Stimulatory effect of gangliosides on phagocytosis, phagosome–lysosome fusion, and intracellular signal transduction system by human polymorphonuclear leukocytes

Gangliosides are known to be differentiation-inducing molecules in mammalian stem cells. We studied the interaction between the molecular structure of glycosphingolipids (GSLs) and their promoting mechanisms of the phagocytic processes in human polymorphonuclear leukocytes (PMN). The effect of various gangliosides from mammalian tissues on adhesion, phagocytosis, phagosome–lysosome (P–L) fusion and superoxide anion production was examined by human PMN using heat-killed cells of Staphylococcus aureus coated with GSLs. Gangliosides GM3, GD1a, GD3 and GT1b showed a marked stimulatory effect on the phagocytosis and P–L fusion in a dose-dependent manner, while ganglioside GM1, asialo GM1 and neutral GSLs did not. The relative phagocytic rate of ganglioside GM3-coated S. aureus was the highest among the tested GSLs. Both P–L fusion rate and phagocytosis of S. aureus were elevated significantly when coated with ganglioside GD1a, GD3 or GT1b, and GT1b gave a five times higher rate than that of the non-coated control. These results suggest that the terminal sialic acid moiety is essential for the enhancement of phagocytosis and that the number of sialic acid molecules in the ganglioside is related to the enhancement of the P–L fusion process. On the other hand, the superoxide anion release from PMN was not affected by ganglioside GM2, GM3, GD1a or GT1b. Furthermore, to clarify the trigger or the signal transduction mechanism of phagocytic processes, we examined the effect of protein kinase inhibitors such as H-7, staurosporine (protein kinase C inhibitor), H-89 (protein kinase A inhibitor), genistein (tyrosine kinase inhibitor), ML-7 (myosin light chain kinase inhibitor), and KN-62 (Ca2+/calmodulin-dependent protein kinase II inhibitor) on ganglioside-induced phagocytosis. H-7, staurosporine and KN-62 inhibited ganglioside-induced phagocytosis in the range of concentration without cell damage, while H-89, genistein and ML-7 did not. Moreover, H-7 and KN-62 inhibited ganglioside-induced P–L fusion. These results suggest that protein kinase C and Ca2+/calmodulin-dependent protein kinase II may be involved in the induction of phagocytosis and P–L fusion stimulated by gangliosides. This revised version was published online in November 2006 with corrections to the Cover Date.     

Direct analysis of sialylated or sulfated glycosphingolipids and other polar and neutral lipids using TLC-MS interfaces

Gangliosides and sulfatides (STs) are acidic glycosphingolipids (GSLs) that have one or more sialic acids or sulfate substituents, in addition to neutral sugars, attached to the C-1 hydroxyl group of the ceramide long chain base. TLC is a widely employed and convenient technique for separation and characterization of GSLs. When TLC is directly coupled to MS, it provides both the molecular mass and structural information without further purification. Here, after development of the TLC plates, the structural analyses of acidic GSLs, including gangliosides and STs, were investigated using the liquid extraction surface analysis (LESA™) and CAMAG TLC-MS interfaces coupled to an ESI QSTAR Pulsar i quadrupole orthogonal TOF mass spectrometer. Coupling TLC with ESI-MS allowed the acquisition of high resolution mass spectra of the acidic GSLs with high sensitivity and mass accuracy, without the loss of sialic acid residues that frequently occurs during low-pressure MALDI MS. These systems were then applied to the analysis of total lipid extracts from bovine brain. This allowed profiling of many different lipid classes, not only gangliosides and STs, but also SMs, neutral GSLs, and phospholipids.     

