Glycoconjugate Journal

, Volume 25, Issue 4, pp 357–374 | Cite as

Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide-mediated superoxide generation and migration in neutrophils

  • Kazuhisa Iwabuchi
  • Alessandro Prinetti
  • Sandro Sonnino
  • Laura Mauri
  • Toshihide Kobayashi
  • Kumiko Ishii
  • Naoko Kaga
  • Kimie Murayama
  • Hidetake Kurihara
  • Hitoshi Nakayama
  • Fumiko Yoshizaki
  • Kenji Takamori
  • Hideoki Ogawa
  • Isao Nagaoka


The neutral glycosphingolipid lactosylceramide (LacCer) forms lipid rafts (membrane microdomains) coupled with the Src family kinase Lyn on the plasma membranes of human neutrophils; ligand binding to LacCer activates Lyn, resulting in neutrophil functions, such as superoxide generation and migration (Iwabuchi and Nagaoka, Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils, Blood 100, 1454–1464, 2002 and Sato et al. Induction of human neutrophil chemotaxis by Candida albicans-derived beta-1,6-long glycoside side-chain-branched beta glycan, J. Leukoc. Biol. 84, 204–211, 2006). Neutrophilic differentiated HL-60 cells (D-HL-60 cells) express almost the same amount of LacCer as neutrophils. However, D-HL-60 cells do not have Lyn-associated LacCer-enriched lipid rafts and lack LacCer-mediated superoxide-generating and migrating abilities. Here, we examined the roles of LacCer molecular species of different fatty acid compositions in these processes. Liquid chromatography-mass spectrometry analyses revealed that the very long fatty acid C24:0 and C24:1 chains were the main components of LacCer (31.6% on the total fatty acid content) in the detergent-resistant membrane fraction (DRM) from neutrophil plasma membranes. In contrast, plasma membrane DRM of D-HL-60 cells included over 70% C16:0-LacCer, but only 13.6% C24-LacCer species. D-HL-60 cells loaded with C24:0 or C24:1-LacCer acquired LacCer-mediated migrating and superoxide-generating abilities, and allowed Lyn coimmunoprecipitation by anti-LacCer antibody. Lyn knockdown by siRNA completely abolished the effect of C24:1-LacCer loading on LacCer-mediated migration of D-HL-60 cells. Immunoelectron microscopy revealed that LacCer clusters were closely associated with Lyn molecules in neutrophils and C24:1-LacCer-loaded D-HL-60 cells, but not in D-HL-60 cells or C16:0-LacCer-loaded cells. Taken together, these observations suggest that LacCer species with very long fatty acids are specifically necessary for Lyn-coupled LacCer-enriched lipid raft-mediated neutrophil superoxide generation and migration.


Fatty acid chain Glycosphingolipid Interdigitation Lactosylceramide Lipid raft 



Dimethyl sulfoxide

D-HL-60 cells

DMSO-treated neutrophilic differentiated human promyelocytic leukemia HL-60 cells


Formyl peptide (N-formyl-methionyl-leucyl-phenylalanine)


Candida albicans-derived β-glucan


Sparassis crispa-derived β-glucan









We are grateful to Dr. Sen-itiroh Hakomori (University of Washington) for his encouragement and invaluable comments throughout this study. We thank Dr. Hiroshi Tamura (Seikagaku Corporation) and Drs. Yoshiyuki Adachi and Naohito Ohno (Tokyo University of Pharmacy and Life Science) for providing Candida albicans-derived β-glucan and Sparassis crispa-derived β-glucan, respectively. We also thank Dr. Irwin D. Bernstein at Fred Hutchinson Cancer Research Center, Seattle, WA, USA, for important contributions.


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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Kazuhisa Iwabuchi
    • 1
    • 2
  • Alessandro Prinetti
    • 3
  • Sandro Sonnino
    • 3
  • Laura Mauri
    • 3
  • Toshihide Kobayashi
    • 4
  • Kumiko Ishii
    • 4
  • Naoko Kaga
    • 5
  • Kimie Murayama
    • 5
  • Hidetake Kurihara
    • 6
  • Hitoshi Nakayama
    • 1
  • Fumiko Yoshizaki
    • 1
  • Kenji Takamori
    • 1
  • Hideoki Ogawa
    • 1
  • Isao Nagaoka
    • 7
  1. 1.Institute for Environmental and Gender-specific MedicineJuntendo University Graduate School of MedicineChibaJapan
  2. 2.Infectious Control NursingJuntendo University Graduate School of Health Care and NursingChibaJapan
  3. 3.Center of Excellence on Neurodegenerative Diseases; Department of Medical Chemistry, Biochemistry and BiotechnologyUniversity of MilanoMilanItaly
  4. 4.Sphingolipid Functions LaboratoryFrontier Research System, RIKENSaitamaJapan
  5. 5.Division of Proteomics and Biomolecular ScienceBioMedical Research Center, Juntendo University Graduate School of MedicineTokyoJapan
  6. 6.Department of AnatomyJuntendo University Graduate School of MedicineTokyoJapan
  7. 7.Department of Host Defense and Biochemical ResearchJuntendo University Graduate School of MedicineTokyoJapan

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