Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation

Abstract

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph-mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.

Keywords: GIPC; Langmuir monolayer; cryo-EM; modeling; neutron reflectivity; phytosterol; plasma membrane; solid-state NMR; sphingolipids; ζ-potential.

Figure 1

Structure and amount of GIPC in plants. A, structure of GIPC series A (two sugars after the inositol group); B, GIPC content of different plant species: Brassica oleracea (cauliflower), Nicotiana tabacum (BY-2 cell culture), Allium porrum (leek), and Oryza sativa (rice cell culture). The GIPC content in mg per g of fresh weight was estimated by calculating the proportion of (h)VLCFA (hydroxylated very-long chain fatty acid) as determined by fatty acid methyl ester (GC-MS). The type of GIPC was defined by HPTLC analysis based on Cacas et al., 2016 (1). Three to five independent samples were processed. Means ± SD are shown. GIPC, glycosyl inositol phosphoryl ceramide; HPTLC, high-performance thin-layer chromatography.

Figure 2

Extraction and purification protocol of GIPCs. A, GIPC purification scheme, adapted from (11, 12, 35). The three steps 1, 2, and 3, respectively, are important milestones in the GIPC isolation steps; B, gas chromatography-mass spectrometry (GC-MS) analysis of fatty acid content after steps 1, 2, and 3 of the extraction and purification process. Aliquots of Bo-cauliflower, Nt-BY-2, Ap-leek, and Os-rice samples at step 1, 2, and 3 underwent transmethylation to release fatty acid before derivatization by BSTFA, and the resulting FAMES were analyzed by GC-MS and the fatty acid content calculated. FA refer to fatty acid of 16 to 18 carbon atoms fatty acids and (h)VLCFA refer to hydroxylated or nonhydroxylated very long chain fatty acid of 20 to 28 carbon atoms. The amount of GIPC in each sample were extrapolated from the (h)VLCFA content. Data shown for three independent replicas. Error bars are SD. BSTFA, N,O-Bis(triméthylsilyl)trifluoroacetamide; FA, fatty acid; FAMES, fatty acid methyl esters; GIPC, glycosyl inositol phosphoryl ceramide; HPTLC, high-performance thin-layer chromatography.

Figure 3

High-performance thin layer chromatography analysis during silica column purification. High-performance thin layer chromatography shows the GIPC content after purification step 3, described in Figure 2. Bo-GIPC purified from cauliflower contains mainly series A. Tobacco cell culture BY-2 (Nt-GIPC) sample were further separated by column chromatography to isolate the different GIPC series. Fraction α contains mainly series A, B, and C, whereas fractions β and γ show presence of polyglycosylated GIPCs (series D, E, F, etc). Ap-GIPC purified from leek and Os-GIPC purified from rice samples contain mainly GIPC series B. GIPC, glycosyl inositol phosphoryl ceramide.

Figure 4

Fatty acid content of GIPC-enriched samples. A, very long-chain fatty acid (VLCFA) and hydroxylated VLCFA (hVLCFA) content of GIPC-enriched samples from cauliflower, BY-2 cell culture, leek and rice cell culture. The fatty acids were released from the GIPC-enriched samples by transmethylation followed by derivatization using BSTFA, before GC-MS analysis. Four to six independent samples were analyzed. Means ± SD are shown. B, Yariv reactivity test of GIPC-enriched samples to detect arabino-galactan content. No arabino-galactan were detected. 50 μg of each sample (1 mg/ml) was deposited in each well, and the picture was taken 48 h after initiating the reaction. BSTFA, N,O-Bis(triméthylsilyl)trifluoroacetamide; GIPC, glycosyl inositol phosphoryl ceramide.

Figure 5

Study of the interaction of GIPC and phytosterols in Langmuir monolayer. A, surface pressure-area (π-A) isotherms, at the air-aqueous phase interface, of pure GIPC and sterol monolayers and of mixed GIPC/sterol monolayer prepared at a molar ratio of 0.80. The isotherms were recorded at 25 °C on an aqueous subphase composed by 10 mM Tris buffer at pH 7. Each compression isotherm is representative of at least two independent experiments, each of them repeated at least three times. B, comparison of the experimental (blue bars) and theoretical (red bars) mean molecular areas at a surface pressure of 30 mN/m for a GIPC/sterol molar ratio of 0.80. The theoretical value is obtained according to the additivity rule: A₁₂ = A₁X₁ + A₂X₂, where A₁₂ is the mean molecular area for ideal mixing of the two components at a given π, A₁ and A₂ are the molecular areas of the respective components in their pure monolayers at the same π, and X₁ and X₂ are the molar ratios of components 1 and 2 in the mixed monolayers. Data are from at least six experiments; Means ± SD are shown; C, excess free energy of mixing (ΔGex; blue bars) and free energy of mixing (ΔGM; red bars) of the mixed monolayer GIPC/sterol at a molar ratio of 0.80 at the surface pressure of 30 mN/m. ΔGex and ΔGM were calculated according to the equations as shown in (41, 77). Data are from at least six experiments; Means ± SD are shown. ASG, acyl steryl glucoside (sitosterol, glucose head group, and C18:2 acyl chain); GIPC, glycosyl inositol phosphoryl ceramide; SG, steryl glucoside (sitosterol, glucose head group).