The Hypothesis on Function of Glycosphingolipids and ABO Blood Groups Revisited

Twenty-five years ago the author proposed new ideas of glycoprotein (GPs) and glycosphingolipid (GSLs) functions at the cell membrane. The GPs, apart from their glycan carrying capacity, were assumed to have specific, protein associated, functions. In contrast, GSLs such as those of globo and neolacto/lacto series, were considered to be energetically cheap membrane packing substances, filling in membrane spaces not covered with functional GPs. The terminal carbohydrate structures of the neolacto/lacto GSLs, i.e., sialic acid residues and ABH glycotopes, were postulated to have either regulatory or protective functions, respectively. A special active role was ascribed to terminal β-galactosyl residues of GSLs and GPs. Gangliosides were considered to be functional GSLs. In the present review the author discusses these old ideas in context of the contemporary knowledge and comes to the conclusion that they have not aged.     

Acidic glycosphingolipids of cock testis elucidated by mass spectrometry and NMR spectroscopy

The main acidic glycosphingolipids (GSLs) of cock testis were identified as GalCer I³-sulfate and gangliosides GM4, GM3, GD3 and GT3. They contained N-acetylneuraminic acid as the major sialic acid, and ceramides composed mainly of sphingosine (dl8:1) and C18–24 non-hydroxy fatty acids. Appreciable amounts of hydroxy fatty acids were detected only in the GM4 preparation.     

Functional Roles of Gangliosides in Neurodevelopment: An Overview of Recent Advances

Gangliosides are sialic acid-containing glycosphingolipids that are most abundant in the nervous system. They are localized primarily in the outer leaflets of plasma membranes and participated in cell–cell recognition, adhesion, and signal transduction and are integral components of cell surface microdomains or lipid rafts along with proteins, sphingomyelin and cholesterol. Ganglioside-rich lipid rafts play an important role in signaling events affecting neural development and the pathogenesis of certain diseases. Disruption of gangloside synthase genes in mice induces developmental defects and neural degeneration. Targeting ganglioside metabolism may represent a novel therapeutic strategy for intervention in certain diseases. In this review, we focus on recent advances on metabolic and functional studies of gangliosides in normal brain development and in certain neurological disorders.     

Specific Synthesis of Neurostatin and Gangliosides O-Acetylated in the Outer Sialic Acids Using a Sialate Transferase

Gangliosides are sialic acid containing glycosphingolipids, commonly found on the outer leaflet of the plasma membrane. O-acetylation of sialic acid hydroxyl groups is one of the most common modifications in gangliosides. Studies on the biological activity of O-acetylated gangliosides have been limited by their scarcity in nature. This comparatively small change in ganglioside structure causes major changes in their physiological properties. When the ganglioside GD1b was O-acetylated in the outer sialic acid, it became the potent inhibitor of astroblast and astrocytoma proliferation called Neurostatin. Although various chemical and enzymatic methods to O-acetylate commercial gangliosides have been described, O-acetylation was nonspecific and produced many side-products that reduced the yield. An enzyme with O-acetyltransferase activity (SOAT) has been previously cloned from the bacteria Campylobacter jejuni. This enzyme catalyzed the acetylation of oligosaccharide-bound sialic acid, with high specificity for terminal alpha-2,8-linked residues. Using this enzyme and commercial gangliosides as starting material, we have specifically O-acetylated the gangliosides’ outer sialic acids, to produce the corresponding gangliosides specifically O-acetylated in the sialic acid bound in alpha-2,3 and alpha-2,8 residues. We demonstrate here that O-acetylation occurred specifically in the C-9 position of the sialic acid. In summary, we present a new method of specific O-acetylation of ganglioside sialic acids that permits the large scale preparation of these modified glycosphingolipids, facilitating both, the study of their mechanism of antitumoral action and their use as therapeutic drugs for treating glioblastoma multiform (GBM) patients.     

