Sialic Acid-containing Glycolipids Research Articles

The ligand interactions of B cell Siglecs are involved in the prevention of autoimmunity to sialylated self-antigens and in the quality control of signaling-competent B cells.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of membrane molecules that recognize sialic acid. Most of them are inhibitory receptors that inhibit immune-cell activation by recognizing sialic acid as a self-motif. Human B cells express CD22 (also known as Siglec-2), Siglec-5, Siglec-6 and Siglec-10 whereas mouse B cells express CD22 and Siglec-G (ortholog of human Siglec-10). Siglecs recognize both sialylated molecules expressed on the same cell (cis-ligands) and those expressed by other cells (trans-ligands). In Guillain-Barré syndrome (GBS), antibody production to gangliosides (which are sialic acid-containing glycolipids) expressed by neurons plays a pathogenic role. A Siglec-10 variant deficient in recognition of gangliosides is genetically associated with GBS, suggesting that Siglec-10 induces self-tolerance to gangliosides by recognizing gangliosides as trans-ligands. Recognition of the BCR as a cis-ligand by Siglec-G and CD22 suppresses BCR signaling in B-1 cells and conventional B cells, respectively. This signal suppression prevents excess expansion of B-1 cells and is involved in the quality control of signaling-competent B cells by setting a threshold for tonic signaling during B cell development. CD22 recognizes other cis-ligands including CD22 and β7 integrin. Interaction of CD22 with other CD22 molecules induces CD22 clustering that suppresses CD22-mediated signal inhibition upon BCR ligation, and interaction with β7 integrin maintains its function in the gut-homing of B cells. Taken together, interactions of B cell Siglecs with multiple trans- and cis-ligands play important roles in B cell homeostasis and immune responses.

Role of inhibitory B cell co-receptors in B cell self-tolerance to non-protein antigens.

Antibodies to non-protein antigens such as nucleic acids, polysaccharides, and glycolipids play important roles in both host defense against microbes and development of autoimmune diseases. Although non-protein antigens are not recognized by T cells, antibody production to non-protein antigens involve T cell-independent mechanisms such as signaling through TLR7 and TLR9 in antibody production to nucleic acids. Although self-reactive B cells are tolerized by various mechanisms including deletion, anergy, and receptor editing, T cell tolerance is also crucial in self-tolerance of B cells to protein self-antigen because self-reactive T cells induce autoantibody production to these self-antigens. However, presence of T cell-independent mechanism suggests that T cell tolerance is not able to maintain B cell tolerance to non-protein self-antigens. Lines of evidence suggest that B cell response to non-protein self-antigens such as nucleic acids and gangliosides, sialic acid-containing glycolipids, are suppressed by inhibitory B cell co-receptors CD72 and Siglec-G, respectively. These inhibitory co-receptors recognize non-protein self-antigens and suppress BCR signaling induced by these antigens, thereby inhibiting B cell response to these self-antigens. Inhibitory B cell co-receptors appear to be involved in B cell self-tolerance to non-protein self-antigens that can activate B cells by T cell-independent mechanisms.

Sialic acid-containing glycolipids mediate binding and viral entry of SARS-CoV-2.

Emerging evidence suggests that host glycans influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal that the receptor-binding domain (RBD) of the spike (S) protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (Sia), with preference for monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind to the RBD. The monomeric affinities (Kd = 100-200 μM) of gangliosides for the RBD are similar to another negatively charged glycan ligand of the RBD proposed as a viral co-receptor, heparan sulfate (HS) dp2-dp6 oligosaccharides. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to angiotensin-converting enzyme 2 (ACE2)-expressing cells is decreased following depletion of cell surface Sia levels using three approaches: sialyltransferase (ST) inhibition, genetic knockout of Sia biosynthesis, or neuraminidase treatment. These effects on RBD binding and both pseudotyped and authentic SARS-CoV-2 viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

Chemoenzymatic Synthesis and Facile Purification of Gangliosides.

