Glycoconj J (2008) 25:357–374 DOI 10.1007/s10719-007-9084-6 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 Received: 11 July 2007 /Revised: 16 October 2007 /Accepted: 1 November 2007 / Published online: 28 November 2007 # Springer Science + Business Media, LLC 2007 Abstract The neutral glycosphingolipid lactosylceramide human neutrophils, Blood 100, 1454–1464, 2002 and Sato (LacCer) forms lipid rafts (membrane microdomains) et al. Induction of human neutrophil chemotaxis by coupled with the Src family kinase Lyn on the plasma Candida albicans-derived beta-1,6-long glycoside side- membranes of human neutrophils; ligand binding to LacCer chain-branched beta glycan, J. Leukoc. Biol. 84, 204–211, activates Lyn, resulting in neutrophil functions, such as 2006). Neutrophilic differentiated HL-60 cells (D-HL-60 superoxide generation and migration (Iwabuchi and cells) express almost the same amount of LacCer as Nagaoka, Lactosylceramide-enriched glycosphingolipid neutrophils. However, D-HL-60 cells do not have Lyn- signaling domain mediates superoxide generation from associated LacCer-enriched lipid rafts and lack LacCer- This study was supported in part by a grant-in-aid for Scientific T. Kobayashi : K. Ishii Research on Priority Areas from the Ministry of Education, Culture, Sphingolipid Functions Laboratory, Sports, Science, and Technology of Japan (16017293) to K.I., by Frontier Research System, RIKEN, COFIN-PRIN 2004 to A.P., and by “High-Tech Research Center” Saitama, Japan Project for Private Universities: matching fund subsidy. N. Kaga : K. Murayama * : : : : K. Iwabuchi ( ) H. Nakayama F. Yoshizaki K. Takamori Division of Proteomics and Biomolecular Science, H. Ogawa BioMedical Research Center, Juntendo University Graduate Institute for Environmental and Gender-specific Medicine, School of Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan 2-1-1 Tomioka, Urayasu-shi, Chiba 279-0021, Japan H. Kurihara e-mail: [email protected] Department of Anatomy, Juntendo University Graduate School of Medicine, K. Iwabuchi Tokyo, Japan Infectious Control Nursing, Juntendo University Graduate School of Health Care and Nursing, I. Nagaoka Chiba, Japan Department of Host Defense and Biochemical Research, : : Juntendo University Graduate School of Medicine, A. Prinetti S. Sonnino L. Mauri Tokyo, Japan Center of Excellence on Neurodegenerative Diseases; Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milano, Milan, Italy DO09084; No of Pages 358 Glycoconj J (2008) 25:357–374 mediated superoxide-generating and migrating abilities. For example, lactosylceramide (LacCer, CDw17; Here, we examined the roles of LacCer molecular species Galβ4Glcβ1Cer) is not expressed on the surface of human of different fatty acid compositions in these processes. metamyelocytes, but is expressed on the plasma membrane Liquid chromatography-mass spectrometry analyses of neutrophils with differentiation [5]. Essential functions revealed that the very long fatty acid C24:0 and C24:1 of GSLs, defining cellular phenotype, are based on their chains were the main components of LacCer (31.6% on the clustering with signal transducers to form lipid rafts total fatty acid content) in the detergent-resistant membrane (membrane microdomains) [6–9]. Lipid rafts are subdo- fraction (DRM) from neutrophil plasma membranes. In mains of the plasma membrane highly enriched in GSLs, contrast, plasma membrane DRM of D-HL-60 cells sphingomyelin, glycosylphosphatidylinositol (GPI)-an- included over 70% C16:0-LacCer, but only 13.6% C24- chored proteins and cholesterol [6, 10]. Several types of LacCer species. D-HL-60 cells loaded with C24:0 or membrane-anchored protein partition into lipid rafts [2, 11]. C24:1-LacCer acquired LacCer-mediated migrating and Although lipid rafts have been implicated in a number of superoxide-generating abilities, and allowed Lyn coimmu- important membrane events [2, 8, 12, 13], the molecular noprecipitation by anti-LacCer antibody. Lyn knockdown mechanisms of GSL-mediated cell functions are still by siRNA completely abolished the effect of C24:1-LacCer unclear. One of the main issues regards the molecular loading on LacCer-mediated migration of D-HL-60 cells. connection of GSLs or GPI-anchored proteins, inserted into Immunoelectron microscopy revealed that LacCer clusters the external leaflet of the membrane bilayer, with signal were closely associated with Lyn molecules in neutrophils transduction molecules located at the cytoplasmic side. and C24:1-LacCer-loaded D-HL-60 cells, but not in D-HL- LacCer is a neutral GSL, and is associated with