Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide-mediated superoxide generation and migration in neutrophils |
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| Authors: | 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 |
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| Affiliation: | (1) Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Tomioka, Urayasu-shi, Chiba 279-0021, Japan;(2) Infectious Control Nursing, Juntendo University Graduate School of Health Care and Nursing, Chiba, Japan;(3) Center of Excellence on Neurodegenerative Diseases; Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milano, Milan, Italy;(4) Sphingolipid Functions Laboratory, Frontier Research System, RIKEN, Saitama, Japan;(5) Division of Proteomics and Biomolecular Science, BioMedical Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan;(6) Department of Anatomy, Juntendo University Graduate School of Medicine, Tokyo, Japan;(7) Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo, Japan |
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| Abstract: | 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. This study was supported in part by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (16017293) to K.I., by COFIN-PRIN 2004 to A.P., and by “High-Tech Research Center” Project for Private Universities: matching fund subsidy. An erratum to this article can be found at |
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| Keywords: | Fatty acid chain Glycosphingolipid Interdigitation Lactosylceramide Lipid raft |
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