Gangliosides are biologically important sialic acid-containing glycolipids found commonly in human and other vertebrates. Isolation of pure gangliosides from cells or tissues is difficult, and chemical synthesis of gangliosides usually involves numerous steps with low synthetic yields. We report here a chemoenzymatic synthesis and purification protocol for two ganglioside cancer antigens, GM3 and GD3. One-pot multienzyme glycosylation reactions are used to sequentially prepare GM3 and GD3 sphingosines from chemically synthesized lactosyl sphingosine. A facile C18-cartridge purification procedure after each glycosylation reaction provides the desired pure glycosyl sphingosine product, which is readily acylated to form the target ganglioside. © 2021 Wiley Periodicals LLC. Basic Protocol: Chemoenzymatic synthesis and purification of GM3 and GD3 gangliosides Support Protocol: Monitoring reactions by thin-layer chromatography.

Monosialyl Ganglioside GM3 Decreases Apolipoprotein B-100 Secretion in Liver Cells.

Some sialic acid-containing glycolipids are known to regulate development of atherosclerosis with accumulated plasma apolipoprotein B-100 (Apo-B)-containing lipoproteins, because Apo-B as an atherogenic apolipoprotein is assembled mainly in VLDL and LDL. Previously, we have elucidated that disialyl GD3 promotes the microsomal triglyceride transfer protein (MTP) gene expression and secretion of triglyceride (TG)-assembled ApoB, claiming the GD3 role in ApoB lipoprotein secretion in liver cells. In the synthetic pathway of gangliosides, GD3 is synthesized by addition of a sialic acid residue to GM3. Thus, there should be some regulatory links between GM3 and GD3. In this study, exogenous and endogenous monosialyl GM3 has been examined how GM3 plays a role in ApoB secretion in Chang liver cells in a view point of MTP and ApoB degradation in the same cells. The level of GM3 ganglioside in the GM3 synthase gene-transfected cells was increased in the cell extract, but not in the medium. In addition, GM3 synthase gene-transfected cells showed a diminished secretion of TG-enriched ApoB with a lower content of TG in the medium. Exogenous GM3 treatment for 24 h exerted a dose dependent inhibitory effect on ApoB secretion together with TG, while a liver-specific albumin was unchanged, indicating that GM3 effect is limited to ApoB secretion. GM3 decreased the mRNA level of MTP gene, too. ApoB protein assembly dysregulated by GM3 indicates the impaired ApoB secretion is caused by a proteasome-dependent pathway. Treatment with small interfering RNAs (siRNAs) decreased ApoB secretion, but GM3-specific antibody did not. These results indicate that plasma membrane associated GM3 inhibits ApoB secretion, lowers development of atherosclerosis by decreasing the secretion of TG-enriched ApoB containing lipoproteins, suggesting that GM3 is an inhibitor of ApoB and TG secretion in liver cells. J. Cell. Biochem. 118: 2168-2181, 2017. © 2017 Wiley Periodicals, Inc.

Detection of Naturally Occurring Human Antibodies Against Gangliosides by ELISA.

Gangliosides are sialic acid-containing glycolipids that have been considered attractive targets for cancer immunotherapy, based on the qualitative and quantitative changes they suffer during malignant transformation and due to their importance for tumor biology. Natural antibodies against gangliosides have been detected not only in cancer patients but also in healthy donors. The presence of these antibodies can be used as diagnostic or prognostic factor. However, these responses are difficult to detect because anti-ganglioside antibodies are usually of IgM isotype and low affinity. Enzyme Linked Immunosorbent Assay (ELISA) is an immunoassay based on the specific binding of antibodies to antigens bound to a solid phase. These antigens can be glycolipids like gangliosides. An enzyme linked to the last reactant allows the detection of specific binding through the development of color after the addition of a suitable substrate. ELISA combines the specificity of antibodies with the sensitivity of enzyme reactions. The ELISA method described herein can be used to detect antibody responses against gangliosides not only related to cancer but also to autoimmune diseases and infections, both in healthy donors, and patients, untreated or receiving specific immunotherapy.

Sialylation regulates brain structure and function.