a 60 cells or C16:0-LacCer-loaded cells. Taken together, number of key cellular processes. It has been demonstrated these observations suggest that LacCer species with very that LacCer activates NADPH oxidase to modulate inter- long fatty acids are specifically necessary for Lyn-coupled cellular adhesion molecule-1 expression on human umbil- LacCer-enriched lipid raft-mediated neutrophil superoxide ical vein endothelial cells [14],andtoinducethe generation and migration. proliferation of human aortic smooth muscle cells [15]. Therefore, it is possible that LacCer activates NADPH Keywords Fatty acid chain . Glycosphingolipid . oxidase, and thereby affects the functions of superoxide- Interdigitation . Lactosylceramide . Lipid raft producing cells. LacCer is also a receptor activator of NF- κB ligand and is essential for osteoclastogenesis mediated Abbreviations by macrophage colony stimulation factor [16]. Recently, DMSO Dimethyl sulfoxide LacCer has been shown to recruit PCKα/ε and phospho- D-HL-60 DMSO-treated neutrophilic differentiated hu- lipase A2 to stimulate PECAM-1 expression in human cells man promyelocytic leukemia HL-60 cells monocytes and adhesion to endothelial cells [17] and to fMLP Formyl peptide (N-formyl-methionyl-leucyl- regulate β1-integrin clustering and endocytosis on cell phenylalanine) surfaces [18]. LacCer has been shown to bind specifically CSBG Candida albicans-derived β-glucan to several types of pathogenic microorganism, including SCG Sparassis crispa-derived β-glucan Escherichia coli, Bordetella pertussis, Bacillus dysenteriae, SM Sphingomyelin Propionibacterium freudenreichii, and Candida albicans PC Phosphatidylcholine [19–25], suggesting that LacCer plays roles in the interactions PE Phosphatidylethanolamine between these microorganisms and host cells. Neutrophils play important roles in innate immunity. On infection with pathogenic microorganisms, circulating neutrophils immediately migrate toward and phagocytose Introduction and kill the microorganisms with microbicidal molecules and by superoxide generation [26]. LacCer accounts for Glycosphingolipids (GSLs) are membrane components about 70% of the GSL in human neutrophils. These consisting of hydrophobic ceramide and hydrophilic sugar immune cells migrate toward C. Albicans-derived β-glucan moieties [1]. More than 400 species of GSL have been (CSBG) [23], and generate superoxide anions via LacCer identified to date based on their sugar chain structures [2]. [12]. Therefore, LacCer is thought to be involved in Furthermore, the ceramide structures of each GSL are also neutrophil microbicidal functions. On neutrophil plasma highly variable [3]. GSL metabolism and composition are membranes, LacCer forms lipid rafts with the Src family specifically altered during cell proliferation and differenti- kinase Lyn; these rafts are also called LacCer-enriched ation in various cell types [2, 4], indicating that expression glycosignaling domains [12]. Dimethyl sulfoxide (DMSO)- patterns of GSLs may reflect the functions of those cells. treated neutrophilic differentiated human promyelocytic Glycoconj J (2008) 25:357–374 359 leukemia HL-60 cells (D-HL-60 cells) possess chemotactic from Matreya (Pleasant Gap, PA). Candida albicans- and superoxide-generating activities induced by formyl derived-βglucan (CSBG) and Sparassis crispa-derived peptide fMLP. Interestingly, D-HL-60 cells do not show β-glucan (SCG), isolated as described previously [23], superoxide-generating or chemotactic activity induced by were kindly provided by Drs. Adachi and Ohno (Tokyo anti-LacCer antibodies, although these cells express almost University of Pharmacy and Life Science) and Dr. Tamura the same amount of LacCer on the plasma membrane as (Seikagaku Corporation), respectively. Dimethylsulfoxide neutrophils. LacCer is localized mainly in the detergent- (DMSO) and N-formylmethionine-leucine-phenylalanine resistant membrane fractions (DRMs) [12, 27, 28], which (fMLP) were purchased from Sigma-Aldrich Japan (Tokyo, are membrane fragments isolated biochemically at low Japan). temperature from cellular membranes using non-ionic detergents, such as Triton X-100; there is thought to be a Synthesis of GSLs and [1-3H]sphingosine close relationship between rafts and DRMs, and isolation of DRMs is one of the most widely used biochemical C16:0-(palmitic acid), C22:0-(behenic acid), C24:0-(ligno- methods for studying lipid rafts [6]. Importantly, the Src ceric acid), and C24:1 (nervonic acid; C24:1ω9)-LacCer family kinase Lyn was immunoprecipitated by anti-LacCer were synthesized from the