Dynamic assembly and interaction of glycosphingolipids in cholesterol-containing model membranes

Introduction

Mammalian cell membranes play a vital role as “protective fences” that separate the inside and outside of cells. Simultaneously, cell membranes selectively connect specific pathways between the inside and outside of cells to maintain homeostasis in signal transduction and cell-to-cell communication. Since Singer and Nicholson’s fluid mosaic model [1], structural and functional studies of lipid bilayers composed of amphiphilic lipids and peripheral proteins surrounding cells have accelerated significantly. The lipid raft model was inspired by the study of biological and model membranes [2,3]. The maintenance and management of mammalian cells can be achieved through dynamic phase separation, in which domain units with various functions on the cell membrane repeatedly dissociate and assemble. Phase separation controls spatiotemporal biological reactions not only in the cell membrane but also inside the cell [4].

Lipid molecules constituting the bilayer of mammalian cell membranes include sphingolipids (sphingomyelin and GSLs), glycerophospholipids, and cholesterol. Sphingomyelin and GSLs with saturated tails de-mix from glycerophospholipids with unsaturated tails, such as palmitoyl-oleoyl phosphatidylcholine (POPC). Membrane cholesterol facilitates the formation of the sphingolipid-enriched liquid-ordered (Lo) domain in model membranes. This Lo phase evokes a relationship with lipid rafts because lipid rafts in cell membranes are enriched with sphingomyelin, glycosphingolipids, and cholesterol [5,6]. Currently, lipid rafts in cell membranes are often considered to be dynamic and multi-shaped domains ranging from 10 to 150 nm in size.

GSLs in mammalian cell membranes have an oligosaccharide head group with a ceramide tail (Fig. 1) and are believed to be distributed in the outer leaflet of GSL-rich membrane domains. Gangliosides are a bioactive class of GSLs that carry sialic acids at the non-reducing terminal of the core lactosylceramide (LacCer) structure [7]. LacCer is also involved in the innate and adaptive immunity triggered by bacterial and fungal infections. The direct interaction of LacCer domains on the immune cell surface with the glycans of pathogenic organisms are followed by the activation of immune signaling through Src family kinases, such as Lyn [8]. Ganglioside GM3 is the simplest ganglioside with sialic acid on the non-reducing terminus of LacCer. The large oligosaccharide head groups with the biphasic nature of the gangliosides normally form micelles in water, except for GM3 with minimal head group size, which forms unilamellar vesicles [9]. These differences in head group size may interplay with membrane cholesterol and alter the interaction with glycan-binding proteins that recognize glycolipid head groups [10]. In particular, GM3 is involved in EGF signaling suppression likely by binding with EGF receptors, which can reduce cancer progression [11,12]. GM3 is also involved in diabetes by regulating the localization of the insulin receptor [13], and innate and adoptive immunities by modulating immune signaling as a ligand for TLR4 [14].

Fig. 1:

Structures of ganglioside GM3, LacCer, and sphingomyelin with stearoyl (C18:0) acyl chains. LacCer is the core structure of complicated GSLs.

In this study, we investigated GSL–GSL and GSL-peptide interactions in model membranes. GSLs carrying relatively small glycan head groups (LacCer and GM3) were incorporated into cholesterol-containing model membranes. The differences in lipid order, motility, and lipid-peptide interaction were examined using solid-state NMR and fluorescence spectroscopy. The effects of cholesterol on GSLs are different from those of sphingomyelin. The difference in the cholesterol effect for membrane LacCers, which reduces the size of the LacCer gel domain but does not form the Lo domain, resulted in forming GSL domains of submicron meter size. We also prepared the transmembrane (TM) α-helix segment of EGFR tagged with the NBD fluorophore and examined the direct interaction with GM3 in the membrane environment. In practice, GM3 destabilizes the assembly of the TM segment through direct interaction, as evidenced by the NBD fluorescence intensities (recovery from self-quenching) on the TM segment in the membrane environment. These physical properties of GSLs in lateral interaction with lipids and proteins highlight their importance in biological membranes.

LacCer domains

LacCer, a neutral GSL with a Galβ(1–4)Glc head group (Fig. 1), is a core structure in the biosynthesis of bioactive gangliosides, globosides, and other types of mammalian GSLs, except for galactosylceramides [15]. LacCer is distributed in the plasma membrane of immune cells, such as human neutrophils, intestinal epithelial cells, and skin, and is involved in stimulating innate and adaptive immunity. In immune cell membranes, LacCer domains are thought to be involved in immune signaling through interaction with glycans of pathogenic organisms, such as yeast β1,3-glucans and mycobacterium α1,2-mannan [16]. In this immune signaling, LacCer carrying very long-chain fatty acyl tail is essential for stimulating lipidated G-protein Gα_i and Src-family kinases Lyn distributed in the inner leaflet of the LacCer domains [16,17]. However, information regarding the assembly state of LacCer in lipid membranes is limited.

