Ganglioside Molecular Species Research Articles

Establishment of a targeted analysis method for gangliosides in mouse tissues by HILIC-ESI-MS/MS.

Gangliosides play an imperative role in cell signaling, neuronal recovery, apoptosis, and other physiological processes. For example, GM3 can regulate hypothalamic leptin resistance and control energy homeostasis, GD3 can mediate cell proliferation and differentiation and induce apoptosis, and GQ1b can stimulate neurogenesis. Therefore, the present study sought to establish and optimize the targeted analysis method for ganglioside subclasses and their molecular species using hydrophilic interaction liquid chromatography-triple quadrupole-MS/MS (HILIC-QQQ-MS/MS). Additionally, the fragmentation pattern of different ganglioside subclasses and their retention time patterns were analyzed, providing more accurate qualitative results. The limit of quantitation (LOQ) was as low as 10-4ng. Moreover, the molecular species of gangliosides in the liver, cortex, and hypothalamus of C57BL/6 mice were analyzed using the established method. A total of 23 ganglioside subclasses with 164 molecular species, including 40 O-acetylated ganglioside molecular species and 28 NeuGc ganglioside molecular species, were identified using the semi-quantitative analysis method of anexternal standard curve corrected by an internal standard. In addition to NeuGc gangliosides, the contents of ganglioside subclasses were more abundant in the mouse brain than those in the mouse liver; especially, the contents of unsaturated gangliosides in the hypothalamus were much higher than those in the liver. Among them, O-acetylated gangliosides were detected only in the cortex and hypothalamus at a concentration of up to 100μg/mg protein (40 molecular species). Overall, the proposed method expanded the detectable number of ganglioside subclasses and molecular species in biological samples and provided more opportunities for further study of the biological functions of gangliosides.

Region-specific mouse brain ganglioside distribution revealed by an improved isobaric aminoxyTMT labeling strategy with automated data processing.

Gangliosides are specialized glycosphingolipids most abundant in the central nervous system. Their complex amphiphilic structure is essential to the formation of membrane lipid rafts and for molecular recognition. Dysfunction of lipid rafts and ganglioside metabolism has been linked to cancer, metabolic disorders, and neurodegenerative disorders. Changes in ganglioside concentration and diversity during the progression of disease have made them potential biomarkers for early detection and shed light on disease mechanisms. Chemical derivatization facilitates whole ion analysis of gangliosides while improving ionization, providing rich fragmentation spectra, and enabling multiplexed analysis schemes such as stable isotope labeling. In this work, we report improvement to our previously reported isobaric labeling methodology for ganglioside analysis by increasing buffer concentration and removing solid-phase extraction desalting for a more complete and quantitative reaction. Identification and quantification of gangliosides are automated through MS-DIAL with an in-house ganglioside derivatives library. We have applied the updated methodology to relative quantification of gangliosides in six mouse brain regions (cerebellum, pons/medulla, midbrain, thalamus/hypothalamus, cortex, and basal ganglia) with 2 mg tissue per sample, and region-specific distributions of 88 ganglioside molecular species are described with ceramide isomers resolved. This method is promising for application to comparative analysis of gangliosides in biological samples.

Pathophysiological Significance of GM3 Ganglioside Molecular Species With a Particular Attention to the Metabolic Syndrome Focusing on Toll-Like Receptor 4 Binding.