Carbohydrate-to-carbohydrate interactions between α2,3-linked sialic acids on α2 integrin subunits and asialo-GM1 underlie the bone metastatic behaviour of LNCAP-derivative C4-2B prostate cancer cells

Complex interplays among proteins, lipids and carbohydrates can alter the phenotype and are suggested to have a crucial role in tumour metastasis. Our previous studies indicated that a complex of the GSLs (glycosphingolipids), AsGM1 (asialo-GM1), which lacks α2,3-linked sialic acid, and α2β1 integrin receptors is responsible for the metastatic behaviour of C4-2B prostate cancer cells. Herein, we identified and addressed the functional significance of changes in sialylation during prostate cancer progression. We observed an increase in α2,3-linked sialic acid residues on α2 subunits of α2β1 integrin receptors, correlating with increased gene expression of α2,3-STs (sialyltransferases), particularly ST3GAL3. Cell surface α2,3-sialylation of α2 subunits was required for the integrin α2β1-dependent cell adhesion to collagen type I and the same α2,3-linked sialic acid residues on the integrin receptor were responsible for the interaction with the carbohydrate moiety of AsGM1, explaining the complex formation between AsGM1 and α2β1 integrin receptors. These results provide novel insights into the role of sialic acids in the organization and function of important membrane components in invasion and metastatic processes.     

Brain gangliosides: functional ligands for myelin stability and the control of nerve regeneration

Gangliosides, sialylated glycosphingolipids which are the predominant glycans on vertebrate nerve cell surfaces, are emerging as components of membrane rafts, where they can mediate important physiological functions. Myelin associated glycoprotein (MAG), a minor constituent of myelin, is a sialic acid binding lectin with two established physiological functions: it is involved in myelin-axon stability and cytoarchitecture, and controls nerve regeneration. MAG is found selectively on the myelin membranes directly apposed to the axon surface, where it has been proposed to mediate myelin-axon interactions. Although the nerve cell surface ligands for MAG remain to be established, evidence supports a functional role for sialylated glycoconjugates. Here we review recent studies that reflect on the role of gangliosides, sialylated glycosphingolipids, as functional MAG ligands. MAG binds to gangliosides with the terminal sequence 'NeuAc alpha 3Gal beta 3GalNAc' which is found on the major nerve gangliosides GD1a and GT1b. Gangliosides lacking that terminus (e.g., GM1 or GD1b), or having any biochemical modification of the terminal NeuAc residue fail to support MAG binding. Genetically engineered mice lacking the GalNAc transferase required for biosynthesis of the 'NeuAc alpha 3Gal beta 3GalNAc' terminus have grossly impaired myelination and progressive neurodegeneration. Notably the MAG level in these animals is dysregulated. Furthermore, removal of NeuAc residues from nerve cells reverses MAG-mediated inhibition of neuritogenesis, and neurons from mice lacking the 'NeuAc alpha 3 Gal beta 3GalNAc' terminus have an attenuated response to MAG. Cross-linking nerve cell surface gangliosides can mimic MAG-mediated inhibition of nerve regeneration. Taken together these observations implicate gangliosides as functional MAG ligands.     

Proton nuclear magnetic resonance of neutral and acidic glycosphingolipids

A number of intact neutral glycosphingolipids (globo, asialoganglio, neolacto, and gala series), gangliosides, and sulfatide were analyzed by proton nuclear magnetic resonance (NMR) using dimethyl-d6 sulfoxide as a solvent at different conditions of measurement. The chemical shifts of amide proton of ceramide, N-acetylhexosamine and sialic acid moieties were positioned with regularity, thus providing their molar composition. The chemical shifts of amide proton in ceramide moiety differed with respect to constituent fatty acids; delta 7.45 to 7.52 ppm at 25 degrees C for the nonhydroxy acids and 7.32 to 7.42 ppm for the hydroxy acids. The chemical shifts of methyl proton in N-acetyl group were distinguished between N-acetylhexosamine and N-acetylneuraminic acid, and those in N-acetylgalactosamine were discriminated between neutral glycolipids and gangliosides. In the presence or absence of D2O in dimethyl sulfoxide at 110 degrees C, the anomeric protons resonated with regularity characteristic of respective monosaccharide linkages, and the anomeric protons of N-acetylgalactosamine in neutral glycolipids and gangliosides were clearly distinguished. The present study thus demonstrates the general applicability of NMR procedure to glycosphingolipids, providing the determination of chemical composition of both the lipophilic and carbohydrate moieties and the structural elucidation.     