Every cell expresses a molecularly diverse surface glycan coat (glycocalyx) comprising its interface with its cellular environment. In vertebrates, the terminal sugars of the glycocalyx are often sialic acids, 9-carbon backbone anionic sugars implicated in intermolecular and intercellular interactions. The vertebrate brain is particularly enriched in sialic acid-containing glycolipids termed gangliosides. Human congenital disorders of ganglioside biosynthesis result in paraplegia, epilepsy, and intellectual disability. To better understand sialoglycan functions in the nervous system, we studied brain anatomy, histology, biochemistry, and behavior in mice with engineered mutations in St3gal2 and St3gal3, sialyltransferase genes responsible for terminal sialylation of gangliosides and some glycoproteins. St3gal2/3 double-null mice displayed dysmyelination marked by a 40% reduction in major myelin proteins, 30% fewer myelinated axons, a 33% decrease in myelin thickness, and molecular disruptions at nodes of Ranvier. In part, these changes may be due to dysregulation of ganglioside-mediated oligodendroglial precursor cell proliferation. Neuronal markers were also reduced up to 40%, and hippocampal neurons had smaller dendritic arbors. Young adult St3gal2/3 double-null mice displayed impaired motor coordination, disturbed gait, and profound cognitive disability. Comparisons among sialyltransferase mutant mice provide insights into the functional roles of brain gangliosides and sialoglycoproteins consistent with related human congenital disorders.

Targeted Delivery of Immunotoxin by Antibody to Ganglioside GD3: A Novel Drug Delivery Route for Tumor Cells

Gangliosides are sialic acid-containing glycolipids expressed on plasma membranes from nearly all vertebrate cells. The expression of ganglioside GD3, which plays essential roles in normal brain development, decreases in adults but is up regulated in neuroectodermal and epithelial derived cancers. R24 antibody, directed against ganglioside GD3, is a validated tumor target which is specifically endocytosed and accumulated in endosomes. Here, we exploit the internalization feature of the R24 antibody for the selective delivery of saporin, a ribosome-inactivating protein, to GD3-expressing cells [human (SK-Mel-28) and mouse (B16) melanoma cells and Chinese hamster ovary (CHO)-K1 cells]. This immunotoxin showed a specific cytotoxicity on tumor cells grew on 2D monolayers, which was further evident by the lack of any effect on GD3-negative cells. To estimate the potential antitumor activity of R24-saporin complex, we also evaluated the effect of the immunotoxin on the clonogenic growth of SK-Mel-28 and CHO-K1GD3+ cells cultured in attachment-free conditions. A drastic growth inhibition (>80–90%) of the cell colonies was reached after 3 days of immunotoxin treatment. By the contrary, colonies continue to growth at the same concentration of the immuntoxin, but in the absence of R24 antibody, or in the absence of both immunotoxin and R24, undoubtedly indicating the specificity of the effect observed. Thus, the ganglioside GD3 emerge as a novel and attractive class of cell surface molecule for targeted delivery of cytotoxic agents and, therefore, provides a rationale for future therapeutic intervention in cancer.

Anti-ganglioside anti-idiotypic vaccination: more than molecular mimicry

Surgery, chemotherapy, and radiation therapy are standard modalities for cancer treatment, but the effectiveness of these treatments has reached a plateau. Thus, other strategies are being explored to combine with the current treatment paradigms in order to reach better clinical results. One of these approaches is the active immunotherapy based on the induction of anti-tumor responses by anti-idiotypic vaccination. This approach arose from Jerne’s idiotypic network theory, which postulates that B lymphocytes forms a functional network, with a role in the establishment of the immune repertoires, in the regulation of natural antibody production and even in the establishment of natural tolerance. Due to the large potential diversity of the immunoglobulin variable regions, the idiotypes repertoire can mimic the universe of self and foreign epitopes, even those of non-protein nature, like gangliosides. Gangliosides are sialic acid-containing glycolipids that have been considered attractive targets for cancer immunotherapy, based on the qualitative and quantitative changes they suffer during malignant transformation and due to their importance for tumor biology. Although any idiotype could be able to mimic any antigen, only those related to antigens involved in functions relevant for organism homeostasis, and that in consequence has been fixed by evolution, would be able not only to mimic, but also to activate the idiotypic cascades related with the nominal antigen. The present review updates the results, failures and hopes, obtained with ganglioside mimicking anti-idiotypic antibodies and presents evidences of the existence of a natural response against gangliosides, suggesting that these glycolipids could be idiotypically relevant antigens.

