Membrane fluidity properties of lipid-coated polylactic acid nanoparticles.

Lipid coating is considered a versatile strategy to equip nanoparticles (NPs) with a biomimetic surface coating, but the membrane properties of these nanoassemblies remain in many cases insufficiently understood. In this work, we apply C-Laurdan generalized polarization (GP) measurements to probe the temperature-dependent polarity of hybrid membranes consisting of a lipid monolayer adsorbed onto a polylactic acid (PLA) polymer core as function of lipid composition and compare the behavior of the lipid coated NPs (LNPs) with that of liposomes assembled from identical lipid mixtures. The LNPs were generated by nanoprecipitation of the polymer in aqueous solutions containing two types of lipid mixtures: (i) cholesterol, dipalmitoylphosphatidylcholine (DPPC), and the ganglioside GM3, as well as (ii) dioleoylphosphatidylcholine (DOPC), DPPC and GM3. LNPs were found to exhibit more distinct and narrower phase transitions than corresponding liposomes and to retain detectable phase transitions even for cholesterol or DOPC concentrations that yielded no detectable transitions in liposomes. These findings together with higher GP values in the case of the LNPs for temperatures above the phase transition temperature indicate a stabilization of the membrane through the polymer core. LNP binding studies to GM3-recognizing cells indicate that differences in the membrane fluidity affect binding avidity in the investigated model system.

Detection of N-glycolyl-neuraminic acid-containing glycolipids in human skin.

Humans lack the enzyme that produces the sialic acid N-glycolyl neuraminic acid (Neu5Gc), but several lines of evidence have shown that Neu5Gc can be taken up by mammalian food sources and replace the common human sialic acid N-acetyl neuraminic acid (Neu5Ac) in glycans. Cancer tissue has been shown to have increased the presence of Neu5Gc and Neu5Gc-containing glycolipids such as the ganglioside GM3, which have been proposed as tumor-specific antigens for antibody treatment. Here, we show that a previously described antibody against Neu5Gc-GM3 is binding to Neu5GC-containing gangliosides and is strongly staining different cancer tissues. However, we also found a strong intracellular staining of keratinocytes of healthy skin. We confirmed this staining on freshly isolated keratinocytes by flow cytometry and detected Neu5Gc by mass spectrometry. This finding implicates that non-human Neu5Gc can be incorporated into gangliosides in human skin, and this should be taken into consideration when targeting Neu5Gc-containing gangliosides for cancer immunotherapy.

Comparison and Possible Binding Orientations of SARS-CoV-2 Spike N-Terminal Domain for Gangliosides GM3 and GM1.

SARS-CoV-2 spike glycoprotein is anchored by gangliosides. The sialic acid in the ganglioside headgroup is responsible for virus attachment and entry into host cells. We used coarse-grained (CG) molecular dynamics simulations to expand on our previous study of GM1 interaction with two different orientations of the SARS-CoV-2 S1 subunit N-terminal domain (NTD) and to confirm the role of sialic acid receptors in driving the viral receptor; GM3 was used as another ganglioside on the membrane. Because of the smaller headgroup, sialic acid is crucial in GM3 interactions, whereas GM1 interacts with NTD via both the sialic acid and external galactose. In line with our previous findings for NTD orientations in GM1 binding, we identified two orientations, "compact" and "distributed", comprising sugar receptor-interacting residues in GM3-embedded lipid bilayers. Gangliosides in closer proximity to the compact NTD orientation might cause relatively greater restrictions to penetrate the bilayer. However, the attachment of a distributed NTD orientation with more negative interaction energies appears to facilitate GM1/GM3 to move quickly across the membrane. Our findings likely shed some light on the orientations that the NTD receptor acquires during the early phases of interaction with GM1 and GM3 in a membrane environment.

[Homeostatic and Pathophysiological Regulation of Toll-like Receptor 4 Signaling by GM3 Ganglioside Molecular Species].

