Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide-mediated superoxide generation and migration in neutrophils.
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.
Iwabuchi
Kazuhisa
K
Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Tomioka, Urayasu-shi, Chiba, 279-0021, Japan. iwabuchi@med.juntendo.ac.jp
Prinetti
Alessandro
A
Sonnino
Sandro
S
Mauri
Laura
L
Kobayashi
Toshihide
T
Ishii
Kumiko
K
Kaga
Naoko
N
Murayama
Kimie
K
Kurihara
Hidetake
H
Nakayama
Hitoshi
H
Yoshizaki
Fumiko
F
Takamori
Kenji
K
Ogawa
Hideoki
H
Nagaoka
Isao
I
D015703:Antigens, CD / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002465:Cell Movement; D003902:Detergents / Q000494:pharmacology; D005227:Fatty Acids / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D007790:Lactosylceramides / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D010713:Phosphatidylcholines / Q000378:metabolism; D021381:Protein Transport; D013109:Sphingomyelins / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D015703:Antigens, CD; D003902:Detergents; D005227:Fatty Acids; D007790:Lactosylceramides; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D013481:Superoxides; C009744:CDw17 antigen; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases