Effect of cholesterol on the lactosylceramide domains in phospholipid bilayers

Abstract

Lactosylceramide (LacCer) in the plasma membranes of immune cells is an important lipid for signaling in innate immunity through the formation of LacCer-rich domains together with cholesterol (Cho). However, the properties of the LacCer domains formed in multicomponent membranes remain unclear. In this study, we examined the properties of the LacCer domains formed in Cho-containing 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) membranes by deuterium solid-state NMR and fluorescence lifetimes. The potent affinity of LacCer-LacCer (homophilic interaction) is known to induce a thermally stable gel phase in the unitary LacCer bilayer. In LacCer/Cho binary membranes, Cho gradually destabilized the LacCer gel phase to form the liquid-ordered phase by its potent order effect. In the LacCer/POPC binary systems without Cho, the ²H NMR spectra of 10',10'-d₂-LacCer and 18',18',18'-d₃-LacCer probes revealed that LacCer was poorly miscible with POPC in the membranes and formed stable gel phases without being distributed in the liquid crystalline domain. The lamellar structure of the LacCer/POPC membrane was gradually disrupted at around 60°C, whereas the addition of Cho increased the thermal stability of the lamellarity. Furthermore, the area of the LacCer gel phase and its chain order were decreased in the LacCer/POPC/Cho ternary membranes, whereas the liquid-ordered domain, which was observed in the LacCer/Cho binary membrane, was not observed. Cho surrounding the LacCer gel domain liberated LacCer and facilitated forming the submicron to nano-scale small domains in the liquid crystalline domain of the LacCer/POPC/Cho membranes, as revealed by the fluorescence lifetimes of trans-parinaric acid and trans-parinaric acid-LacCer. Our findings on the membrane properties of the LacCer domains, particularly in the presence of Cho, would help elucidate the properties of the LacCer domains in biological membranes.

Figure 1

Chemical structures of N-stearoyl-lactosylceramide (LacCer) and its analogs, 10′,10′-d₂-LacCer and 18′,18′,18′-d₃-LacCer, for solid-state ²H NMR and tPA-LacCer to probe the fluorescence lifetimes.

Figure 2

DSC thermograms of LacCer membranes with Cho at 100:0, 90:10, 80:20, and 50:50 molar ratios of LacCer/Cho.

Figure 3

Solid-state ²H NMR spectra of membranes with a 10′,10′-d₂-LacCer/Cho 50:50 ratio to examine the chain ordering and gradual phase transition from gel phase to the Lo phase. The intensity of the isotropic peaks adjusted the vertical axes. The center peak originates from the remaining deuterium in water.

Figure 4

Microscopic images of GUVs composed of membranes with LacCer/POPC/Cho ratios of 26:65:9 (A), 22:65:13 (B), and 15:65:20 (C), and LacCer/POPC ratio of 25:75 (D). Bodipy-PC was included at 0.2 mol% to visualize the GUVs. To see this figure in color, go online.

Figure 5

The ²H NMR spectra of 10′,10′-d₂-LacCer (A) and 18′,18′,18′-d₃-LacCer (B) in membranes with a LacCer/POPC ratio of 25:75. The inset spectra in panel B provide enlarged views of the center peak. The spectral intensity was normalized to that at 60°C except for 30°C in panel B, which was magnified two times. The sharp center peaks in panels A and B likely originate from residual deuterium in water and/or the deuterated lipids in micelles or a cubic phase.

Figure 6

²H NMR spectra of 18′,18′,18′-d₃-LacCer in membranes with an 18′,18′,18′-d₃-LacCer/Cho ratio of 50:50. The poor S/N ratios at 30°C and 40°C show that the ²H signal intensity was very low, where LacCer largely forms in the gel phase.

Figure 7

Solid-state ²H NMR spectra and the quadrupolar coupling widths (Δν) of 10′,10′-d₂-LacCer (A–C) and 18′,18′,18′-d₃-LacCer (D–F) in LacCer/POPC/Cho bilayers at 30°C. The other spectra using 10′,10′-d₂-LacCer and 18′,18′,18′-d₃-LacCer are shown in Figs. S4–S7. (G) The Δν values of 10′,10′-d₂-LacCer in membranes with a 10′,10′-d₂-LacCer/POPC/Cho ratio of 26:65:9 (diamond), 10′,10′-d₂-LacCer/POPC/Cho ratio of 22:65:13 (triangle), and 10′,10′-d₂-LacCer/POPC/Cho ratio of 15:65:20 (circle) in each temperature. The center peak was largely originated from the residual HDO.

Figure 8

The temperature-dependent change in fluorescence lifetimes of the tPA in the membranes with a LacCer/POPC ratio of 25:75 (A), LacCer/POPC/Cho ratio of 22:65:13 (B), and LacCer/POPC/Cho ratio of 15:65:20 (C). Long lifetimes are shown as filled circles, and average lifetimes are shown as blank squares. tPA at 1 mol% was included in the membranes. Each value is the mean ± SEM for n = 3.

Figure 9

The long (A) and average (B) lifetimes of tPA-LacCer in membranes with a LacCer/POPC ratio of 75:25 and LacCer/POPC/Cho ratios of 22:65:13 and 15:65:20. The lifetimes were collected at 23°C (white bar), 33°C (black bar), and 50°C (gray bar), respectively. tPA-LacCer at 1 mol% was included. Standard deviations are shown with a gray line.

Figure 10

Models of the LacCer-lipid interaction and domain formations. (A) The addition of Cho destabilizes the LacCer gel phase and induces the Lo phase at ≥40°C in the binary membrane. The potent ordering effect of Cho on the LacCer acyl chain was clearly observed. (B) The LacCer gel phase is highly stable and not miscible well with POPC in bilayers even at 40°C. (C) In the LacCer/POPC/Cho ternary membranes, Cho reduces the cooperativity of the LacCer gel phase and facilitates the formation of small and ordered LacCer domains, which are distributed to the POPC-rich Ld area.