Advances in the separation of gangliosides by counter-current chromatography (CCC).

Gangliosides play critical roles in the development of many progressive diseases. Due to their structural diversity, efficient methods are needed to separate individual gangliosides for studies of their functions, and for use as standards in the analysis of ganglioside mixtures. This proof-of-concept study reports a useful analytical-semi-preparative scale counter-current chromatography (CCC) enrichment of multiple ganglioside homologues of various species and classes at the milligram level. Since few individual ganglioside standards were available, this research aimed to achieve analytical-semi-preparative scale separation of gangliosides by differences in saccharide monomer compositions (classes), their arrangements (species), or ceramide compositions (homologues), using CCC. The solvent system composition, addition of solvent modifiers, and elution modes were all adjusted to separate porcine gangliosides, mainly GM1 (d36:1), GD1a (d36:1), GD1b (d36:1) and their (d38:1) homologues as a demonstration. The eluted compounds were analyzed by flow-injection analysis (FIA)-MS and LC-MS/MS. A two-phase solvent system, consisting of butanol/methyl t-butyl ether/acetonitrile/water at a ratio of 2:4:3:8 (v/v/v/v) with 0.5% (v/v) acetic acid added to the lower phase, was used to separate mg-levels of porcine gangliosides under dual-mode elution. The relative abundances of the above 6 gangliosides increased from 10 to 21% in the ganglioside extract to 55-73% in the collected fractions through the purification.

Ganglioside Extraction, Purification and Profiling.

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Hp-s1 Ganglioside Suppresses Proinflammatory Responses by Inhibiting MyD88-Dependent NF-κB and JNK/p38 MAPK Pathways in Lipopolysaccharide-Stimulated Microglial Cells.

Hp-s1 ganglioside is isolated from the sperm of sea urchin (Hemicentrotus pulcherrimus). In addition to neuritogenic activity, the biological function of Hp-s1 in neuroinflammation is unknown. In this study, we investigated the anti-neuroinflammatory effect of Hp-s1 on lipopolysaccharide (LPS)-stimulated microglial cells. MG6 microglial cells were stimulated with LPS in the presence or absence of different Hp-s1 concentrations. The anti-inflammatory effect and underlying mechanism of Hp-s1 in LPS-activated microglia cells were assessed through a Cell Counting kit-8 assay, Western blot analysis, and immunofluorescence. We found that Hp-s1 suppressed not only the expression of inducible nitric oxide synthase and cyclooxygenase-2 but also the expression of proinflammatory cytokines, such as TNF-α, IL-1ß, and IL-6. Hp-s1 inhibited the LPS-induced NF-κB signaling pathway by attenuating the phosphorylation and translocation of NF-κB p65 and by disrupting the degradation and phosphorylation of inhibitor κB-α (IκBα). Moreover, Hp-s1 inhibited the LPS-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Hp-s1 also reduced the expression of myeloid differentiation factor 88 (MyD88) and TNF receptor-associated factors 6 (TRAF6), which are prerequisites for NF-κB and MAPKs activation. These findings indicated that Hp-s1 alleviated LPS-induced proinflammatory responses in microglial cells by downregulating MyD88-mediated NF-κB and JNK/p38 MAPK signaling pathways, suggesting further evaluation as a new anti-neuroinflammatory drug.

Comprehensive Profiling of Surface Gangliosides Extracted from Various Cell Lines by LC-MS/MS.