Reply to Mangold and Ankersmit

We appreciate the comments [1] of Drs Mangold and Ankersmit on our article [2]. Our group proved that genipin fixation is a novel alternative to conventional glutaraldehyde fixation, and the addition of decellularization, organic solvent treatment and detoxification prevent calcification of glutaraldehyde/genipinfixed bovine pericardium. We also found that the titer of anti-α-Gal(Galα1-3Galβ1-4GlcNAc-R) antibodies (immunoglobulin G) increases after the implantation of bovine pericardium to rabbit, and in vivo calcification results are consistent with the increase in anti-α-Gal antibodies titer [2]. Although the cause and significance of the xenoreactive antibody response between the different species in α-Gal concordant animal model have not been exactly known, some possible suggestions may exist: (i) by a species-specific expression of these conjugated compounds including α-Gal epitopes; (ii) a tissue-specific expression of these conjugated compounds including α-Gal epitopes and (iii) the differences in the fine specificity of natural anti-Gal antibodies in various species which recognize various ‘facets’ of the α-Gal epitope in its three-dimensional form. A species-specific expression of these conjugated compounds including α-Gal epitopes has been well demonstrated for so long. Thyroglobulin, fibrinogen and immunoglobulins from various species express varying amounts of Galα1-3Galβ14GlcNAc residues between 0.01 and 11 residues per molecule [3]. Rabbit red blood cells contain a range of glycolipids of varying lengths that terminate with the Galα1-3Galβ1-4GlcNAc-R structure. These glycolipids vary in size by increments of five monosaccharides, and each increment forms an additional branch. Although rat did not contain Galα1-3Galβ1-4GlcNAc neutral glycosphingolipids in the kidney, pig and sheep contained sialic acid-containing glycolipids (gangliosides) with the Galα1-3Galβ1-4GlcNAc-R structure, most likely the same branched, ganglioside identified in bovine red blood cells [4]. Thymus tissue from sheep, pig and rabbit also contained a range (compounds with 5–11 sugars) of neutral glycolipids terminating with the Galα1-3Galβ1-4GlcNAc-R structure demonstrating a species-specific expression of these compounds. Although the kidney and thymus from sheep, pig and rabbit express Galα1-3Galβ1-4GlcNAc-R glycolipids, these glycolipids were not detected in brain tissue demonstrating a tissue-specific expression of these compounds [5]. The detailed analysis of N-linked glycans from porcine kidney demonstrated that Galα1-3Galβ14GlcNAc-R termini were found on a variety of complex bi-, triand tetra-antennary N-glycans [6]. As we evaluated recently the immune response in the implantation of bovine pericardium to mice in which the α1,3-galactosyltransferase gene was knocked out (Gal/ mice), the titer of anti-α-Gal antibodies much increased. But interestingly, we also observed the increase in anti-α-Gal antibodies in wild-type mouse and rat as well as rabbit, although much less than Gal/ mice. We think that further transplantation or implantation experiments are needed to validate more exact xenoreactive immunologic responses between the different species.

Modified GM3 gangliosides produced by metabolic oligosaccharide engineering

Metabolic oligosaccharide engineering is powerful approach to altering the structure of cellular sialosides. This method relies on culturing cells with N-acetylmannosamine (ManNAc) analogs that are metabolized to their sialic acid counterparts and added to glycoproteins and glycolipids. Here we employed two cell lines that are deficient in ManNAc biosynthesis and examined their relative abilities to metabolize a panel of ManNAc analogs to sialosides. In addition to measuring global sialoside production, we also examined biosynthesis of the sialic acid-containing glycolipid, GM3. We discovered that the two cell lines differ in their ability to discriminate among the variant forms of ManNAc. Further, our data suggest that modified forms of sialic acid may be preferentially incorporated into certain sialosides and excluded from others. Taken together, our results demonstrate that global analysis of sialoside production can obscure sialoside-specific differences. These findings have implications for downstream applications of metabolic oligosaccharide engineering, including imaging and proteomics.