The LacCer domains that formed in the model bilayers were investigated from a physicochemical perspective. The unitary membranes of LacCer with a stearoyl tail showed an extremely high phase transition temperature at 79 °C, similar to the previous report [18]. In contrast, an Lo phase was formed in LacCer/cholesterol 50:50 membranes (Fig. 2c) [19]. We were highly interested in phase-separated ternary membrane systems composed of LacCer, cholesterol, and POPC. Microscopic observation of Bodipy-PC distributed in giant unilamellar vesicles (GUVs) composed of LacCer/cholesterol 3:1 in 65 mol% POPC membranes suggested that the potent homophilic LacCer–LacCer interaction stabilized LacCer gel domains that were separated from the liquid disordered (Ld) domain (Fig. 2). The solid-state ²H NMR using 18′,18′,18′-d ₃-LacCer in the membrane system, showing broadened spectra, consistently revealed the gel phase of LacCer in the membrane system. Moreover, an increase in cholesterol concentration decreased the size of the LacCer gel domains, whereas the macroscopic Lo phase did not appear. The fluorescence probe trans-parinaric acid (tPA) is preferentially distributed in the ordered membrane domains of the bilayer membranes. Therefore, tPA fluorescence lifetimes of tens of nanoseconds were successfully used to examine ordered sub-nanometer scale domains in bilayers [20]. The fluorescence lifetime of tPA in LacCer/cholesterol 3:2 and 3:4 in 65 mol% POPC membranes suggested that LacCer formed nanoscopic ordered domains by the action of cholesterol (Fig. 2e) [21].

Fig. 2:

LacCer domains in LacCer/POPC/cholesterol membranes were composed of 65 mol% POPC and 35 mol% LacCer/cholesterol at molar ratios of 3:1, 3:2, and 3:4. a: GUV images of LacCer/POPC/cholesterol membranes. The Ld domain was visualized using Bodipy-PC. b: Structure of 18′,18′,18′-d ₃-LacCer used as the ²H NMR probe. c and d: Solid-state ²H NMR spectra of 18′,18′,18′-d ₃-LacCer in LacCer/cholesterol 50:50 (c) and LacCer/POPC/cholesterol (d) membranes. Images of the GUVs and NMR spectra were obtained at 30 °C. e: Cholesterol decreased the size of LacCer gel domains and facilitated LacCer nanodomains formation. Cho: cholesterol. Reproduced from Ref. [19] with permission from ELSEVIER (225479041246903).

Phosphatidylserine (PS) interaction with N-terminal unique domain of Src family kinase Lyn

Lyn is a Src family kinase that harbors the inner leaflet of the LacCer domain, and is involved in LacCer domain-mediated immune signaling [16]. Lyn undergoes N-myristoylation and S-palmitoylation at its unique N-terminal domain. The membrane association of Lyn carrying N-myristoylation at the N-terminal Gly is regulated through reversible S-palmitoylation of the next Cys residue, known as the S-palmitoyl-switch. There is no doubt that the increasing number of hydrophobic acyl chains on Lyn facilitates membrane partitioning, whereas details of its increased distribution in the ordered LacCer domain are unclear.

We prepared N-terminal Lyn peptides with lipidations via chemical synthesis and adopted solid-state NMR using model membranes consisting of 10 mol% peptides in unitary POPC membranes or POPC/palmitoyl-oleoyl PS (POPS)/palmitoyl-oleoyl phosphatidyl ethanolamine (POPE) 50:25:25 membranes (Fig. 3) [22]. As a result, S-palmitoylation of the N-myristoylated Lyn peptide decreased the ³¹P spin-lattice (T ₁) relaxation time of POPS/POPE more than that of POPC, suggesting preferable interaction of the doubly lipidated peptide with POPS (or POPE) head group on the membrane surfaces. Lys residues enriched in the N-terminal region of this unique domain were assumed to interact with the head group of the anionic POPS. Solid-state ²H NMR spectra were acquired using N-terminal Lyn peptides with perdeuterated N-myristoyl chains. S-palmitoylation of the N-myristoylated Lyn peptide increased the ²H quadrupolar coupling widths, originating from CD₂ segments in the perdeuterated N-myristoyl chain. This increase indicates that the N-myristoyl chain likely extended through interaction with the neighboring S-palmitoyl chain. This chain extension assists in increasing the preference for lipidated Lyn distribution in the ordered domain as opposed to the LacCer domain. Moreover, Lyn with an extended lipid chain, is prone to interdigitate with the very long-chain fatty acyl chain of LacCer, and the interdigitated structure assists in transmitting transmembrane signals.