Gangliosides act as a surface marker at the outer cellular membrane and play key roles in cancer cell invasion and metastasis. Despite the biological importance of gangliosides, they have been still poorly characterized due to the lack of effective analytical tools. Herein, we performed molecular profiling and structural elucidation of intact gangliosides in various cell lines including CFPAC1, A549, NCI-H358, MCF7, and Caski. We identified and quantified a total of 76 gangliosides on cell membrane using C18 LC-MS/MS. Gangliosides found in each cell line exhibited high complexity and diversity both qualitatively and quantitatively. The most abundant species was GM3(d34:1) in CFPAC1, NCI-H358, and MCF7, while GM2(d34:1) and GM1(d34:1) were major components in A549 and Caski, respectively. Notably, glycan moieties showed more diversity between cancer cell lines than ceramide moieties. In addition, noncancerous pancreatic cell line (hTERT/HPNE) could be distinguished by gangliosides containing different levels of sialic acid compared with cancerous pancreatic cell line (CFPAC1). These results clearly demonstrated the feasibility of our analytical platform to comprehensive profile of cell surface gangliosides for identifying cell types and subgrouping cancer cell types.

Improved approach for comprehensive profiling of gangliosides and sulfatides in rat brain tissues by using UHPLC-Q-TOF-MS.

Gangliosides (GAs) and sulfatides (STs) are major acidic glycosphingolipids (GSLs) that are particularly abundant in the central nervous system and associated with substantial neurodegenerative diseases. In this study, we developed an improved approach for the comprehensive profiling of GAs and STs in rat brain tissues by adopting a pre-fractionation step before the LC-MS analysis. The pre-fractionation step allows the efficient enrichment of different types of acidic GSLs and the removal of high-abundance interferences, thereby greatly enhanced the detection sensitivity and accuracy of low-abundance acidic GSLs. By using this improved approach, a total of 340 acidic GSLs (from 281 compositions) were characterized in rat brain tissues, including 277 GAs (from 230 compositions) and 63 STs (from 51 compositions), among which 57 GAs and 14 STs were novel acidic GSLs that have not been reported previously. This study represented the most comprehensive profiling of acidic GSLs in rat brain tissues. The result of this study greatly enlarged our understanding of the structural diversity of natural acidic GSLs, and provided important chemical information for the exploration of biological function of acidic GSLs in the central nervous system.

Ion mobility mass spectrometry provides novel insights into the expression and structure of gangliosides in the normal adult human hippocampus.

Clustered into the so-called "glycosynaptic" microdomains in the central nervous system (CNS), gangliosides (GGs) are involved in the formation of functional synapses and neural circuits. Therefore, GGs are important biomarkers in the early diagnosis of CNS pathologies, which are the focus of our research as potential therapeutic targets. A series of neuropsychiatric disorders, including Alzheimer's disease and schizophrenia, are characterized by amnesia and disorientation caused by hippocampal atrophy and diminished cholinergic activity. Based on ion mobility mass spectrometry (IM-MS) capability for the reliable determination of glycopatterns, the changes in the diversity and number of GGs with age and the occurrence of neurological disorders, we report here the development of a high performance IM-MS strategy for assessing the GG profile in a complex mixture extracted from a 20 year old hippocampus. IM separation of GGs based on the charge state, carbohydrate chain length and degree of sialylation led to the detection and identification of 140 species, the largest number of GGs ever reported in an adult hippocampus. Moreover, the obtained data support the concept of GG cholinergic activity. IM tandem MS experiments using collision induced dissociation (CID) confirmed the incidence of GD1b(d18:1/24:1) in the investigated hippocampus specimen.

Imaging mass spectrometry identifies prognostic ganglioside species in rodent intracranial transplants of glioma and medulloblastoma.

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (MALDI-MSI) allows us to investigate the distribution of lipid molecules within tissues. We used MALDI-MSI to identify prognostic gangliosides in tissue sections of rat intracranial allografts of rat glioma and mouse intracranial xenografts of human medulloblastoma. In the healthy adult rodent brain, GM1 and GD1 were the main types of glycolipids. Both gangliosides were absent in both intracranial transplants. The ganglioside GM3 was not present in the healthy adult brain but was highly expressed in rat glioma allografts. In combination with tandem mass spectrometry GM3 (d18:1/C24:0) was identified as the most abundant ganglioside species in the glioma allotransplant. By contrast, mouse xenografts of human medulloblastoma were characterized by prominent expression of the ganglioside GM2 (d18:0/C18:0). Together, these data demonstrate that tissue-based MALDI-MSI of gangliosides is able to discriminate between different brain tumors and may be a useful clinical tool for their classification and grading.