Figure 6

Modeling of the interaction between GIPC and sterols. Theoretical interactions calculated by HyperMatrix docking method with one molecule of GIPC series A t18:0/h24:0 and one molecule of either A, β-sitosterol or B, stigmasterol or C, steryl Glucoside, SG (β-sitosterol, with glucose head group), or D, acyl steryl glucoside, ASG (β-sitosterol, with glucose head group/18:2 acyl chain). GIPC, glycosyl inositol phosphoryl ceramide.

Figure 7

Study of GIPC containing-liposomes in binary and ternary. A, phase contrast microscopy images of Bo-GIPC containing-liposomes in buffer solution after three cycles of freeze and thaw. Enriched Bo-GIPC (cauliflower) underwent freeze (−20 °C, 20 min) and thaw (60 °C, 20 min) cycles three times GIPC in TBS buffer pH 5.8 with or without phospholipid and β-sitosterol at a concentration of 1 mg/ml. (I) GIPCs alone form crystals in a saline buffer solution. A lipid mix, at a concentration of 1 mg/ml, of GIPC/PLPC/β-sitosterol or GIPC/POPC/β-sitosterol (1:1:1, mol/mol), shown in (II) and (III) respectively, forms vesicles of approx. 2 μm. B, fluorescence and phase contrast microscopy images of Giant unilamellar vesicles (GUVs) of GIPC/DOPC/β-sitosterol (1:1:1, mol/mol). The lipid mix was labeled by NBD-PC at 0.1% mol. C, dynamic light scattering (DLS) and ζ-potential of liposomes containing DOPC/β-sitosterol (7:3, mol ratio) (yellow) and GIPC/DOPC/β-sitosterol (1:1:1, mol ratio) (green), respectively, provide the size which is around 100 nm and ζ-potential values of −28 mV in the presence of GIPC. Three to four replica using independent GIPC purification was measured. Means ± SD are shown. DOPC, dioleoyl-sn-glycero-3-phosphocholine; GIPC, glycosyl inositol phosphoryl ceramide; NBD-PC, 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino]hexanoyl}-sn-glycero-3-phosphocholine.

Figure 8

Membrane thickness vary with the presence of GIPC.A, cryo-EM images of liposomes. POPC-d31 that is a deuterated POPC on the carbon of the palmitoyl chain: 16:0-d31 to 18:1 PC, in the presence of sterols (ii: sitosterol and iv: stigmasterol) are mainly present as vesicles, showing one to few bilayers. In the presence of GIPC, these liposomes are still observed, but at the same time, rigid bilayers structures appearing as flat entities are also observed (white arrows in i and iii). Scale bar, 100 nm; B, membrane thickness measurements. Measurements were made using ImageJ software to compare the membrane thickness with or without GIPC. For each lipid system, the width of the bilayer was measured in two different ROI per liposomes in ten independent liposomes from two different cryoEM grids. Error bars are SD (n = 20). Significance was determined by Student’s t test. ∗∗∗p < 0.0001. cryo-EM, cryo-electron microscopy; GIPC, glycosyl inositol phosphoryl ceramide; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

Figure 9

Reflectivity profiles of GIPC-containing lipid bilayer. Reflectivity profiles and calculated scattering length density (SLD) following lipid bilayer deposition of (i) POPC/β-sitosterol (70:30, mol/mol) and (ii) GIPC/POPC/β-sitosterol (55:15:30, mol/mol). A, the multilayer model was composed from the silicon substrate (SLD = 2.07 10⁻⁶ Å⁻²) covered with a layer of silicon oxide (SLD = 3.47 10⁻⁶ Å⁻²); B, structural parameters after multilayer model fitting of reflectivity profiles of lipid bilayer; C, scheme showing the SLD profile overlaid on the multilayer model as obtained for POPC/GIPC membrane. GIPC, glycosyl inositol phosphoryl ceramide; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

Figure 10

Solid state NMR studies of GIPC-containing liposomes. A, ²H-NMR powder spectra of lipid mix, and B, first, spectral moment of ²H-NMR spectra showing membrane ordering versus temperature POPC-(2)H₃₁system alone, in binary systems of POPC-(2)H₃₁/β-sitosterol (1:1 mol/mol) and POPC-(2)H31/Stigmasterol (1:1 mol/mol) and ternary systems of GIPC/POPC-(2)H31/β-sitosterol (1:1:1 mol ratio) and GIPC/POPC-(2)H31/Stigmasterol (1:1:1 mol ratio). Error bars are mean ± SD from three independent measurements. GIPC, glycosyl inositol phosphoryl ceramide; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.