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. 2010 Dec 24;285(52):40864-78.
doi: 10.1074/jbc.M110.167296. Epub 2010 Oct 7.

Novel anti-carbohydrate antibodies reveal the cooperative function of sulfated N- and O-glycans in lymphocyte homing

Jotaro Hirakawa  1 , Koichiro Tsuboi, Kaori Sato, Motohiro Kobayashi, Sota Watanabe, Atsushi Takakura, Yasuyuki Imai, Yuki Ito, Minoru Fukuda, Hiroto Kawashima
Affiliations

Novel anti-carbohydrate antibodies reveal the cooperative function of sulfated N- and O-glycans in lymphocyte homing

Jotaro Hirakawa et al. J Biol Chem. .

Abstract

Cell surface glycans play pivotal roles in immune cell trafficking and immunity. Here we present an efficient method for generating anti-carbohydrate monoclonal antibodies (mAbs) using gene-targeted mice and describe critical glycans in lymphocyte homing. We immunized sulfotransferase GlcNAc6ST-1 and GlcNAc6ST-2 doubly deficient mice with sulfotransferase-overexpressing Chinese hamster ovary cells and generated two mAbs, termed S1 and S2. Both S1 and S2 bound high endothelial venules (HEVs) in the lymphoid organs of humans and wild-type mice, but not in those of doubly deficient mice. Glycan array analysis indicated that both S1 and S2 specifically bound 6-sulfo sialyl Lewis X and its defucosylated structure. Interestingly, S2 inhibited lymphocyte homing to peripheral lymph nodes by 95%, whereas S1 inhibited it by only 25%. S2 also significantly inhibited contact hypersensitivity responses and L-selectin-dependent leukocyte adhesion to HEVs. Immunohistochemical and Western blot analyses indicated that S1 preferentially bound sulfated O-glycans, whereas S2 bound both sulfated N- and O-glycans in HEVs. Furthermore, S2 strongly inhibited the N-glycan-dependent residual lymphocyte homing in mutant mice lacking sulfated O-glycans, indicating the importance of both sulfated N- and O-glycans in lymphocyte homing. Thus, the two mAbs generated by a novel method revealed the cooperative function of sulfated N- and O-glycans in lymphocyte homing and immune surveillance.