Increased Expression of GM1 Detected by Electrospray Mass Spectrometry in Rat Primary Embryonic Cortical Neurons Exposed to Glutamate Toxicity.

Neurons within different brain regions have varying levels of vulnerability to external stress and respond differently to injury. A potential reason to explain this may lie within a key lipid class of the cell's plasma membrane called gangliosides. These glycosphingolipid species have been shown to play various roles in the maintenance of neuronal viability. The purpose of this study is to use electrospray ionization mass spectrometry (ESI-MS) and immunohistochemistry to evaluate the temporal expression profiles of gangliosides during the course of neurodegeneration in rat primary cortical neurons exposed to glutamate toxicity. Primary embryonic (E18) rat cortical neurons were cultured to DIV (days in vitro) 14. Glutamate toxicity was induced for 1, 3, 6, and 24 h to injure and kill neurons. Immunofluorescence was used to stain for GM1 and GM3 species, and ESI-MS was used to quantify the ganglioside species expressed within these injured neurons. ESI-MS data revealed that GM1, GM2, and GM3 were up-regulated in neurons exposed to glutamate. Interestingly, using immunofluorescence, we demonstrated that the GM1 increase following glutamate exposure occurred in viable neurons, possibly indicating a potential intrinsic neuroprotective response. To test this potential neuroprotective property, neurons were pretreated with GM1 for 24 h prior to glutamate exposure. Pretreatment with GM1 conferred significant neuroprotection against glutamate-induced cell death. Overall, work from this study validates the use of ESI-MS for cell-derived gangliosides and supports the further development of lipid based strategies to protect against neuron cell death.

Effects of Ganglioside on Working Memory and the Default Mode Network in Individuals with Subjective Cognitive Impairment: A Randomized Controlled Trial.

This randomized, double-blind, placebo-controlled trial examined whether the administration of ganglioside, an active ingredient of deer bone extract, can improve working memory performance by increasing gray matter volume and functional connectivity in the default mode network (DMN) in individuals with subjective cognitive impairment. Seventy-five individuals with subjective cognitive impairment were chosen to receive either ganglioside (330[Formula: see text][Formula: see text]g/day or 660[Formula: see text][Formula: see text]g/day) or a placebo for 8 weeks. Changes in working memory performance with treatment of either ganglioside or placebo were assessed as cognitive outcome measures. Using voxel-based morphometry and functional connectivity analyses, changes in gray matter volume and functional connectivity in the DMN were also assessed as brain outcome measures. Improvement in working memory performance was greater in the ganglioside group than in the placebo group. The ganglioside group, relative to the placebo group, showed greater increases in gray matter volume and functional connectivity in the DMN. A significant relationship between increased functional connectivity of the precuneus and improved working memory performance was observed in the ganglioside group. The current findings suggest that ganglioside has cognitive-enhancing effects in individuals with subjective cognitive impairment. Ganglioside-induced increases in gray matter volume and functional connectivity in the DMN may partly be responsible for the potential nootropic effects of ganglioside. The clinical trial was registered with ClinicalTrials.gov (identifier: NCT02379481).

Enhanced capabilities for imaging gangliosides in murine brain with matrix-assisted laser desorption/ionization and desorption electrospray ionization mass spectrometry coupled to ion mobility separation.