GM3 ganglioside, the first molecule in ganglioside family biosynthesis, is formed by transfer of sialic acid to lactosylceramide. Several dozen GM3 molecular species exist, based on diversity of ceramide structures. Among ceramide structures composed of sphingosine and fatty acids, there is a great diversity resulting from different combinations of chain length, hydroxylation, and unsaturation of fatty acid chains. Expression patterns of GM3 species in serum vary during pathogenesis of metabolic syndrome. Physiological activity of each species, and significance of the variability, are poorly understood. Our studies revealed that GM3 species with differing fatty acid structures act as pro- or anti-inflammatory endogenous Toll-like receptor 4 (TLR4) ligands. Very long-chain fatty acid (VLCFA) and α-hydroxyl VLCFA GM3 variants strongly enhanced TLR4 activation. In contrast, long-chain fatty acid (LCFA) and ω-9 unsaturated VLCFA GM3 variants suppressed TLR4 activation. GM3 interacted with extracellular TLR4/myeloid differentiation factor 2 (MD-2) complex, thereby promoting dimerization/oligomerization. In obesity and metabolic syndrome, VLCFA-variant GM3 species were elevated in serum and adipose tissue, whereas LCFA-variant species were reduced, and such imbalances were correlated with disease progression. Our findings summarized in this review demonstrate that GM3 molecular species are disease-related endogenous TLR4 ligands and modulate homeostatic and pathogenic innate immune responses.

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 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.

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.

Ganglioside Profiling of the Human Retina: Comparison with Other Ocular Structures, Brain and Plasma Reveals Tissue Specificities.

Gangliosides make a wide family of glycosphingolipids, highly heterogeneous in both the ceramide moiety and the oligosaccharide chain. While ubiquitously expressed in mammalian tissues, they are particularly abundant in the brain and the peripheral nervous system. Gangliosides are known to play a crucial role in the development, maintenance and functional integrity of the nervous system. However, the expression and roles of gangliosides in the retina, although often considered as a window on the brain, has been far less studied. We performed an in-depth analysis of gangliosides of the human retina, especially using powerful LC/MS methods. We compared the pattern of ganglioside classes and ceramide molecular species of this tissue with other ocular structures and with brain and plasma in elderly human individuals. About a hundred of ganglioside molecular species among 15 distinct classes were detected illustrating the huge structural diversity of these compounds. The retina exhibited a very diverse ganglioside profile and shared several common features with the brain (prominence of tetraosylgangliosides, abundance of d20:1 long chain base and 18:0 fatty acid…). However, the retina stood out with the specific expression of GD3, GT3 and AcGT3, which further presented a peculiar molecular species distribution. The unique ganglioside pattern we observed in the human retina suggests that these ganglioside species play a specific role in the structure and function of this tissue. This lipidomic study, by highlighting retina specific ganglioside species, opens up novel research directions for a better understanding of the biological role of gangliosides in the retina.

Alzheimer’s disease is associated with disordered localization of ganglioside GM1 molecular species in the human dentate gyrus

Alzheimer’s disease (AD) is a progressive dementia associated with loss of memory and cognitive dysfunction. In a previous study, we demonstrated a decrease in b-series gangliosides along with a change in ganglioside molecular species in the hippocampal grey matter of patients with AD. The present study demonstrates the use of imaging mass spectrometry for analyzing the spatial arrangement of ganglioside GM1 (GM1) molecular species in the hippocampus. In AD patients, we found a decrease in the ratio of GM1(d20:1/C18:0) to GM1 d18:1/C18:0) in the outer molecular layer (ML) of the dentate gyrus. Because the outer ML is the region of main input into the hippocampus, our findings may have a direct relationship to the mechanism of dysfunction in AD.

Lactational changes in concentration and distribution of ganglioside molecular species in human breast milk from Chinese mothers.

Gangliosides play a critical role in human brain development and function. Human breast milk (HBM) is an important dietary source of gangliosides for the growing infant. In this study, ganglioside concentrations were measured in the breast milk from a cross-sectional sample of Chinese mothers over an 8-month lactation period. The average total ganglioside concentration increased from 13.1 mg/l during the first month to 20.9 mg/l by 8 months of lactation. The average concentration during the typically solely breast-feeding period of 1‒6 months was 18.9 mg/l. This is the first study to report the relative distribution of the individual ganglioside molecular species through lactation for any population group. The ganglioside molecular species are made up of different fatty acid moieties that influence the physical properties of these gangliosides, and hence affect their function. The GM(3) molecular species containing long-chain acyl fatty acids had the most prominent changes, increasing in both concentration and relative distribution. The equivalent long-chain acyl fatty acid GD(3) molecular species typically decreased in concentration and relative distribution. The lactational trends for both concentration and relative distribution for the very long-chain acyl fatty acid molecular species were more varied. The major GM(3) and GD(3) molecular species during lactation were d40:1 and d42:1, respectively. An understanding of ganglioside molecular species distribution in HBM is essential for accurate application of mass spectrometry methods for ganglioside quantification.

