments. B, cryostat-cut sections of lungs from N. brasiliensis-infected mice. Sections were stained with Siglec-F-Fc (green), anti-eMBP (red), anti-proSP-C (blue), and DAPI (white). Low power (top) and high power (bottom) fields are shown. Eos, eosinophils; Neut, neutrophils; Mono, classical monocytes; NK, natural killer cells.
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