Disialoganglioside GD3 increases in the secretion of apoB-containing lipoproteins

The function of gangliosides, sialic acid-containing glycolipids, on the secretion and assembly of apoB-containing lipoproteins is poorly understood. Here, we report that the GD3 synthase is involved in apoB secretion in retinoic acid (RA)-treated Chang liver cells via transcriptional induction of microsomal triglyceride transfer protein (MTP). The overexpression of GD3 synthase in Chang liver cells increases the expression of the MTP gene, but GM3 synthase-transfected cells did not. The levels of GM3 and GD3 gangliosides in each of the transfected cells were increased in the cell extract as well as the medium. In addition, GD3 synthase-transfected cells showed an increased secretion of triglyceride-enriched apoB. In contrast, the triglyceride content in GM3 synthase-transfected cells was relatively lower. Treatment with small interfering RNAs (siRNAs) and GD3 antibody decreased apoB secretion. These results indicate that plasma membrane associated GD3 play important roles in apoB secretion, and that an enhancement in GD3 levels might be a risk factor for the development of atherosclerosis by increasing the secretion of triglyceride-enriched apoB containing lipoproteins.

Biosynthesis and degradation of mammalian glycosphingolipids.

Glycolipids are a large and heterogeneous family of sphingolipids that form complex patterns on eukaryotic cell surfaces. This molecular diversity is generated by only a few enzymes and is a paradigm of naturally occurring combinatorial synthesis. We report on the biosynthetic principles leading to this large molecular diversity and focus on sialic acid-containing glycolipids of the ganglio-series. These glycolipids are particularly concentrated in the plasma membrane of neuronal cells. Their de novo synthesis starts with the formation of the membrane anchor, ceramide, at the endoplasmic reticulum (ER) and is continued by glycosyltransferases of the Golgi complex. Recent findings from genetically engineered mice are discussed. The constitutive degradation of glycosphingolipids (GSLs) occurs in the acidic compartments, the endosomes and the lysosomes. Here, water-soluble glycosidases sequentially cleave off the terminal carbohydrate residues from glycolipids. For glycolipid substrates with short oligosaccharide chains, the additional presence of membrane-active sphingolipid activator proteins (SAPs) is required. A considerable part of our current knowledge about glycolipid degradation is derived from a class of human diseases, the sphingolipidoses, which are caused by inherited defects within this pathway. A new post-translational modification is the attachment of glycolipids to proteins of the human skin.

Gangliosides GD1b, GT1b, and GQ1b enhance IL-2 and IFN-gamma production and suppress IL-4 and IL-5 production in phytohemagglutinin-stimulated human T cells.

Gangliosides are sialic acid-containing glycolipids. We studied the in vitro effects of gangliosides on Th1 and Th2 cytokine production in PHA-stimulated human T cells. Gangliosides GD1b, GT1b, and GQ1b (each 100 nM) enhanced PHA-induced IL-2 secretion of peripheral blood T cells approximately 4-fold and enhanced that of IFN-gamma 3- to 4-fold compared with controls. These gangliosides decreased PHA-induced IL-4 secretion by 50-53% and that of IL-5 by 53-63% compared with controls, respectively. The other gangliosides did not alter the secretion of Th1 or Th2 cytokines. RT-PCR showed that GD1b, GT1b, and GQ1b enhanced PHA-induced IL-2 and IFN-gamma transcription and suppressed that of IL-4 and IL-5. Transient transfection assays of Jurkat T cells showed that GD1b, GT1b, and GQ1b enhanced PHA-induced IL-2 and IFN-gamma promoter activities but suppressed those of IL-4 and IL-5. The cAMP analogue dibutyryl cAMP and the cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine each reversed GD1b-, GT1b-, and GQ1b-induced stimulation of IL-2 and IFN-gamma production and inhibition of IL-4 and IL-5 production at the levels of proteins, transcription, and promoter activities. GD1b, GT1b, and GQ1b suppressed PHA-induced increase in cAMP level in T cells. These gangliosides suppressed PHA-stimulated adenylate cyclase activity in T cells. These results suggest that GD1b, GT1b, and GQ1b may enhance Th1 cytokine production while suppressing Th2 production by inhibiting adenylate cyclase activity.