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-galactofuranosyl-(1→3)-α-galactopyranosyl-(1→9)-N-acetyl-α-neuraminosyl-(2→3)-β-galactopyranosyl]-ceramide for PNG-2B. PNG-2A and PNG-2B represent the first GM4 elongation products in nature.

Determination of disialoganglioside GD3 and monosialoganglioside GM3 in infant formulas and whey protein concentrates by ultra‐performance liquid chromatography/electrospray ionization tandem mass spectrometry

A method of ultra-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UPLC-ESI-MS/MS) has been established for simultaneous determination of major disialoganglioside 3 (GD3) and monosialoganglioside 3 (GM3) in infant formulas and whey protein concentrates. Gangliosides were extracted by using the technique of Svennerholm and Fredman and then cleaned up with OASIS HLB solid-phase extraction (SPE) cartridges. The various molecular species of gangliosides were separated on an Acquity UPLC BEH C8 column and analyzed under the negative ion mode. GD3 and GM3 were rapidly quantified using internal standard (IS) method. The developed method was further validated by determining the linearity, average recovery, sensitivity (limit of quantification), and precision. The results presented high correlation coefficients (R(2) > 0.993) of the selected 16 gangliosides molecular species and provided the respective linear ranges. The limit of quantification was 0.325-0.734 mg/100 g for eight molecular species of GD3 and 0.008-0.312 mg/100 g for eight molecular species of GM3, respectively. The reasonable average recoveries (81-95%) and precision (relative standard deviation [RSD] ≤15%) were also demonstrated in three different spiked levels. This new method would be very useful in the quantitative determination of gangliosides in infant formulas and whey protein concentrates.

Imaging Technology of Complex Lipid Molecular Species by a Combination of TLC-Blot and MALDI-TOF –Special Reference to Human Brain Ganglioside Molecular Species

Glycosphingolipids and phospholipids from white matter of human hippocampus, were analyzed by a sequential procedure of two-dimensional TLC (2d-TLC), transference of separated lipids to a PVDF membrane by a TLC-Blot equipment and Mass spectrometry (MS) analysis with an ion-trap type MALDI-TOF equipment. The method is simple and quick; very small amount of sample (0.1 mg of brain tissue) is enough to analyze all lipid components. The 2d-TLC provided excellent separation and the MS analyses allowed identifying the characteristic profile of molecular species for individual glycolipids and phospholipids. The results of MS analyses on gangliosides showed that di- and tri-sialogangliosides are richer in d20:1 sphingosine-containing ceramide than monosialogangliosides, suggesting the presence of sialylation selection after GM1 gangliosides. Then we analyzed ganglioside molecular species obtained from different brain regions by using MS imaging technology. The MS images of individual gangliosides provided clear visual profiles in terms of molecular species distribution. The imaging profiles were region dependent and also indicated that the sialyltransferase toward GM1 ganglioside prefers to select d20:1 sphingosine containing molecule. This technology provides visual characterization of individual phospholipid and glycosphingolipid molecular species and informs us about the metabolic characterization of target tissue, opening a new gate for colorful lipidomics research.