Apoptosis of human carcinoma cells in the presence of inhibitors of glycosphingolipid biosynthesis: I. Treatment of Colo-205 and SKBR3 cells with isomers of PDMP and PPMP

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells by anti-cancer drugs and biosynthetic inhibitors of cells surface glycolipids in the human colon carcinoma cells (Colo-205) are of interest in recent years. In our present studies, we have employed different stereoisomers of PPMP and PDMP (inhibit GlcT-glycosyltransferase (GlcT-GLT)) to initiate apoptosis in Colo-205 cells grown in culture in the presence of ³H-TdR and ³H/or ¹⁴C-L-Serine. Our analysis showed that the above reagents (between 1 to 20 μM) initiated apoptosis with induction of Caspase-3 activities and phenotypic morphological changes in a dose-dependent manner. We have observed an increase of radioactive ceramide formation in the presence of a low concentration (1–4 μM) of these reagents in these cell lines. However, high concentrations (4–20 μM) inhibited incorporation of radioactive serine in the higher glycolipids. Colo-205 cells were treated with L-threo-PPMP (0–20 μM) and activities of different GSL: GLTs were estimated in total Golgi-pellets. The cells contained high activity of GalT-4 (UDP-Gal: LcOse3Cer β1-4galactosyltransferase), whereas negligible activity of GalT-3 (UDP-Gal: GM2 β1-3galactosyltransferase) or GM2-synthase activity of the ganglioside pathway was detected. Previously, GLTs involved in the biosynthetic pathway of SA-Le^x formation had been detected in these colon carcinoma (or Colo-205) cells (Basu M et al. Glycobiology 1, 527–35 (1991)). However, during progression of apoptosis in Colo-205 cells with increasing concentrations of L-PPMP, the GalT-4 activity was decreased significantly. These changes in the specific activity of GalT-4 in the total Golgi-membranes could be the resultant of decreased gene expression of the enzyme. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Glycosphingolipids of purified human lymphocytes.

Biochemical analysis of the glycosphingolipids (GSLs) of human lymphocytes revealed qualitative and quantitative variations among purified lymphocytes from different tissues. The major neutral GSLs of tonsil lymphocytes are glucosyl ceramide (CMH), lactosyl ceramide (CDH), trihexosyl ceramide (CTH), and globoside. Thymocytes and peripheral blood lymphocytes (PBL) contain only traces of CTH and globoside, and PBL contain more CMH and CDH per cell than tonsil lymphocytes. Thymocytes and PBL contain relatively large amounts of more complex neutral GSLs that are present in only trace amounts in tonsil lymphocytes. Peripheral blood lymphocytes contained three and five times more lipid-bound sialic acid than thymocytes and toncil lymphocytes, respectively. Thymocytes and PBL contained mostly hematoside, whereas tonsil lymphocytes contained more complex gangliosides in addition to hematoside. The observed differences in GSL content among these cells may be related to their content of B cells, which comprise approximately 50% of tonsil lymphocytes, 10% of PBL and 0-2% of thymus cells, and/or the known differences in functional capacities of cells in different lymphoid organs. These findings suggest that cell surface GSLs may serve as markers for identification of functional subpopulations of human lymphocytes.     

Metabolic pathways and intracellular trafficking of gangliosides

Gangliosides constitute a large and heterogeneous family of acidic glycosphingolipids that contain one or more sialic acid residues and are expressed in nearly all vertebrate cells. Their de novo synthesis starts at the endoplasmic reticulum and is continued by a combination of glycosyltransferase activities at the Golgi complex, followed by vesicular delivery to the plasma membrane. At the cell surface, gangliosides participate in a variety of physiological as well as pathological processes. The cloning of genes for most of the glycosyltransferases responsible for ganglioside biosynthesis has produced a better understanding of the cellular and molecular basis of the ganglioside metabolism. In addition, the ability to delete groups of glycosphingolipid structures in mice has been enormously important in determining their physiological roles. Recently, a number of enzymes for ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane, which might contribute to modulate local glycolipid composition, and consequently, the cell function.     