Polyglycosylceramides recognized by Helicobacter pylori: analysis by matrix-assisted laser desorption/ionization mass spectrometry after degradation with endo-beta-galactosidase and by fast atom bombardment mass spectrometry of permethylated undegraded material.

Human erythrocyte polyglycosylceramides (PGCs) are recognized by the gastric pathogen Helicobacter pylori and are based on a successively extended and highly branched N-acetyllactosamine core linked to ceramide and substituted by fucose and sialic acid. As a step in the identification of the binding epitope we earlier characterized intact PGCs by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS (Karlsson,H., Johansson,L., Miller-Podraza,H., and Karlsson,K-A. [1999] Glycobiology, 9, 765-778). In the present work, PGCs from human blood group O erythrocytes were digested with endo-ss-galactosidase (Bacterioides fragilis), an enzyme which cleaves the bond 3Galss1-4GlcNAc in linear but not branched poly-N-acetyllactosamine chains. The enzymatic digestion resulted in a mixture of neutral and sialic acid-containing glycolipids together with terminal and internal sequences of mainly neutral oligosaccharides. The products were analyzed by MALDI-TOF MS in both positive and negative ion mode which gave spectra where the ions could be assigned to structures of the neutral and acidic components, respectively. Among glycolipids found were [structure in text] where R could be H, Fuc or NeuAc. Also observed were structures as [structure in text] which indicated linear extension along both branches. Observed at higher masses were fully branched structures obtained by stepwise extension with [structure in text] where R could be H, Fuc or NeuAc. Most probably further branching may occur along both the (1-->3)- and the (1-->6)-linked branches to give a partly dendritic structure. Structures with more than one sialic acid substituted could not be observed in the MALDI spectrum. Complementary information of the terminal sequences was obtained by FAB-MS analysis of permethylated undegraded PGCs. High-temperature gas chromatography/mass spectrometry of reduced and permethylated products from enzyme hydrolysis documented that Fuc was present in a blood group O sequence, Fuc-Hex-HexN-. Fucose may be placed on short (monolactosamine) or longer branches, while sialic acid seems to be restricted to monolactosamine branches. The conclusion is that human erythrocyte PGCs display microheterogeneity within terminal and internal parts of the poly-N-acetyllactosamine chains. The first branch from the ceramide end may be located at the second or third Gal and possibly also on the first Gal. Other branches may occur on every N-acetyllactosamine unit in fully branched domains, or there may be linear extensions between branches resulting in incompletely branched structures. The extended linear sequences may be present in both 3- and 6-linked antennae. Terminal structures are based on one, two or maybe higher number of N-acetyllactosamine units.

Ganglioside GD1b suppresses immunoglobulin production by human peripheral blood mononuclear cells

Gangliosides are sialic acid-containing glycolipids, that have various immunomodulatory effects. We previously reported that various gangliosides in vitro either inhibited or enhanced spontaneous immunoglobulin (Ig) production by human peripheral blood mononuclear cells (PBMC). GD1b was one of the inhibitory gangliosides. In this study, we further examined the mechanism for the inhibitory effect of GD1b. The inhibitory effect of GD1b was revealed at 0.1 mM, increased dose dependently, and was maximized at 10 mM, which reduced spontaneous IgG, IgM, and IgA production of human PBMC by 50.5%, 52.0%, and 48.3% compared with controls, respectively. GD1b did not affect the proliferation and viability of PBMC. GD1b did not alter Ig production of B cells alone. Interleukin 6 (IL-6) and IL-10 each partially reversed the GD1b-induced inhibition of Ig production by PBMC, and the addition of both cytokines completely reversed the inhibition. When endogenous IL-6 and IL-10 were neutralized by specific antibodies, GD1b did not reveal inhibitory effects on the Ig production. GD1b inhibited IL-6 and IL-10 production of CD4 + T cells, without affecting those of CD8 + T cells, monocytes, or B cells. When CD4 + T cells were preincubated with GD1b and washed and cultured with B cells and monocytes, Ig production was also suppressed. These results suggest that GD1b may indirectly suppress Ig production of B cells in whole PBMC by reducing IL-6 and IL-10 production of CD4 + T cells. GD1b may act as an important inhibitor of human humoral immune responses.