An Approach to Glycobiology from Glycolipidomics: Ganglioside Molecular Scanning in the Brains of Patients with Alzheimer’s Disease by TLC-Blot/Matrix Assisted Laser Desorption/Ionization-Time of Flight MS

We have established a new approach to glyco- and lipidomics using a thin layer chromatography (TLC)-Blot/matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) system. This new approach consists of a combination of a method for transferring lipids separated on a TLC-plate to a poly-vinylidene difluoride (PVDF) membrane and direct mass spectrometric analysis of the individual lipids on the membrane by ion trap-type MALDI-TOF MS. This technology was applied to the analysis of individual lipids from the human brain. Then, based on the results of this analysis, ganglioside molecular species in neural diseases were analyzed. The levels of gangliosides GD1b and GT1b were lower in the hippocampal gray matter of patients with Alzheimer's disease than in the hippocampal gray matter of patients with Parkinson's disease or the control patients. The molecular scanning of individual ganglioside molecular species showed a significant reduction of d20 : 1/C18 : 0 ceramide-containing gangliosides in patients with Alzheimer's disease. These findings suggest that Alzheimer's disease is a kind of ganglioside metabolic disease affecting the hippocampal area. A new approach to glycobiology by the TLC-Blot/MALDI-TOF MS system is proposed.

New approach for glyco‐ and lipidomics – Molecular scanning of human brain gangliosides by TLC‐Blot and MALDI‐QIT‐TOF MS

We have developed a TLC-Blot system that makes possible the direct analysis of blotted glycosphingolipids on a polyvinylidene difluoride membrane from a high-performance TLC plate by immunological staining, chemical staining, enzymatic treatment and mass spectrometric (MS) analysis. An ion trap type matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF) MS apparatus improved not only the molecular identification but also the analysis of molecular species of lipids on the polyvinylidene difluoride membrane. A new approach for glyco- and lipidomics, molecular scanning technology by a combination of TLC-Blot and MALDI-QIT-TOF MS, was developed and applied to human brain gangliosides separated from the tissues of patients with neural diseases and control patients. The results clearly showed a change of ganglioside composition, in addition to identifying individual ganglioside molecular species, in the hippocampus gray matter of patients with Alzheimer's disease. The results strongly suggested that metabolic changes of gangliosides played an important role in the progression of this disease. The present technology with molecular imaging should provide valuable information for elucidating the significance of molecular species in neuronal functions such as neural transmission, memory, and learning.

Highly sensitive localization analysis of gangliosides and sulfatides including structural isomers in mouse cerebellum sections by combination of laser microdissection and hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry with theoretically expanded multiple reaction monitoring

Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is suitable for analysis of glycosphingolipids such as fragile gangliosides avoiding the use of the sialic acid elimination. However, it was not possible to distinguish the structural isomers such as GD1a and GD1b with reversed-phase LC/ESI-MS by hydrophobic interaction. Here we report an effective method for targeted analysis of theoretically expanded ganglioside molecular species including structural isomers by hydrophilic interaction liquid chromatography (HILIC)/ESI-MS with multiple reaction monitoring (MRM). As a result of MRM analysis of glycosphingolipid mixtures from porcine brain, each of the lipid classes was detected within 25 min in the following order: sulfatides > GM3 > GM2 > GM1 > GD3 > GD1a > GD2 > GD1b > GT1a > GT1b > GQ1b. For the advanced application, localization analysis of postnatal day 15 (P15) mouse cerebellum layered structures was carried out by combination of MRM and laser microdissection (LMD). As a result, GM3, GD1a, GT1b and GQ1b were abundantly detected in the molecular and granular layers, whereas GM1 was widely presented in each layered structure. These gangliosides were mainly composed of d18:1-18:0 and d18:1-20:0, but GM3 was d18:1-16:0 and d18:1-20:0. Meanwhile, sulfatide molecular species were mostly localized in the myelinated fibers and scarcely found in the molecular layer. These results suggested that our method is suitable to detect a variety of ganglioside classes and sulfatides with high sensitivity at the molecular species level and effective for localization analysis of these glycosphingolipids from mouse brain sections.

