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Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment

Nature Immunology volume 8, pages 409–418 (2007)Cite this article

Abstract

Lymphocyte homing is mediated by specific interaction between L-selectin on lymphocytes and the carbohydrate ligand 6-sulfo sialyl Lewis X on high endothelial venules. Here we generated mice lacking both core 1 extension and core 2 branching enzymes to assess the functions of O-glycan-borne L-selectin ligands in vivo. Mutant mice maintained robust lymphocyte homing, yet they lacked O-glycan L-selectin ligands. Biochemical analyses identified a class of N-glycans bearing the 6-sulfo sialyl Lewis X L-selectin ligand in high endothelial venules. These N-glycans supported the binding of L-selectin to high endothelial venules in vitro and contributed in vivo to O-glycan-independent lymphocyte homing in wild-type and mutant mice. Our results demonstrate the critical function of N-glycan-linked 6-sulfo sialyl Lewis X in L-selectin-dependent lymphocyte homing and recruitment.

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Figure 1: Core1-β3GlcNAcT-deficient mice have no MECA-79 antigen.
Figure 2: Lymphocyte trafficking in Core1-β3GlcNAcT-deficient mice.
Figure 3: Expression of L-selectin and E-selectin ligands.
Figure 4: Lymphocyte trafficking in double-knockout mice.
Figure 5: N-glycan-based L-selectin ligands in double-knockout and wild-type mice.
Figure 6: N-glycans in double-knockout and wild-type mice contain 6-sulfo sLex and support lymphocyte rolling.
Figure 7: N-glycan-based L-selectin ligands and contact hypersensitivity.

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Acknowledgements

We thank S. Rosen, R. Cummings and M.N. Fukuda for discussions; Y. Altman for cell sorting; K. Sun for technical assistance; E. Lamar for critical reading of the manuscript; and A. Morse and T. Mabry for organizing the manuscript. S. Rosen (University of California at San Francisco) provided 38C13 B lymphoma cells. Supported by the National Institutes of Health (CA48737 to M.F.; P01 CA71932 to M.F. and J.B.L.; AI061663, AI069259 and AR42689 to U.H.v.A.; and DK48247 to J.D.M.) and the Taiwan National Science Council (95-3112-B-001-014 to the National Core Facilities for Proteomics (for mass spectrometry)).

Author information

Author notes

  1. Junya Mitoma

    Present address: Present address: Division of Glyco-Signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba, Sendai, Miyagi 981-8558, Japan.,

  2. Junya Mitoma and Xingfeng Bao: These authors equally contributed to this work.

Authors and Affiliations

  1. Glycobiology Program, Cancer Research Center, Burnham Institute for Medical Research, La Jolla, 92037, California, USA

    Junya Mitoma, Xingfeng Bao, Hiroto Kawashima, Hideo Saito & Minoru Fukuda

  2. Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, 44106, Ohio, USA

    Bronislawa Petryanik & John B Lowe

  3. CBR Institute for Biomedical Research and Department of Pathology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Patrick Schaerli, Jean-Marc Gauguet & Ulrich H von Andrian

  4. Institute of Biological Chemistry, Academia Sinica, 11529, Taipei, Taiwan

    Shin-Yi Yu & Kay-Hooi Khoo

  5. Howard Hughes Medical Institute and Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, 92093, California, USA

    Kazuaki Ohtsubo & Jamey D Marth

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Contributions

J.M. and X.B. did most of the experiments; B.P. did the rolling assays; P.S. and J.-M.G. did intravital microscopy; S.-Y.Y. did mass spectrometry; H.K. and H.S. assisted with the lymphocyte homing assay; K.O. and J.D.M. did embryonic stem cell culture; J.M., X.B., K.-H.K., U.H.v.A. and J.B.L. contributed to the preparation of the manuscript; and M.F. conceptualized the work, organized all experiments and wrote the manuscript.

Corresponding author

Correspondence to Minoru Fukuda.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Structure and biosynthesis of L-selectin ligands and Core1-β3GlcNAc knockout. (PDF 401 kb)

Supplementary Fig. 2

MECA-79 antibody inhibited lymphocyte homing in WT but not in Core1-β3GlcNAcT (C1-E) KO mice. (PDF 235 kb)

Supplementary Fig. 3

Effects of enzymatic digestions on binding of L-selectin-IgM to HEV. (PDF 162 kb)

Supplementary Fig. 4

Binding of N-glycan-specific lectins in HEV and their effects in inhibiting L-selectin-IgM binding. (PDF 439 kb)

Supplementary Fig. 5

MALDI-MS analysis of permethylated, sulfated N-glycans in negative ion mode. (PDF 663 kb)

Supplementary Fig. 6

Specificity of anti-CD34 antibody and number of leukocytes and CD3+ lymphocytes in contact hypersensitivity (CHS). (PDF 353 kb)

Supplementary Table 1

Tentative assignment of the MALDI-MS molecular ion signals afforded by the permethylated sulfated N-glycans in negative ion mode. (PDF 74 kb)

Supplementary Methods (PDF 119 kb)

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Mitoma, J., Bao, X., Petryanik, B. et al. Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment. Nat Immunol 8, 409–418 (2007). https://doi.org/10.1038/ni1442

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