Fig. 3:

The membrane interaction mechanism of lipidated Lyn peptides was assumed based on the results of solid-state NMR using unitary POPC or POPC/POPE/POPS 50:25:25 membranes including 10 mol% lipidated peptides. a: Difference in ³¹P T ₁ relaxation time originating from the head group of membrane phospholipids with or without lipidated peptides. b: Extension of peptide lipid chains derived from ²H NMR quadrupolar coupling widths of the perdeuterated acyl chains of the peptides. c: Chain extension model of lipidated Lyn peptides, which is dependent on membrane lipids and the number of peptide lipid tails. M-Lyn and MP-Lyn are Lyn peptides with an N-myristoyl chain and both N-myristoyl and S-palmitoyl chains. “d” means perdeuterated chain. Reproduced from Ref. [22] with permission from the Royal Society of Chemistry.

GM3 structure and interaction with EGFR-TM peptide

Lateral lipid-protein interaction in membranes play key roles in forming biologically functional domains in cell membranes. Ganglioside has an oligosaccharide head group with at least one sialic acid, and thus, the negative charge often contributes to lateral interaction with the membrane proteins via cationic side chains at the appropriate positions close to the membrane surface [13]. We synthesized the transmembrane (TM) domain of EGFR and examined its interaction with GM3 carrying a stearoyl chain. The experiments were carried out in dimyristoyl-PC (DMPC) bilayers (Fig. 4) [23]. The synthetic peptide with the NBD fluorophore was prone to oligomerization in the membrane environment, resulting in NBD fluorescence suppression by self-quenching. This self-quenching was partially recovered in the presence of GM3, suggesting that GM3 and TM segments of EGFR interact to stabilize the TM monomer. The interaction between GM3 and the EGFR TM segment was further examined by lateral FRET using a FRET pair consisting of the NBD-labeled TM peptide (donor) and ATTO594-labeled GM3 (acceptor) [24]. Lateral FRET with ATTO594-GM3 was more efficient compared to ATTO594-sphingomyelin [25]. Furthermore, the FRET efficiency of ATTO594-GM3 was higher compared to ATTO594-GM1. Therefore, the EGFR TM segment selectively interacts with GM3 under membrane conditions. The Van’t Hoff plot afforded ΔG = −12 kJ/mol for the TM-GM3 interaction. It was assumed that the stabilization energy was insufficient to form a stable complex between glycosylated full-length EGFR and GM3; thus, additional interaction points, such as the distal ectodomain and glycans of EGFR, may also be involved in this interaction event occurring on cell membranes. EGFR can attain various conformations [26], and some of which may interact with membrane GM3; however, membrane structural information near lipid membranes is still limited.

Fig. 4:

Lateral interaction between GM3 and the TM segment of EGFR in unitary DMPC membranes. a: Lateral FRET model between the NBD-labeled TM segment and ATTO594-labeled lipid probes. b: Dose-dependent FRET curves of ATTO594-GM3, ATTO594-GM1, and ATTO594-sphingomyelin (SM). These fluorescent lipid probes have an identical ceramide tail with C18:0 acyl chains. The theoretical FRET efficiency derived from nonspecific interaction is indicated by a dotted curve [27]. c: Interaction mode between GM3 and TM segments in membranes. Panel c was reproduced from Ref. [23] with permission from ELSEVIER (5479050236512).

The conformation and orientation of the GSL head groups in the domains may contribute to the interaction with proteins in cis- and trans-binding modes. The torsional ensemble structures of GM3 glycans in solution have been studied in combination with NMR and MD simulations [28], [29], [30]. GM3 glycan torsions were similar to those of GM1 and larger GSLs, except for the ϕ ₁ angle, because GM3 potentially forms additional hydrogen bonds between Neu5Ac and Gal using free Gal4-OH [31]. The conformation of the oligosaccharide moiety in membrane environments is extremely important for understanding GSL-protein interactions, which depend on membrane properties and the amount of membrane cholesterol [10,32]. A solid-state NMR study of GM3 in bilayer membranes of POPC suggests that GM3 affects the motion of the surrounding POPC bilayer because GM3 glycans have slow dynamics in bilayer environments [33]. Additionally, sphingomyelin in model membranes with cholesterol changes the interaction with sphingomyelin-binding proteins, likely due to a partial change of the head group conformation [34,35]. Further research progress in this field is needed to elucidate the biological function of GSLs.

Conclusions

We examined the lateral interaction of GSLs in bilayer membranes in phase-separated model membranes composed of unsaturated lipids and cholesterol. Cholesterol affected the gel domain of LacCer in the POPC membrane, which reduced the domain size, but did not form macroscopic LacCer-cholesterol Lo domains. The interaction of cholesterol with LacCer forms very small, likely submicron size, ordered LacCer domains, believed to be a minimum unit of the LacCer domains that induce immune signals in immune cells. TM segment of EGFR preferentially interacts with GM3, rather than with sphingomyelin or GM1 in the model membranes. This selective interaction can partially inhibit oligomerization by stabilizing an inactive monomeric form and transferring the TM segment into the GM3 enriched lipid rafts in the cell membranes. These studies for lateral interaction of GSLs in a homophilic manner, or with other lipids and membrane proteins, will help elucidate the essential molecular mechanisms of biological events occurring in lipid membranes.