The increased interest in lipidomics calls for improved yet simplified methods of lipid analysis. Over the past two decades, mass spectrometry imaging (MSI) has been established as a powerful technique for the analysis of molecular distribution of a variety of compounds across tissue surfaces. Matrix-assisted laser desorption/ionization (MALDI) MSI is widely used to study the spatial distribution of common lipids. However, a thorough sample preparation and necessity of vacuum for efficient ionization might hamper its use for high-throughput lipid analysis. Desorption electrospray ionization (DESI) is a relatively young MS technique. In DESI, ionization of molecules occurs under ambient conditions, which alleviates sample preparation. Moreover, DESI does not require the application of an external matrix, making the detection of low mass species more feasible due to the lack of chemical matrix background. However, irrespective of the ionization method, the final information obtained during an MSI experiment is very complex and its analysis becomes challenging. It was shown that coupling MSI to ion mobility separation (IMS) simplifies imaging data interpretation. Here we employed DESI and MALDI MSI for a lipidomic analysis of the murine brain using the same IMS-enabled instrument. We report for the first time on the DESI IMS-MSI of multiply sialylated ganglioside species, as well as their acetylated versions, which we detected directly from the murine brain tissue. We show that poly-sialylated gangliosides can be imaged as multiply charged ions using DESI, while they are clearly separated from the rest of the lipid classes based on their charge state using ion mobility. This represents a major improvement in MSI of intact fragile lipid species. We additionally show that complementary lipid information is reached under particular conditions when DESI is compared to MALDI MSI.

Structure-Activity Relationship Study of the Neuritogenic Potential of the Glycan of Starfish Ganglioside LLG-3 (‡).

LLG-3 is a ganglioside isolated from the starfish Linchia laevigata. To clarify the structure-activity relationship of the glycan of LLG-3 toward rat pheochromocytoma PC12 cells in the presence of nerve growth factor, a series of mono- to tetrasaccharide glycan derivatives were chemically synthesized and evaluated in vitro. The methyl group at C8 of the terminal sialic acid residue was crucial for neuritogenic activity, and the terminal trisaccharide moiety was the minimum active motif. Furthermore, the trisaccharide also stimulated neuritogenesis in human neuroblastoma SH-SY5Y cells via mitogen-activated protein kinase (MAPK) signaling. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was rapidly induced by adding 1 or 10 nM of the trisaccharide. The ratio of phosphorylated ERK to ERK reached a maximum 5 min after stimulation, and then decreased gradually. However, the trisaccharide did not induce significant Akt phosphorylation. These effects were abolished by pretreatment with the MAPK inhibitor U0126, which inhibits enzymes MEK1 and MEK2. In addition, U0126 inhibited the phosphorylation of ERK 1/2 in response to the trisaccharide dose-dependently. Therefore, we concluded that the trisaccharide promotes neurite extension in SH-SY5Y cells via MAPK/ERK signaling, not Akt signaling.

Apprehending ganglioside diversity: a comprehensive methodological approach.

Gangliosides (GGs) make a wide family of glycosphingolipids ubiquitously expressed in mammalian tissues and particularly abundant in the brain and nervous system. They exhibit a huge diversity due to structural variations in both their oligosaccharidic chain and ceramide moiety, which represent a real analytical challenge. Since their discovery in the 1940s, methods have persistently improved until the emergence of LC/MS, which offers a high level of specificity and sensitivity and is suitable with high-throughput profiling studies. We describe here a comprehensive approach relying on various techniques and aiming at fully characterizing GGs in biological samples. First, total GG content was determined by a biochemical assay. Second, GG class composition was assessed by high-performance thin-layer chromatography followed by colorimetric revelation. Then, ceramide types of GG classes were identified, and their relative quantification was performed thanks to the development of a powerful and reliable LC/MS method. Finally, ceramides were structurally characterized, and minor and less common GG classes were identified using high-resolution MS. These methods were applied to the rat retina to provide an exhaustive description of its GG composition, giving the base for a better understanding of the precise roles of GGs in this tissue.

Partial synthesis of ganglioside and lysoganglioside lipoforms as internal standards for MS quantification.

Within recent years, ganglioside patterns have been increasingly analyzed by MS. However, internal standards for calibration are only available for gangliosides GM1, GM2, and GM3. For this reason, we prepared homologous internal standards bearing nonnatural fatty acids of the major mammalian brain gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, and of the tumor-associated gangliosides GM2 and GD2. The fatty acid moieties were incorporated after selective chemical or enzymatic deacylation of bovine brain gangliosides. For modification of the sphingoid bases, we developed a new synthetic method based on olefin cross metathesis. This method was used for the preparation of a lyso-GM1 and a lyso-GM2 standard. The total yield of this method was 8.7% for the synthesis of d17:1-lyso-GM1 from d20:1/18:0-GM1 in four steps. The title compounds are currently used as calibration substances for MS quantification and are also suitable for functional studies.