Chronic inflammation plays an important role in the pathogenesis of obesity and metabolic disorders. In obesity, pattern-recognition receptors in innate immune system, such as Toll-like receptor 4 (TLR4), cause chronic inflammation through prolonged activation by various endogenous ligands, including fatty acids and its metabolites. Gangliosides and other glycosphingolipids are important metabolites of fatty acids and saccharides. GM3, the simplest ganglioside comprising α2,3-sialyllactose, is expressed in insulin-sensitive peripheral tissues such as liver and adipose tissue, and furthermore secreted abundantly into serum. It has been shown that GM3 regulates the signal transduction of insulin receptor in adipose tissue as a component of membrane microdomains, and elevation in GM3 level causes insulin resistance. However, the homeostatic and pathophysiological functions of extracellularly secreted GM3 are poorly understood. We recently reported that GM3 species with differing fatty acid structures act as pro- and anti-inflammatory endogenous TLR4 ligands. GM3 with very long-chain fatty acid (VLCFA) and α-hydroxyl VLCFA strongly enhanced TLR4 activation. Conversely, GM3 with long-chain fatty acid (LCFA) and ω-9 unsaturated VLCFA inhibited TLR4 activation, counteracting the VLCFA species. GM3 interacted with the extracellular complex of TLR4 and promoted dimerization/oligomerization. In obesity and metabolic disorders, VLCFA species were increased in serum and adipose tissue, whereas LCFA species was relatively decreased; their imbalances were correlated to disease progression. Our findings suggest that GM3 species are disease-related endogenous TLR4 ligands, and "glycosphingolipid sensing" by TLR4 controls the homeostatic and pathological roles of innate immune signaling.

Homeostatic and pathogenic roles of the GM3 ganglioside.

Two decades ago, we achieved molecular cloning of ganglioside GM3 synthase (GM3S; ST3GAL5), the enzyme responsible for initiating biosynthesis of complex gangliosides. The efforts of our research group since then have been focused on clarifying the physiological and pathological roles of gangliosides, particularly GM3. This review summarizes our long-term studies on the roles of GM3 in insulin resistance and adipogenesis in adipose tissues, cholesterol uptake in intestine, and leptin resistance in hypothalamus. We hypothesized that GM3 plays a role in innate immune function of macrophages and demonstrated that molecular species of GM3 with differing acyl-chain structures and modifications functioned as pro- and anti-inflammatory endogenous Toll-like receptor 4 (TLR4) modulators in macrophages. Very-long-chain and α-hydroxy GM3 species enhanced TLR4 activation, whereas long-chain and unsaturated GM3 species counteracted this effect. Lipidomic analyses of serum and adipose tissues revealed that imbalances between such pro- and anti-inflammatory GM3 species promoted progression of metabolic disorders. GM3 thus functions as a physiological regulatory factor controlling the balance between homeostatic and pathological states. Ongoing studies based on these findings will clarify the mechanisms underlying ganglioside-dependent control of energy homeostasis and innate immune responses.

Key role of a structural water molecule for the specificity of 14F7-An antitumor antibody targeting the NeuGc GM3 ganglioside.

Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. 14F7 is a monoclonal IgG1 with high clinical potential in cancer immunotherapy as it displays extraordinary specificity for NeuGc GM3, while it does not recognize the very similar, ubiquitous NeuAc GM3. Here we present the 2.3 Å crystal structure of the 14F7 antigen-binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. Modeling analysis and previous mutagenesis data suggest that 14F7 may also bind to an alternative NeuGc GM3 conformation, not observed in the crystal structure. The most intriguing finding, however, was that a water molecule centrally placed in the complementarity-determining region directly mediates the specificity of 14F7 to NeuGc GM3. This has profound impact on the complexity of engineering in the binding site and provides an excellent example of the importance in understanding the water structure in antibody-antigen interactions.

FRET detects lateral interaction between transmembrane domain of EGF receptor and ganglioside GM3 in lipid bilayers.

Ganglioside GM3 in the plasma membranes suppresses cell growth by preventing the autophosphorylation of the epidermal growth factor receptor (EGFR). Biological studies have suggested that GM3 interacts with the transmembrane segment of EGFR. Further biophysical experiments are particularly important for quantitative evaluation of the peptide-glycolipid interplay in bilayer membranes using a simple reconstituted system. To examine these interactions in this way, we synthesized the transmembrane segment of EGFR bearing a nitrobenzoxadiazole fluorophore (NBD-TM) at the N-terminus. The affinity between EGFR and GM3 was evaluated based on Förster resonance energy transfer (FRET) between NBD-TM and ATTO594-labeled GM3 in bilayers where their non-specific interaction due to lateral proximity was subtracted by using NBD-labeled phospholipid. This method for selectively detecting the specific lipid-peptide interactions in model lipid bilayers disclosed that the lateral interaction between GM3 and the transmembrane segment of EGFR plays a certain role in disturbing the formation of active EGFR dimers.

Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity.