Ganglioside GT1b suppresses immunoglobulin production by human peripheral blood mononuclear cells.

Gangliosides are sialic acid-containing glycolipids and have various immunomodulatory effects. We previously reported that various gangliosides in vitro either inhibited or enhanced spontaneous immunoglobulin production by human peripheral blood mononuclear cells (PBMC). Among them, GT1b was the most inhibitory. In this study, we further examined the mechanism for the inhibitory effect of GT1b. The inhibitory effect of GT1b was apparent at 0.1 micrometers, increased dose dependently, and was maximal at 10 micrometers. In the presence of 10 micrometers GT1b, spontaneous production of immunoglobulin (Ig)G, IgM and IgA in human PBMC was reduced by 60%, 59.5% and 58%, respectively, compared with controls. GT1b did not affect the proliferation and viability of PBMC, and did not enhance their apoptosis. GT1b did not alter immunoglobulin production of B cells alone. Interleukin (IL)-6 and IL-10 each partially reversed the GT1b-induced inhibition of immunoglobulin production by PBMC, and the presence of both cytokines completely reversed the inhibition. GT1b inhibited IL-6 and IL-10 production in monocytes, without affecting that in T or B cells. When monocytes were preincubated with GT1b, washed and then cultured with B and T cells, the immunoglobulin production was also suppressed. These results suggest that GT1b may indirectly suppress immunoglobulin production of B cells in whole PBMC via reducing the production of IL-6 and IL-10 in monocytes. It is thus indicated that GT1b may act as an important inhibitor for human humoral immune responses.

Gangliosides in cerebrospinal fluid in children with autism spectrum disorders.

Gangliosides are sialic acid-containing glycolipids found in all cells, especially abundant in nerve cells and mainly situated on outer-membrane surfaces. The aim of this study was to provide data on the concentration of gangliosides in the CSF of children and adolescents with autism spectrum disorders (ASD) - 66 with autistic disorder, and 19 with other autism spectrum disorders. The comparison group consisted of 29 children and adolescents, whose CSF had been sampled to exclude acute infectious CNS disorder. The concentrations of the gangliosides GM1, GD1a, GD1b, and GT1b were determined using a microimmunoaffinity technique. The ASD group had a significantly higher concentration of ganglioside GM1 compared with the comparison group. The GM1 increase could not be explained as secondary to other clinical factors. Mean ganglioside levels did not differentiate subgroups with autistic disorder and those with a more atypical clinical picture, nor subgroups with known medical disorders and those with idiopathic autism. Altered patterns of gangliosides in the CNS might reflect important correlates of pathogenesis in autism.

Dependence of Cardiac Cell Ca2+Permeability on Sialic Acid-containing Sarcolemmal Gangliosides

The specific removal of negatively-charged sialic acid by neuraminidase produces a large increase in cardiac myocyte Ca uptake (17.3±1.1 mmol Ca/kg dry weight) and marked cell contracture. Importantly, the insertion of the negatively-charged amphiphile dodecyl sulfate in the sarcolemma eliminates the increased calcium uptake and preserves contractile function. In the present study, we further examine the role of sialic acid–Ca interaction and, specifically, the role of gangliosides (sialic acid-containing glycolipids) in cardiac cells» Ca permeability. Neonatal cell culture and adult ventricular myocytes were used. The major findings of this study are: (1) while dodecyl sulfate inhibits cellular calcium uptake and contracture development induced by sialic acid removal, cationic and neutral amphiphiles are without effect. (2) Ca channel blockers (nifedipine and protamine) and the Na/Ca exchange inhibitor Ni do not modify the effect of sialic acid removal. (3) A non-classical-channel related whole-cell current appears and increases after 21±2.2 min treatment with 0.02 U/ml neuraminidase (n=4). Incubation with neuraminidase in the presence of dodecyl sulfate greatly delays the appearance of these currents to 44.4±6.1 min (n=4). (4) The use of a specific probe for GM1 ganglioside, the cholera toxin B subunit (3μg/ml), induces a moderate but clear increase in cellular Ca (1.63±0.3 mmol Ca kg dry weight;n=8). However, this increase was not modified by treatment with dodecyl sulfate. (5) Finally, 50 mU/ml endoglycoceramidase, an enzyme which specifically cleaves the link between the sialic acid-containing oligosaccharide and ceramide of gangliosides, induced a significant increase in Ca uptake (4.4±0.9 mmol Ca kg dry weight,n=4). These results indicate the importance of negatively charged sialic acid-containing gangliosides in the maintenance of cardiac cell physiological Ca permeability. The increase in Ca uptake induced by the removal of sialic acid seems to be mediated by development of a Ca “leak” via other than classical cation channels.