Gangliosides in human, cow and goat milk, and their abilities as to neutralization of cholera toxin and botulinum type A neurotoxin

To elucidate the potential of mammalian milk as to protection of infants from infections, we determined the ganglioside compositions of human, cow and goat milk in relation with cholera toxin and botulinum type A neurotoxin-receptors. Gangliosides accounted for 1 to 2 μmol of lipid-bound sialic acid (LSA) in 100 ml of milk, and GD3 comprised about 69% of LSA in all milk samples. Among the milk samples examined, goat milk was found to contain an amount of gangliosides belonging to the b-pathway representing 15.8% of the total LSA. Accordingly, botulinum neurotoxin bound to GT1b and GQ1b in goat milk, but not to any gangliosides in human or cow milk. On the other hand, GM1, the cholera toxin receptor, was found to be present in all milk samples at concentrations of 0.02% to 0.77% of the total LSA and to be maintained at a relatively constant level in human milk during the postpartum period. Gangliosides from 1 ml of pooled human milk exhibited the ability to attenuate the binding of cholera toxin (30 ng) to GM1 by 93%, and those from 500 μl of goat milk completely inhibited the binding of botulinum type A neurotoxin 1.5 μg to GT1b. The glycolipid nomenclature is based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [1]. The ganglioside nomenclature of Svennerholm is employed throughout [2]. PVP, polyvinylpyrrolidone; LSA, lipid-bound sialic acid.     

Epigenetic regulation of ganglioside expression in neural stem cells and neuronal cells

The structural diversity and localization of cell surface glycosphingolipids (GSLs), including gangliosides, in glycolipid-enriched microdomains (GEMs, also known as lipid rafts) render them ideally suited to play important roles in mediating intercellular recognition, interactions, adhesion, receptor function, and signaling. Gangliosides, sialic acid-containing GSLs, are most abundant in the nerve tissues. The quantity and expression pattern of gangliosides in brain change drastically throughout development and these changes are mainly regulated through stage-specific expression of glycosyltransferase genes. We previously demonstrated for the first time that efficient histone acetylation of the glycosyltransferase genes in mouse brain contributes to the developmental alteration of ganglioside expression. We further demonstrated that acetylation of histones H3 and H4 on the N-acetylgalactosaminyltransferase I (GalNAcT, GA2/GM2/GD2/GT2-synthase; B4galnt1) gene promoter resulted in recruitment of trans-activation factors. In addition, we showed that epigenetic activation of the GalNAcT gene was detected and accompanied by an apparent induction of neuronal differentiation of neural stem cells (NSCs) responding to an exogenous supplement of ganglioside GM1. Most recently, we found that nuclear GM1 binds with acetylated histones on the promoters of the GalNAcT as well as on the NeuroD1 genes in differentiated neurons. Here, we will introduce epigenetic regulation of ganglioside synthase genes in neural development and neuronal differentiation of NSCs.     

Gangliosides in the nervous system

Gangliosides, sialic acid-containing glycosphingolipids, are embedded in neural plasma membranes to provide cell surface recognition sites with negative charges. Exogenous ligands such as bacterial toxins, hormones, growth factors, antibodies, viruses, and interferons bind to specific gangliosides to induce sequential activations of cellular metabolisms. Predominant expression of some of ganglioside series (A, B, C or hematosides) occurs during cell differentiation and transformation. Gangliosides may regulate cell growth and nerve sprouting, suggesting the potential therapeutic value for some neurological disorders. Current ganglioside research is more rapidly growing largely due to advancing methodologies.     