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Figures

FIGURE 1.
FIGURE 1.
Structures of the O- and N-glycans that are modified with 6-sulfo sialyl Lewis X structures, and a novel strategy for generating anti-carbohydrate mAbs. A, 6-sulfo sialyl Lewis X structures on O- and N-glycans. Core 2 branched O-glycan (upper left), extended core 1 structure (upper middle), biantennary O-glycan containing both a core 2 branch and extended core 1 structure (upper right), and N-glycans (bottom) can be modified with 6-sulfo sialyl Lewis X (shown in gray). The extended core 1 structure modified with GlcNAc-6-O-sulfate (shown in yellow) is recognized by the mAb MECA-79 (16). B, functional analysis of glycogenes. Glycogene-deficient (Gene A KO) mice and glycogene-overexpressing transfectant cells have been widely used for functional analyses. C, a novel strategy for generating anti-carbohydrate mAbs. Glycogene-deficient mice are immunized with glycogene-overexpressing transfected cells. The carbohydrate structures formed by the glycogene-encoded enzyme are expected to be highly antigenic in glycogene-deficient mice.
FIGURE 2.
FIGURE 2.
Specific binding of S1 and S2 to sulfated glycans in HEVs. Immunofluorescence of acetone-fixed frozen sections of PLNs, MLNs, and PPs from WT and GlcNAc6ST-1 and GlcNAc6ST-2 DKO mice reacted with S1 and S2 (red). Nuclear staining was performed with DAPI (blue). The data are representative of three independent experiments. Bar, 50 μm.
FIGURE 3.
FIGURE 3.
Binding of S1 and S2 to sulfated and sialylated glycoproteins but not glycolipids. A, parental CHO (CHO-K1), CHO/CD34/F7/C1/C2/GlcNAc6ST-2, and CHO/CD34/F7/GlcNAc6ST-2 cells were stained with purified S1, S2, or MECA-79 using FITC-conjugated secondary Abs. B–D, the GlcNAc6ST-2 gene was transiently transfected into CHO-K1 (B), Lec1 (C), or Lec2 (D) cells with a pcDNA3.1/EGFP expression vector. Forty-eight hours after transfection, the binding of S1, S2, and MECA-79 was determined by flow cytometry, as described under “Experimental Procedures.” None, untransfected cells. Mock, cells mock-transfected with pcDNA3.1 empty vector together with the pcDNA3.1/EGFP expression vector. The data are representative of three (A and B) or two (C and D) independent experiments.
FIGURE 4.
FIGURE 4.
Inhibition of lymphocyte homing and CHS responses by S2. A and B, CMFDA-labeled lymphocytes (2.0 × 107 cells) were injected into the tail vein of WT (A) or GlcNAc6ST-1 and GlcNAc6ST-2 DKO mice (B). One hour after injection, CMFDA-labeled lymphocytes in lymphocyte suspensions from the PLNs, MLNs, PPs, and spleens were quantified by flow cytometry. The mice were preinjected with MECA-79, S1, or S2 (200 μg/mouse) or PBS 2 h before the injection of CMFDA-labeled lymphocytes. Lymphocyte homing to different lymphoid organs in the mAb-injected animals is shown as a percentage of that observed in PBS-injected animals, which was set as 100%. At least three recipient mice were used in each experiment. *, p < 0.0001; **, p < 0.001; ***, p < 0.02 versus PBS-injected control mice. C, ear swelling 24 h after challenge with oxazolone or vehicle alone in WT mice intraperitoneally injected twice with 200 μg/mouse of S1, S2, or PBS alone, as described under “Experimental Procedures.” Open bars, change in thickness of the left ear treated with vehicle alone; closed bars, change in thickness of the right ear treated with oxazolone (OX (+)). *, p < 0.01; N.S., not significant. n = 4. D, hematoxylin-and-eosin staining of ear sections 24 h after oxazolone challenge. *, ear cartilage. Scale bar, 100 μm. Each bar in A–C represents the means ± S.D. The data are representative of three (A) or two (B–D) independent experiments.
FIGURE 5.
FIGURE 5.
Inhibition of the l-selectin-dependent adhesion of leukocytes to HEVs and leukocyte rolling by mAbs. A and B, binding of CMTMR-labeled leukocytes incubated with or without 10 μg/ml rat IgG or MEL-14 to PLN tissue sections incubated with or without 10 μg/ml mouse IgM, S1, S2, rat IgM, or MECA-79. A, photomicrographs of leukocyte binding to HEVs. Dotted lines, outline of HEV. Bar, 40 μm. B, number of bound leukocytes per HEV. The number of cells bound to an HEV is plotted. The number of HEVs analyzed per sample (n) is indicated at the bottom. The horizontal red lines represent the average number of leukocytes bound per HEV. N.S., not significant; *, p < 0.001. C, number of rolling leukocytes per 30 s on CHO transfectants stably expressing the 6-sulfo-sialyl Lewis X structure on N- and O-glycans (CHO/CD34/F7/C1/C2/GlcNAc6ST-2), pretreated with or without 10 μg/ml S1 or S2. Leukocytes applied to the flow chamber were treated with or without 10 μg/ml MEL-14. The data are representative of four (A and B) or three (C) independent experiments.
