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Direct interaction, instrumental for signaling processes, between lactosylceramide and Lyn in the lipid rafts of neutrophil-like cells

Significant statement

 Glycosphingolipids (GSL) are expressed on the outer layer of plasma membranes, and participate in transducing signals from outside to inside cells. More than 400 different oligosaccharide chains associated with GSL have been identified to date, with the number of GSL molecular species being at least 10-fold higher, due to the heterogeneity of their ceramide moieties. Several studies have demonstrated that the molecular varieties and expression patterns of GSL reflect their biological functions. In mammalian cells, ceramides are synthesized by a family of six enzymes, ceramide synthase (CerS) 1–6, each of which uses a restricted subset of fatty acyl-CoAs to N-acylate the sphingoid long chain base. The levels of expression of each CerS-encoding gene differ among tissues, suggesting that molecular variations and expression patterns of fatty acid chains of GSLs reflect the functions of these cells. GSLs cluster to form lipid rafts, which have been implicated in a number of important membrane events. However, the molecular mechanisms by which GSLs mediate cell functions remain unclear. One major issue is the association of GSLs with signal transducer molecules localized to the cytosolic side of the plasma membrane. We have addressed this issue using human neutrophilic lineage cells. High levels of lactosylceramide (LacCer) are expressed on the plasma membranes of human neutrophils. lactosylceramide forms Lyn coupled lipid rafts, which mediate neutrophil chemotaxis, phagocytosis and superoxide generation. The main types of lactosylceramide in human neutrophil plasma membranes contain the very long C24 fatty acid chains. In contrast, over 70% of LacCer on plasma-membranes of neutrophilic differentiated human promyelocytic leukemia HL-60 cells (D-HL-60 cells) consists of C16:0-LacCer, with only 13.6% being C24-LacCer. D-HL-60 cells loaded with C24-LacCer showed lactosylceramide-mediated functional activity and allowed Lyn coimmunoprecipitation by anti-LacCer antibody. Lyn knockdown by siRNA completely abolished the effect of C24-LacCer loading on the LacCer mediated functions of D-HL-60 cells. Experiments using azide-photoactivatable tritium-labeled C24- and C18-LacCer showed that C24- but not C16-LacCer was directly associated with Lyn and Gαi. These results confirm a specific direct interaction between C24-LacCer and the signal transduction molecules Lyn and Gαi, which associate with the cytoplasmic layer via palmitic chains. lactosylceramide species with very long fatty acids are indispensable for Lyn-coupled LacCer-enriched lipid raft-mediated neutrophil functions.


Figure Legend:

1. Sizes of lactosylceramide containing C16:0 and C24:0 fatty acid chains.

2. lactosylceramide forms lipid rafts on plasma membranes of human neutrophils and acts as a pattern recognition receptor. The C24 fatty acid chains of lactosylceramide interdigitate into the inner leaflet of plasma membranes and directly interact with Lyn and Gαi to allow association of these molecules with lactosylceramide to mediate signaling from outside to inside the cells, resulting in neutrophil chemotaxis, migration and phagocytosis.

Direct interaction, instrumental for signaling processes, between LacCer and Lyn in the lipid rafts of neutrophil-like cells. Global Medical Discovery



















Journal Reference

Chiricozzi E1, Ciampa MG1, Brasile G1, Compostella F1, Prinetti A1, Nakayama H2, Ekyalongo RC2, Iwabuchi K2, Sonnino S1, Mauri L1. J Lipid Res. 2015;56(1):129-41.

1Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy.

2Institute for Environmental Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Chiba, Japan.


 Lactosylceramide [LacCer; β-Gal-(1-4)-β-Glc-(1-1)-Cer] has been shown to contain very long fatty acids that specifically modulate neutrophil properties. The interactions between lactosylceramide and proteins and their role in cell signaling processes were assessed by synthesizing two molecular species of azide-photoactivable tritium-labeled LacCer having acyl chains of different lengths. The lengths of the two acyl chains corresponded to those of a short/medium and very long fatty acid, comparable to the lengths of stearic and lignoceric acids, respectively. These derivatives, designated C18-[(3)H]LacCer-(N3) and C24-[(3)H]LacCer-(N3), were incorporated into the lipid rafts of plasma membranes of neutrophilic differentiated HL-60 (D-HL-60) cells. C24-[(3)H]LacCer-(N3), but not C18-[(3)H]LacCer-(N3), induced the phosphorylation of Lyn and promoted phagocytosis. Incorporation of C24-[(3)H]LacCer-(N3) into plasma membranes, followed by illumination, resulted in the formation of several tritium-labeled LacCer-protein complexes, including the LacCer-Lyn complex, into plasma membrane lipid rafts. Administration of C18-[(3)H]LacCer-(N3) to cells, however, did not result in the formation of the LacCer-Lyn complex. These results suggest that lactosylceramide derivatives mimic the biological properties of natural lactosylceramide species and can be utilized as tools to study lactosylceramide-protein interactions, and confirm a specific direct  interaction between lactosylceramide species containing very long fatty acids, and Lyn protein, associated  with the cytoplasmic layer via myristic/palmitic chains.

Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

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