Ganglioside/glycosphingolipid turnover: New concepts

In this review focus is given to the metabolic turnover of gangliosides/glycosphingolipids. The metabolism and accompanying intracellular trafficking of gangliosides/glycosphingolipids is illustrated with particular attention to the following events: (a) the de novo biosynthesis in the endoplasmic reticulum and Golgi apparatus, followed by vesicular sorting to the plasma membrane; (b) the enzyme-assisted chemical modifications occurring at the plasma membrane level; (c) the internalization via endocytosis and recycling to the plasma membrane; (d) the direct glycosylations taking place after sorting from endosomes to the Golgi apparatus; (e) the degradation at the late endosomal/lysosomal level with formation of fragments of sugar (glucose, galactose, hexosamine, sialic acid) and lipid (ceramide, sphingosine, fatty acid) nature; (f) the metabolic recycling of these fragments for biosynthetic purposes (salvage pathways); and (g) further degradation of fragments to waste products. Noteworthy, the correct course of ganglioside/glycosphingolipid metabolism requires the presence of the vimentin intracellular filament net work, likely to assist intracellular transport of sphingoid molecules. Out of the above events those that can be quantitatively evaluated with acceptable reliability are the processes of de novo biosynthesis, metabolic salvage and direct glycosylation. Depending on the cultured cells employed, the percentage of distribution of de novo biosynthesis, salvage pathways, and direct glycosylation, over total metabolism were reported to be: 35% (range: 10–90%) for de novo biosynthesis, 7% (range: 5–10%) for direct glycosylation, and 58% (range: 10–90%) for salvage pathways. The attempts made to calculate the half-life of overall ganglioside turnover provided data of unsure reliability, especially because in many studies salvage pathways were not taken into consideration. The values of half-life range from 2 to 6.5 h to 3 days depending on the cells used. Available evidence for changes of ganglioside/glycosphingolipid turnover, due to extracellular stimuli, is also considered and discussed. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Life without gangliosides

Gangliosides are sialic acid containing glycosphingolipids that are present on all plasma cell membranes. Although they represent the major sialoconjugates in the central nervous system, their precise functions remain obscure. We have generated a series of knockout mice with gene disruptions in the ganglioside biosynthetic pathway. Individually these mice lack subsets of gangliosides allowing investigations into their specific functions. By breeding mice to contain multiple mutations in the biosynthetic pathway we have now produced mice without neuronal gangliosides. These mice display an extremely severe phenotype and neuronal pathology highlighting the essential function of gangliosides in the central nervous system.     

Occurrence of an alpha-galacturonosyl-ceramide in the dioxin-degrading bacterium Sphingomonas wittichii

The chemical structure of two glycosphingolipids (GSLs) found in the dioxin-degrading bacterium Sphingomonas wittichii strain RW1 was investigated by means of mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. One of the GSLs was alpha-D-glucuronosyl-ceramide, commonly present in Sphingomonas spp., and the other was proved to be alpha-D-galacturonosyl-ceramide, whose sugar configuration has not been reported before. In both GSLs the ceramide portion was composed of myristic acid or 2-hydroxy-myristic acid as the fatty acid, and 2-amino-1,3-octadecanediol or 2-amino-cis-13,14-methylene-1,3-eicosanediol as the dihydrosphingosine.     

Gangliosides GD1a and GM3 induce interleukin-10 production by human T cells

Gangliosides are sialic acid-containing glycosphingolipids and exhibit various physiologic functions. Gangliosides GD1a and GM3 strongly induced interleukin-10 (IL-10) protein secretion and mRNA expression in T cells from normal human subjects while the other gangliosides were ineffective. IL-10 induction by both gangliosides was completely blocked by protein tyrosine kinase (PTK) inhibitors, herbimycin A, genistein, and tyrphostin AG 1288, but not by other signal transduction inhibitors. These results suggest that GD1a and GM3 may induce IL-10 production in T cells by regulating the PTK-dependent signaling pathway. These gangliosides may thus act as important immunoregulators via IL-10.