ChemInform Abstract: Biologically Active Glycosides from Asteroidea. Part 25. Glycosphingolipids from the Starfish Asterina pectinifera. Part 2. Structure of Three Ganglioside Molecular Species and a Homogeneous Ganglioside, and Biological Activity of the

ChemInform Abstract: Biologically Active Glycosides from Asteroidea. Part 25. Glycosphingolipids from the Starfish Asterina pectinifera. Part 2. Structure of Three Ganglioside Molecular Species and a Homogeneous Ganglioside, and Biological Activity of the

ChemInform Abstract: Constituents of Holothuroidea. Part 9. Isolation and Structure of a New Ganglioside Molecular Species from the Sea Cucumber Holothuria pervicax.

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Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-MS/MS with theoretically expanded multiple reaction monitoring

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has been applied primarily to the analysis of glycosphingolipids separated from other complex mixtures by TLC, but it is difficult to obtain quantitative profiling of each glycosphingolipid among the different spots on TLC by MALDI-MS. Thus, the development of a convenient approach that utilizes liquid chromatography/electrospray ionization (LC/ESI)-MS has received interest. However, previously reported methods have been insufficient to separate and distinguish each ganglioside class. Here we report an effective method for the targeted analysis of theoretically expected ganglioside molecular species by LC/ESI tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). MRM detection specific for sialic acid enabled us to analyze ganglioside standards such as GM1, GM2, GM3, GD1, and GT1 at picomolar to femtomolar levels. Furthermore, other gangliosides, such as GD2, GD3, GT2, GT3, and GQ1, were also detected in glycosphingolipid standard mixtures from porcine brain and acidic glycolipid extract from mouse brain by theoretically expanded MRM. We found that this approach was also applicable to sulfatides contained in the glycosphingolipid mixtures. In addition, we established a method to separate and distinguish regioisomeric gangliosides, such as GM1a and -1b, GD1a, -1b, and -1c, and GT1a, -1b, and -1c with diagnostic sugar chains in the MRM.

The ceramide structure of GM1 ganglioside differently affects its recovery in low-density membrane fractions prepared from HL-60 cells with or without triton-X100

Gangliosides are characteristically enriched in various membrane domains that can be isolated as low density membrane fraction insoluble in detergents (detergent-resistant membranes, DRMs) or obtained after homogenization and sonication in 0.5 M sodium carbonate (low-density membranes, LDMs). We assessed the effect of the ceramide structure of four [3H]-labeled GM1 ganglioside molecular species (GM1s) taken up by HL-60 cells on their occurrence in LDMs, and compared it with our previous observations for DRMs. All GM1s contained C18 sphingosine, which was acetylated in GM1(18:1/2) or acylated with C14, C18 or C18:1 fatty acids (Fas)

Imaging Mass Spectrometry Technology and Application on Ganglioside Study; Visualization of Age-Dependent Accumulation of C20-Ganglioside Molecular Species in the Mouse Hippocampus

Gangliosides are particularly abundant in the central nervous system (CNS) and thought to play important roles in memory formation, neuritogenesis, synaptic transmission, and other neural functions. Although several molecular species of gangliosides have been characterized and their individual functions elucidated, their differential distribution in the CNS are not well understood. In particular, whether the different molecular species show different distribution patterns in the brain remains unclear. We report the distinct and characteristic distributions of ganglioside molecular species, as revealed by imaging mass spectrometry (IMS). This technique can discriminate the molecular species, raised from both oligosaccharide and ceramide structure by determining the difference of the mass-to-charge ratio, and structural analysis by tandem mass spectrometry. Gangliosides in the CNS are characterized by the structure of the long-chain base (LCB) in the ceramide moiety. The LCB of the main ganglioside species has either 18 or 20 carbons (i.e., C18- or C20-sphingosine); we found that these 2 types of gangliosides are differentially distributed in the mouse brain. While the C18-species was widely distributed throughout the frontal brain, the C20-species selectively localized along the entorhinal-hippocampus projections, especially in the molecular layer (ML) of the dentate gyrus (DG). We revealed development- and aging-related accumulation of the C-20 species in the ML-DG. Thus it is possible to consider that this brain-region specific regulation of LCB chain length is particularly important for the distinct function in cells of CNS.