Rapid and sensitive MRM-based mass spectrometry approach for systematically exploring ganglioside-protein interactions.

Gangliosides are ubiquitous components of cell membranes. Their interactions with bacterial toxins and membrane-associated proteins (e.g. receptor tyrosine kinases) have important roles in the regulation of multiple cellular functions. Currently, an effective approach for measuring ganglioside-protein interactions especially in a large-scale fashion is largely missing. To this end, we report a facile MS-based approach to explore gangliosides extracted from cells and measure their interactions with protein of interest globally. We optimized a two-step protocol for extracting total gangliosides from cells within 2 h. Easy-to-use magnetic beads conjugated with a protein of interest were used to capture interacting gangliosides. To measure ganglioside-protein interaction on a global scale, we applied a high-sensitive LC-MS system, containing hydrophilic interaction LC separation and multiple reaction monitoring-based MS for ganglioside detection. Sensitivity for ganglioside GM1 is below 100 pg, and the whole analysis can be done in 20 min with isocratic elution. To measure ganglioside interactions with soluble vascular endothelial growth factor receptor 1 (sFlt1), we extracted and readily detected 36 species of gangliosides from perivascular retinal pigment epithelium cells across eight different classes. Twenty-three ganglioside species have significant interactions with sFlt1 as compared with IgG control based on p value cutoff <0.05. These results show that the described method provides a rapid and high-sensitive approach for systematically measuring ganglioside-protein interactions.

Effect of tumor gangliosides on tyrosine phosphorylation of p125FAK in platelet adhesion to collagen.

The exact mechanisms as to how platelets influence blood-borne metastasis remain poorly understood. Gangliosides, sialic acid-containing glycosphingolipids, are associated with tumor progression and metastasis in humans. Gangliosides isolated from tumor cells promote collagen-stimulated platelet aggregation and ATP secretion and enhance platelet adhesion to immobilized collagen. Gangliosides interact with a number of cell surface receptors including integrin receptors. In this study, we examined the effects of α2ß1 integrin-mediated platelet adhesion to collagen and phosphotyrosine signaling of focal adhesion kinase, p125FAK (FAK). platelets pre-incubated with neuroblastoma tumor gangliosides (NBTGs) or their major component GD2 (disialoganglioside) were more adherent to immobilized collagen (OD570 0.43±0.12, 0.39±0.13) compared to platelets pre-incubated with MTB (0.14±0.06, p<0.001); the effect of NBTGs was blocked by F-17 anti-α2 antibody. Pre-incubation of platelets with NBTGs resulted in a marked increase in the phosphotyrosine content of p125FAK in the adherent platelets compared to the MTB-pre-incubated adherent platelets. F-17 anti-α2 antibody decreased protein tyrosine phosphorylation of NBTG-incubated platelets adherent to collagen. These results indicate that the tumor gangliosides enhance platelet adhesion to extracellular matrix collagen by upregulating integrin α2ß1-mediated tyrosine phosphorylation of p125FAK, thereby providing insight into how this interaction may be involved in neuroblastoma metastasis.

Isolation and structure elucidation of GM4-type gangliosides from the Okinawan starfish Protoreaster nodosus.

