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

Elena Chiricozzi; Maria Grazia Ciampa; Giuseppina Brasile; Federica Compostella; Alessandro Prinetti; Hitoshi Nakayama; Roudy C. Ekyalongo; Kazuhisa Iwabuchi; Sandro Sonnino; Laura Mauri

doi:10.1194/jlr.M055319

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

^*Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy
^†Institute for Environmental Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Chiba, Japan

Abstract

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 LacCer 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-[³H]LacCer-(N₃) and C24-[³H]LacCer-(N₃), were incorporated into the lipid rafts of plasma membranes of neutrophilic differentiated HL-60 (D-HL-60) cells. C24-[³H]LacCer-(N₃), but not C18-[³H]LacCer-(N₃), induced the phosphorylation of Lyn and promoted phagocytosis. Incorporation of C24-[³H]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-[³H]LacCer-(N₃) to cells, however, did not result in the formation of the LacCer-Lyn complex. These results suggest that LacCer derivatives mimic the biological properties of natural LacCer species and can be utilized as tools to study LacCer-protein interactions, and confirm a specific direct interaction between LacCer species containing very long fatty acids, and Lyn protein, associated with the cytoplasmic layer via myristic/palmitic chains.

Footnotes

↵1 To whom correspondence should be addressed. e-mail: iwabuchi{at}juntendo.ac.jp (K.I.)
Abbreviations:

Bu₃N

tributylamine

CerS

ceramide synthase

DDQ

2,3-dichloro-5,6-dicyano-1,4-benzoquinone

DFP

diisopropyl fluorophosphate

D-HL-60 cell

DMSO-treated human promyelocytic leukemia cell

DMF

dimethylformamide

DRM

detergent-resistant membrane

Et₃N

triethylamine

fMLP

formyl peptide (N-formylmethionine-leucine-phenylalanine)

GD1b

Galb1-3GalNAcb1-4(NeuAca2-8NeuAca2-3)Galb1-4Glcb1-1Cer

GM1

Galb1-3GalNAcb1-4(NeuAca2-3)Galb1-4Glcb1-1’-Cer

GM3

NeuAca2-3Galb1-4Glcb1-1’-Cer

GSL

glycosphingolipid

H₂O

water

LacCer

lactosylceramide [β-Gal-(1-4)-β-Glc-(1-1)-Cer]

MeOH

methanol

MsCl

methane sulfonyl chloride

PNS

post nuclear supernatant

PVDF

polyvinylidene difluoride
This study was supported in part by a grant-in-aid (S1201013, S1311011) from the Foundation of Strategic Research Projects in Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and by Matsumae Foundation (for K.I.). S.S. was supported by funds obtained through performance in tariff of the Department of Medical Biotechnology and Translational Medicine, University of Milan.