Abstract
The lipidome of mammalian cells not only contain sphingomyelin (SM) but also, as a minor component, dihydrosphongomyelin (DHSM), in which the double bond at C4–C5 in the sphingosine base is reduced to a single-bond linkage. It has been indicated that DHSM forms ordered domains more effectively than SM due to its greater potential to induce intermolecular hydrogen bonds. However, direct information on partition and dynamic behaviors of DHSM in raft-like liquid-ordered (Lo) and non-raft-like liquid-disordered (Ld) phase-segregated membranes has been lacking. In the present study, we prepared fluorescent derivatives of DHSM and compared their behaviors to those of fluorescent SM and phosphatidylcholine (PC) derivatives. Fluorescence microscopy showed that DHSM is more preferentially localized to the Lo domains in the Lo/Ld phase-segregated giant unilamellar vesicles than SM and PC. Most importantly, diffusion coefficient measurements indicated that DHSM molecules form DHSM-condensed assembly inside the SM-rich Lo domain of the SM/dioleoylphosphatidylcholine/cholesterol system even when DHSM accounts for 1–3.3 mol% of total lipids. Such heterogeneous distribution of DHSM in the SM-rich Lo domains was further confirmed by inter-lipid FRET experiments. This study provides new insights into the biological functions and significance of minor component DHSM in lipid rafts.
Highlights
The lipidome of mammalian cells contain sphingomyelin (SM) and, as a minor component, dihydrosphongomyelin (DHSM), in which the double bond at C4–C5 in the sphingosine base is reduced to a single-bond linkage
The physicochemical properties of lipid rafts have been examined with artificial membrane systems consisting of SM, cholesterol, and unsaturated phospholipids such as dioleoylphosphatidylcholine (DOPC) or palmitoyloleoylphosphatidylcholine (POPC), because these mixtures undergo macroscopic phase separation between raft-like liquid-ordered (Lo) and non-raft-like liquid-disordered ( Ld) domains[3,4,5,6,7,8]
In this study, applying the procedure used for the development of fluorescent S Ms10, we prepared novel fluorescent derivatives of DHSM (488negDHpSM and 594neg-DHpSM, inclusively termed neg-DHpSMs; Fig. 1) and compared their properties with those of fluorescent SM and PC to discuss the functional role of DHSM in ordered membranes
Summary
The lipidome of mammalian cells contain sphingomyelin (SM) and, as a minor component, dihydrosphongomyelin (DHSM), in which the double bond at C4–C5 in the sphingosine base is reduced to a single-bond linkage. Diffusion coefficient measurements indicated that DHSM molecules form DHSM-condensed assembly inside the SM-rich L o domain of the SM/ dioleoylphosphatidylcholine/cholesterol system even when DHSM accounts for 1–3.3 mol% of total lipids. We developed excellent fluorescent derivatives of SM: 488neg-SM and 594neg-SM (inclusively termed neg-SMs; Fig. 1)[10], which reproduce partition and dynamic behaviors of native SM and enable the visualization of SM in artificial and biological m embranes[10,11,12]. DHSMs are more likely than SMs to undergo lateral phase separation from DOPC m embranes[23] and may contribute to the formation of laterally condensed domains in biomembranes[24] These results considered in combination suggest that DHSM forms more condensed and ordered domains than SM via effective hydrogen bond formation. In this study, applying the procedure used for the development of fluorescent S Ms10, we prepared novel fluorescent derivatives of DHSM (488negDHpSM and 594neg-DHpSM, inclusively termed neg-DHpSMs; Fig. 1) and compared their properties with those of fluorescent SM and PC to discuss the functional role of DHSM in ordered membranes
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