Innate immune signaling via TLR4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM3 ganglioside in human serum is composed of a variety of fatty acids, including long-chain (LCFA) and very-long-chain (VLCFA). Analysis of circulating levels of human serum GM3 species from patients at different stages of insulin resistance and chronic inflammation reveals that levels of VLCFA-GM3 increase significantly in metabolic disorders, while LCFA-GM3 serum levels decrease. Specific GM3 species also correlates with disease symptoms. VLCFA-GM3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR4-mutant mice. In cultured monocytes, GM3 by itself has no effect on TLR4 activation; however, VLCFA-GM3 synergistically and selectively enhances TLR4 activation by LPS/HMGB1, while LCFA-GM3 and unsaturated VLCFA-GM3 suppresses TLR4 activation. GM3 interacts with the extracellular region of TLR4/MD2 complex to modulate dimerization/oligomerization. Ligand-molecular docking analysis supports that VLCFA-GM3 and LCFA-GM3 act as agonist and antagonist of TLR4 activity, respectively, by differentially binding to the hydrophobic pocket of MD2. Our findings suggest that VLCFA-GM3 is a risk factor for TLR4-mediated disease progression.

A combined NMR, MD and DFT conformational analysis of 9-O-acetyl sialic acid-containing GM3 ganglioside glycan and its 9-N-acetyl mimic.

O-Acetylation of carbohydrates such as sialic acids is common in nature, but its role is not clearly understood due to the lability of O-acetyl groups. We demonstrated previously that 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc) is a chemically and biologically stable mimic of the 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) of the corresponding sialoglycans. Here, a systematic nuclear magnetic resonance (NMR) spectroscopic and molecular dynamics (MD) simulation study was undertaken for Neu5,9Ac2-containing GM3 ganglioside glycan (GM3-glycan) and its Neu5Ac9NAc analog. GM3-glycan with Neu5Ac as the non-O-acetyl form of Neu5,9Ac2 was used as a control. Complete 1H and 13C NMR chemical shift assignments, three-bond 1H-13C trans-glycosidic coupling constants (3JCH), accurate 1H-1H coupling constants (3JHH), nuclear Overhauser effects and hydrogen bonding detection were carried out. Results show that structural modification (O- or N-acetylation) on the C-9 of Neu5Ac in GM3 glycan does not cause significant conformational changes on either its glycosidic dihedral angles or its secondary structure. All structural differences are confined to the Neu5Ac glycerol chain, and minor temperature-dependent changes are seen in the aglycone portion. We also used Density Functional Theory (DFT) quantum mechanical calculations to improve currently used 3JHH Karplus relations. Furthermore, OH chemical shifts were assigned at -10°C and no evidence of an intramolecular hydrogen bond was observed. The results provide additional evidence regarding structural similarities between sialosides containing 9-N-acetylated and 9-O-acetylated Neu5Ac and support the opportunity of using 9-N-acetylated Neu5Ac as a stable mimic to study the biochemical role of 9-O-acetylated Neu5Ac.

Lipid-specific interactions determine the organization and dynamics of membrane-active peptide melittin.

The cell membranes of different cells deviate significantly in lipid compositions and thus provide varying biological environments to modulate the diffusion, organization and the resultant function of biomacromolecules. However, the detailed modulation mechanism remains elusive especially in consideration of the current overuse of the simplified membrane models such as the pure phosphatidylcholine (PC) membrane. In this work, with the typical membrane-active peptide melittin, we demonstrated that a more complicated membrane environment, such as the bacterial (IME) or plasma membrane (PM), would significantly change the organization and dynamics of melittin, by using molecular dynamics simulations as a "computational microscope". It was found that in these membrane systems, adding melittin would cause a varying degree of reduction in the lateral diffusion of lipids due to the different assembly states of peptides. Melittin tended to aggregate to oligomers in the pure PC membrane, mostly as a tetramer or trimer, while in IME or PM, its degree of oligomerization was significantly reduced. More surprisingly, melittin displayed a strong affinity with ganglioside GM3 in PM, leading to the formation of melittin-GM3 nanoclusters, which hindered its diffusion and further oligomerization. Additionally, small changes in the residue sequence of melittin could modulate the degree or structure of the peptide oligomer. Our work provides a typical example of a study on the organization and dynamics of pore-forming peptides in specific membrane environments and has great significance on the optimization of peptide sequences and the design of helix bundles in the membrane for target biological function.

Effects of Gangliosides on Spermatozoa, Oocytes, and Preimplantation Embryos.