Three new ganglioside molecular species, termed PNG-1, PNG-2A, and PNG-2B were isolated from pyloric caeca of the starfish Protoreaster nodosus. Their structures were elucidated using a combination of spectroscopic and chemical methods, and characterized as 1-O-[8-O-methyl-N-acetyl-α-neuraminosyl-(2→3)-β-galactopyranosyl]-ceramide for PNG-1, 1-O-[β-galactofuranosyl-(1→3)-α-galactopyranosyl-(1→4)-8-O-methyl-N-acetyl-α-neuraminosyl-(2→3)-β-galactopyranosyl]-ceramide for PNG-2A, and 1-O-[β-galacto furanosyl-(1→3)-α-galactopyranosyl-(1→9)-N-acetyl-α-neuraminosyl-(2→3)-β-galactopyr anosyl]-ceramide for PNG-2B. PNG-2A and PNG-2B represent the first GM4 elongation products in nature.

Large scale biosynthesis of ganglioside analogues by RERF-LC-AI cells cultured in HYPERFlask.

The efficient production of ganglioside analogues was accomplished using RERF-LC-AI cells cultured in HYPERFlask (High Yield PERformance Flask). Eight kinds of ganglioside analogues (GM3, GM2, sialylparagloboside, GD3, di-sialylated lacto-N-tetraose, and another three kinds of analogues with intricate structures) were synthesized by the saccharide primer method using lung squamous-cell carcinoma line RERF-LC-AI and 12-azidododecyl ß-lactoside primer. The yield for each analogue obtained using HYPERFlask was higher than yields obtained from 100-mm dishes.

Molecular identification of GD3 as a suppressor of the innate immune response in ovarian cancer.

Tumors often display mechanisms to avoid or suppress immune recognition. One such mechanism is the shedding of gangliosides into the local tumor microenvironment, and a high concentration of circulating gangliosides is associated with poor prognosis. In this study, we identify ganglioside GD3, which was isolated from the polar lipid fraction of ovarian cancer-associated ascites, as an inhibitory factor that prevents innate immune activation of natural killer T (NKT) cells. Purified GD3 displayed a high affinity for both human and mouse CD1d, a molecule involved in the presentation of lipid antigens to T cells. Purified GD3, as well as substances within the ascites, bound to the CD1d antigenic-binding site and did not require additional processing for its inhibitory effect on NKT cells. Importantly, in vivo administration of GD3 inhibited α-galactosylceramide (α-GalCer)-induced NKT cell activation in a dose-dependent manner. These data therefore indicate that ovarian cancer tumors may use GD3 to inhibit the antitumor NKT cell response as an early mechanism of tumor immune evasion.

Distribution of mono-, di- and trisialo gangliosides in the brain of Actinopterygian fishes.

BACKGROUND: Mono-, di- and trisialo gangliosides are major glycosphingolipids in the brain of higher vertebrates involved in lipid raft assembly. In contrast, the fish brain is abundant in polisialo-gangliosides, whose function is implicated in the modulation of repulsive and attractive intercellular interactions during embryonic development and a temperature adaptation process. The histological distribution of gangliosides is usually studied in rodent and mammalian brains, but to date it has not been described in the case of fish brain. METHODS: Gangliosides were extracted from adult brains of trout, carp and zebrafish and separated by TLC. High-affinity anti-ganglioside (GM1, GD1a, GD1b, GT1b) IgG antibodies were used for immunohistochemistry. RESULTS: In trout and carp brains GM1 and GT1b are expressed in the same neuronal cell bodies from the telencephalon to the spinal cord. In zebrafish brain GM1 was not detected, whereas GT1b is a general neuropil staining. GD1a is specific for unmyelinated parallel fibers in carp and zebrafish brains as well as parallel fibers in the molecular layer of all cerebellar divisions. In trout brain GD1b is found in parallel fibers of the cerebellum, but not in the tectum mesencephali. GD1b is expressed in zebrafish neuronal cell bodies. CONCLUSIONS: Each studied species has a different expression of complex gangliosides. GT1b is widely present, whereas GD1a and GD1b appear in a specific group of unmyelinated fibers and could be used as their specific marker. GENERAL SIGNIFICANCE: This is the first report on mono-, di- and trisialo ganglioside (GM1, GD1a, GD1b and GT1b) distribution in the brain of adult Actinopterygian fishes. This article is part of a Special Issue entitled Glycoproteomics.