Gangliosides and autoimmune diabetes.

Gangliosides are sialic acid-containing glycolipids which are formed by a hydrophobic portion, the ceramide, and a hydrophilic part, i.e. the oligosaccharide chain. First described in neural tissue, several studies have shown that gangliosides are almost ubiquitous molecules expressed in all vertebrate tissues. Within cells, gangliosides are usually associated with plasma membranes, where they can act as receptors for a variety of molecules and have been shown to take part in cell-to-cell interaction and in signal transduction. In addition, gangliosides are expressed in cytosol membranes like those of secretory granules of some endocrine cells (adrenal medulla, pancreatic islets). As far as the role of gangliosides in diseases is concerned, there are some cases in which an aberrant ganglioside expression plays a crucial role in the disease pathogenetic process. These diseases include two major forms of ganglioside storage, namely GM2-gangliosidosis (Tay-Sachs and its beta-hexosaminidase deficiency) and GM1-gangliosidosis (beta-galactosidase deficiency), where the most prominent pathological characteristic is the lysosomal ganglioside accumulation in neurons. Other inflammatory or degenerative diseases both within and outside the nervous system have been shown to be associated with an altered pattern of ganglioside expression in the target organ. Since monoclonal antibodies have been discovered and used in immunology, a large variety of ganglioside antigens has been described both as blood group antigens and as tumour-related antigens. Several studies have also indicated that gangliosides can act not only as antigens, but also as autoantigens. As a matter of fact, auto-antibodies to gangliosides, detected by immunostaining methods performed directly on TLC plates or by ELISA, have been described in several autoimmune disorders such as Guillain-Barré syndrome, multiple sclerosis, lupus erythematosus, Hashimoto's thyroiditis and, last but not least, insulin-dependent (type 1) diabetes mellitus. This last disease is caused by the autoimmune destruction of insulin-producing pancreatic islet cells in genetically predisposed individuals. Autoantibodies and T lymphocytes directed towards multiple islet autoantigens have been detected in the circulation, well before the clinical onset of the disease, in a prodromal phase during which pancreatic islet beta-cells are presumably destroyed. Among the target autoantigens, some are of protein nature but others are acidic glycolipids such as sulphatides158 and the gangliosides GT3, GD3 and especially GM2-1. This last component is specifically expressed in pancreatic islets and has been shown to represent a target of IgG autoantibodies highly associated with diabetes development in first-degree relatives of type 1 diabetic individuals. In addition, the GM2-1 ganglioside appears to be one of the antigens recognized by cytoplasmic ICA, a heterogeneous group of antibodies which specifically react with islets on pancreatic frozen sections. In conclusion, studies performed in the last decade have clearly indicated that gangliosides represent a heterogeneous class of molecules that are involved in several cellular processes that are of crucial importance in physiological as well as in pathological conditions. Interestingly, these molecules, despite their small size, have been shown to represent not only important antigens in tumour immunology but are also able to elicit a specific autoimmune response, thus representing important autoantigens in some autoimmune disorders. It is of interest that, in addition to neurological autoimmune disorders where autoimmunity to gangliosides is frequent and usually of considerable magnitude, an autoimmune response to this class of molecules has been observed in autoimmune diabetes. (ABSTRACT TRUNCATED)