Gangliosides are sialic acid-containing glycosphingolipids, which are the most abundant family of glycolipids in eukaryotes. Gangliosides have been suggested to be important lipid molecules required for the control of cellular procedures, such as cell differentiation, proliferation, and signaling. GD1a is expressed in interstitial cells during ovarian maturation in mice and exogenous GD1a is important to oocyte maturation, monospermic fertilization, and embryonic development. In this context, GM1 is known to influence signaling pathways in cells and is important in sperm-oocyte interactions and sperm maturation processes, such as capacitation. GM3 is expressed in the vertebrate oocyte cytoplasm, and exogenously added GM3 induces apoptosis and DNA injury during in vitro oocyte maturation and embryogenesis. As a consequence of this, ganglioside GT1b and GM1 decrease DNA fragmentation and act as H2O2 inhibitors on germ cells and preimplantation embryos. This review describes the functional roles of gangliosides in spermatozoa, oocytes, and early embryonic development.

GM3(Neu5Gc) ganglioside: an evolution fixed neoantigen for cancer immunotherapy.

Numerous molecules have been considered as targets for cancer immunotherapy because of their levels of expression on tumor cells, their putative importance for tumor biology, and relative immunogenicity. In this review we focus on the ganglioside GM3(Neu5Gc), a glycosphingolipid present on the outer side of the plasma membrane of vertebrate cells. The reasons for selecting GM3(Neu5Gc) as a tumor-specific antigen and its use as a target for cancer immunotherapy are discussed, together with the development of antitumor therapies focused on this target by the Center of Molecular Immunology (CIM, Cuba).

Conjugation of a GM3 lactone mimetic on carbon nanotubes enhances the related inhibition of melanoma-associated metastatic events.

GM3-ganglioside is known to be involved in melanoma proliferation. In order to modulate metastatic-related events, we have functionalized multi-walled carbon nanotubes (MWCNTs) with multiple copies of a GM3-lactone mimetic. The MWCNTs proved to guarantee the appropriate spatial arrangement of the mimetic allowing a stronger inhibition of migration and invasiveness of human melanoma (A375) cells compared to other multivalent constructs reported before. In addition, the effect of the multivalent tubular conjugate on the inhibition of specific tyrosine kinases, which are associated with the ganglioside complexes within the membrane domains, was demonstrated. Finally, the short-term fate of the conjugate was assessed, for the first time, by means of the 1H NMR relaxometry technique by exploiting the signal arising from the CNTs.

Crystal structure of an L chain optimised 14F7 anti-ganglioside Fv suggests a unique tumour-specificity through an unusual H-chain CDR3 architecture.

Targeted cancer immunotherapy offers increased efficacy concomitantly with reduced side effects. One antibody with promising clinical potential is 14F7, which specifically recognises the NeuGc GM3 ganglioside. This antigen is found in the plasma membrane of a range of tumours, but is essentially absent from healthy human cells. 14F7 can discriminate NeuGc GM3 from the very similar NeuAc GM3, a common component of cell membranes. The molecular basis for this unique specificity is poorly understood. Here we designed and expressed 14F7-derived single-chain Fvs (scFvs), which retained the specificity of the parent antibody. Detailed expression and purification protocols are described as well as the synthesis of the NeuGc GM3 trisaccharide. The most successful scFv construct, which comprises an alternative variable light chain (VLA), allowed structure determination to 2.2 Å resolution. The structure gives insights into the conformation of the important CDR H3 loop and the suspected antigen binding site. Furthermore, the presence of VLA instead of the original VL elucidates how this subdomain indirectly stabilises the CDR H3 loop. The current work may serve as a guideline for the efficient production of scFvs for structure determination.

Altered Expression of Ganglioside Metabolizing Enzymes Results in GM3 Ganglioside Accumulation in Cerebellar Cells of a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis.

Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene. Most JNCL patients exhibit a 1.02 kb genomic deletion removing exons 7 and 8 of this gene, which results in a truncated CLN3 protein carrying an aberrant C-terminus. A genetically accurate mouse model (Cln3Δex7/8 mice) for this deletion has been generated. Using cerebellar precursor cell lines generated from wildtype and Cln3Δex7/8 mice, we have here analyzed the consequences of the CLN3 deletion on levels of cellular gangliosides, particularly GM3, GM2, GM1a and GD1a. The levels of GM1a and GD1a were found to be significantly reduced by both biochemical and cytochemical methods. However, quantitative high-performance liquid chromatography analysis revealed a highly significant increase in GM3, suggesting a metabolic blockade in the conversion of GM3 to more complex gangliosides. Quantitative real-time PCR analysis revealed a significant reduction in the transcripts of the interconverting enzymes, especially of ß-1,4-N-acetyl-galactosaminyl transferase 1 (GM2 synthase), which is the enzyme converting GM3 to GM2. Thus, our data suggest that the complex a-series gangliosides are reduced in Cln3Δex7/8 mouse cerebellar precursor cells due to impaired transcription of the genes responsible for their synthesis.