FIGURE 6.
FIGURE 6.
Glycan array analysis of S1 and S2. Version 4.1 of the printed array of the Consortium for Functional Glycomics was probed with 10 μg/ml S1 or S2. The error bars represent the means ± S.D. of four measurements. Glycan numbers are shown in the parentheses after their structures.
FIGURE 7.
FIGURE 7.
Binding of mAbs to PLN sections from various gene-targeted mice. A, frozen sections (7 μm) of PLNs from WT, FucT-IV, and FucT-VII DKO, C1β3GnT and C2GnT-I DKO, GlcNAc6ST-1 and GlcNAc6ST-2 DKO, GlcNAc6ST-1-deficient, and GlcNAc6ST-2-deficient mice were incubated with 5 μg/ml biotinylated S1, S2, or MECA79 for 2 h (red). B, effects of N-glycosidase F treatment on the binding of mAbs or E-PHA to HEVs. Frozen sections from the PLNs of WT and C1β3GnT and C2GnT-I DKO mice were fixed with cold acetone and treated for 24 h at 37 °C with or without 100 milliunits/ml N-glycosidase F (Calbiochem) in 10 mm HEPES-NaOH (pH 7.4), 0.1% Triton X-100, and complete protease inhibitor (Roche Applied Science) and incubated with biotinylated E-PHA (0.1 μg/ml), MECA-79 (1 μg/ml), or S2 (1 μg/ml) for 1 h. The binding of biotinylated E-PHA and mAbs was detected with Alexa Fluor 594-conjugated streptavidin using a confocal laser scanning microscope (LSM510 META). Bar, 50 μm. The data are representative of four (A) or three (B) independent experiments.
FIGURE 8.
FIGURE 8.
Western blot analysis of PLN lysates with S2 and MECA-79. A, immunoblot of lysates (20 μg) from WT or GlcNAc6ST-1 and GlcNAc6ST-2 DKO (ST DKO) mice probed with 0.5 μg/ml biotinylated MECA-79 or -S2. B, immunoblot of lysates from WT or C1β3GnT and C2GnT-I DKO (C1/C2 DKO) mice treated with buffer alone (−) or with N-glycosidase F (+) probed with 0.5 μg/ml biotinylated E-PHA, -MECA-79, or -S2. N-glycosidase F treatment (100 units/ml; Roche Applied Science) was performed in PBS containing 0.9% Triton X-100 and a protease inhibitor mixture (1:40 dilution; Sigma-Aldrich) for 2 h at 37 °C. Ten micrograms of lysate proteins were applied to each lane, except that 100 μg of lysate proteins from the C1β3GnT and C2GnT-I DKO mice were applied for the blot with S2. The data are representative of three independent experiments.
FIGURE 9.
FIGURE 9.
Inhibition of N-glycan-dependent lymphocyte homing by S2. A, CMFDA-labeled lymphocytes (2.0 × 107 cells) were injected into the tail vein of WT or C1β3GnT and C2GnT-I DKO mice (C1/C2 DKO), and the lymphocyte homing to different lymphoid organs is shown as a percentage of that observed in WT mice, which was set as 100%. Three to four recipient mice were used in each experiment. *, p < 0.005; **, p < 0.03. B, CMFDA-labeled lymphocytes (2.0 × 107 cells) were injected into the tail vein of C1β3GnT and C2GnT-I DKO mice. One hour after injection, CMFDA-labeled lymphocytes in the lymphocyte suspensions from PLNs, MLNs, and PPs were quantified by flow cytometry. The C1β3GnT and C2GnT-I DKO mice were preinjected with 200 μg/mouse of MECA-79, S1, S2, or PBS 2 h before the injection of CMFDA-labeled lymphocytes. In some cases (S2 + AAA), C1β3GnT and C2GnT-I DKO mice were preinjected with 200 μg/mouse of S2 together with 100 μg/mouse of AAA lectin (Seikagaku Kogyo Co.) before the injection of CMFDA-labeled lymphocytes. Lymphocyte homing to different lymphoid organs in the mAb-injected animals is shown as a percentage of that observed in PBS-injected animals, which was set as 100%. Three to four recipient mice were used in each experiment. *, p < 0.001; **, p < 0.025. The data are representative of two independent experiments.
FIGURE 10.
FIGURE 10.
Immunohistochemistry of human tissue sections with the MECA-79, S1, and S2 mAbs. HEVs in the human tonsil were stained well with the MECA-79, S1, and S2 mAbs (top row). HEV-like vessels induced in the gastrointestinal lamina propria, particularly in and around the muscularis mucosae, in chronic H. pylori gastritis (middle row) and ulcerative colitis (bottom row) specimens also showed positive signals. Bar, 50 μm. The data are representative of two independent experiments.
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