Chemoenzymatically synthesized GM3 analogues as potential therapeutic agents to recover nervous functionality after injury by inducing neurite outgrowth.

Ganglioside GM3 is implicated in a variety of physiological and pathological processes. Due to GM3 exposes on the outer surface of cell membranes, it is strongly associated with cell adhesion, motility and differentiation. Neurite outgrowth is a key process in the development of functional neuronal circuits and regeneration of the nervous system after injury. In the present study, we used enzymatic hydrolysis and chemical synthesis to obtain novel galactose containing GM3 analogues. By enzymatic hydrolysis to prepare GM3 building block, we can avoid multiple chemical procedures. Next, we employed the PC12 cells as a model to evaluate the effects of GM3 analogues on neurite outgrowth with or without NGF induction. The biological tests showed that GM3 analogues could induce neurite outgrowth, which provides the valuable sights for potential nervous system treatment after injury.

Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods.

Syntheses of Fluorescent Gangliosides for the Studies of Raft Domains.

Gangliosides, glycosphingolipids containing one or more sialic acids in the glycan chain, are involved in various important biological processes in cell plasma membranes (PMs). However, the behaviors and functions of gangliosides are poorly understood, primarily because of the lack of fluorescent analogs that are equivalent to native gangliosides that can be used as chemical and physical probes. In this study, we developed entirely chemical methods to synthesize fluorescent gangliosides (GM3, GM2, GM1, and GD1b) in which the glycan components are site-specifically labeled with various fluorescent dyes. The functional evaluations of the synthesized fluorescent gangliosides demonstrated the great influence of fluorescent dye on the physical properties of gangliosides in PMs and revealed the fluorescent ganglioside analogs which show similar behaviors to the native gangliosides.

Altered expression of ganglioside GM3 molecular species and a potential regulatory role during myoblast differentiation.

Gangliosides (sialic acid-containing glycosphingolipids) help regulate many important biological processes, including cell proliferation, signal transduction, and differentiation, via formation of functional microdomains in plasma membranes. The structural diversity of gangliosides arises from both the ceramide moiety and glycan portion. Recently, differing molecular species of a given ganglioside are suggested to have distinct biological properties and regulate specific and distinct biological events. Elucidation of the function of each molecular species is important and will provide new insights into ganglioside biology. Gangliosides are also suggested to be involved in skeletal muscle differentiation; however, the differential roles of ganglioside molecular species remain unclear. Here we describe striking changes in quantity and quality of gangliosides (particularly GM3) during differentiation of mouse C2C12 myoblast cells and key roles played by distinct GM3 molecular species at each step of the process.

Glycosylation Changes Triggered by the Differentiation of Monocytic THP-1 Cell Line into Macrophages.

The human acute monocytic leukemia cell line THP-1 is widely used as an in vitro phagocytic cell model because it exhibits several immune properties similar to native monocyte-derived macrophages. In this study, we investigated the alteration of N- and O-linked glycans as well as glycosphingolipids, during THP-1 differentiation, combining mass spectrometry, flow cytometry, and quantitative real-time PCR. Mass spectrometry revealed that macrophage differentiation led to a marked upregulation of expression of GM3 ganglioside as well as an increase in complex-type structures, particularly triantennary glycans, occurring at the expense of high-mannose N-glycans. Moreover, we observed a slight decrease in the proportion of multifucosylated N-glycans and α2,6-sialylation. The uncovered changes in glycosylation correlated with variations of gene expression of relevant glycosyltransferases and glycosidases including sialyltransferases, ß-N-acetylglucosaminyltransferases, fucosyltransferases, and neuraminidase. Furthermore, using flow cytometry and antibodies directed against glycan structures, we confirmed that the alteration of glycosylation occurs at the cell surface of THP-1 macrophage-like cells. Altogether, we established that macrophagic maturation of THP-1 induces dramatic modifications of the surface glycosylation pattern that may result in differential interaction of monocytic and macrophagic THP-1 with immune or bacterial lectins.