Interaction of fibroblast growth factor-2 (FGF-2) with free gangliosides: biochemical characterization and biological consequences in endothelial cell cultures

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, or GM1. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b > GD1b > GM1 = GM2 = sulfatide > GM3 = galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF-2/GM1 interaction occurs with a Kd equal to 6 microM. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112-129) and, to a lesser extent, FGF-2(130-155), whereas peptides FGF-2(10-33), FGF-2(39-59), FGF-2(86-96), and the basic peptide HIV-1 Tat(41-60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT1b > GD1b > GM1 > sulfatide a = sialo-GM1. Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 and asialo-GM1, prevent the binding of 125I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM1 to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT1b was the most effective among the gangliosides tested while asialo-GM1, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.     

Structural characterization of the disialogangliosides of murine peritoneal macrophages

Sialoglycosphingolipids (gangliosides) have been increasingly implicated as regulators of membrane signaling events. Macrophage ganglioside patterns dramatically increase in complexity when murine peritoneal macrophages are stimulated in vivo with the appearance of the sialidase-sensitive monosialoganglioside GM1b (cisGM1) as a major component. Gangliosides from stimulated murine peritoneal macrophages were separated into monosialo and polysialo fractions and the polysialo fraction structurally characterized by enzymatic, chemical, and mass spectra methods. All detectable components of the polysialo fraction were determined to be disialogangliosides. Treatment of the polysialo fraction with Clostridium perfringens sialidase produced mostly the sialidase-resistant monosialoganglioside, GM1a, and a minor amount of asialoGM1. Periodate oxidation and mass spectrometry analyses demonstrated the lack of tandem disialo moieties which indicated the absence of GD1b or GD1c (GD1) entities. The combined data showed the major disialogangliosides consisted of GD1a entities comprising IV3-NeuAc,II3NeuAc-GgOse4Cer, IV3-NeuGc,II3NeuAc-GgOse4Cer, IV3NeuAc,II3NeuGc-GgOse4Cer, and IV3-NeuGc,II3NeuGc-GgOse4Cer. Minor components consisted of GD1alpha entities, IV3NeuAc, III6NeuAcGgOse4Cer, IV3NeuGc, III6NeuGc-GgOse4Cer, and also positional isomer(s) of GD1alpha(NeuAc, NeuGc). These isomeric components were identified by collision analysis and tandem mass spectrometry. Consistent with previous analyses, the ceramide portion of all polysialo (disialo) gangliosides contained solely C18 sphingosine with C16 and C24 fatty acid moieties. These results, combined with the previous characterization of macrophage monosialogangliosides, indicate normal murine macrophage ganglioside biosynthesis proceeds along the "a" ganglioside pathway, e.g., GM3-->GM2-->GM1a-->GD1a, and the proposed asialoganglioside or "alpha" pathway, asialoGM1-->GM1b-->GD1alpha. The presence of totally sialidase-sensitive gangliosides appears to be characteristic of functional murine peritoneal macrophages while they are reduced in genetically impaired cells.     

Differential expression of gangliosides and galactolipids in fetal human oligodendrocytes and astrocytes in culture

The phenotypic expression of gangliosides and galactolipids was investigated using primary cultures of fetal human oligodendrocytes and astrocytes. These glial cells were isolated from fetal human brains of 12-18 weeks' gestation. Expression of gangliosides and galactolipids in oligodendrocytes and astrocytes was investigated by double labeling immunocytochemistry using rabbit antibodies specific for galactocerebroside (GalC, a cell type-specific marker for oligodendrocyte) and glial fibrillary acidic protein (GFAP, a cell type-specific marker for astrocyte) in combination with a panel of mouse monoclonal antibodies which react with specific gangliosides or galactolipids. A considerable number of GalC+ oligodendrocytes expressed intense immunoreactivities specific for GM3 (19%) and GM2 (45%) gangliosides. Approximately 11% of GalC+ oligodendrocytes expressed GM4 immunoreactivity, and smaller numbers of GalC+ oligodendrocytes expressed GD3 (4%), GD2 (1%), GT1b (5%) and A2B5 (3%) immunoreactivities. However, GalC+ oligodendrocytes did not express GM1, GD1a, GT1b or GQ1c. Major populations of GalC+ oligodendrocytes immunolabeled by rabbit anti-GalC antibody reacted with anti-GalC mAb (Ranscht mAb, 81%) or by anti-sulfatide mAb (O4 mAb, 91%). A considerable number of GFAP+ astrocytes expressed intense GM2 (26%) and GD2 (15%) immunoreactivities, while a smaller population expressed intense GM3 (3%), GD3 (6%) and GM4 (4%) immunoreactivities. Weak immunoreactions specific for GD1b, A2B5 and sulfatide were found in less than 1% each of GFAP+ astrocytes, while GFAP+ astrocytes did not express GM1, GD1a, GT1a, GT1b or GQ1b. These results indicate that GM3, GM2 and sulfatide are expressed in a major population of GalC+ oligodendrocytes, while GM3, GM2, GD3, GD2, and GM4 are expressed in a small but distinctive population of GFAP+ astrocytes. Our results suggest that GM4, GM1 and GD3, which are utilized as markers for adult human oligodendrocytes and myelin, are not the major ganglioside constituents in cultured fetal human oligodendrocytes.     

Altered ganglioside expression by SH-SY5Y cells upon retinoic acid-induced neuronal differentiation

SH-SY5Y Neuroblastoma cells were used to study the effect of retinoic acid (RA)-induced differentiation on the expression of gangliosides and neuronal markers. In the presence of 10 microM RA, more than 70% of the cells differentiate to a neuronal phenotype within 8 days. They extend long neuritic processes and show an enhanced immuno-expression of neurone-specific enolase (NSE), neurofilament protein (NF-M), and polysialic acid (PSA). SH-SY5Y cells were found to express at least 12 different gangliosides. RA-induced neuronal differentiation led to a decrease in the content of GM2, GD3, and GD2 and to a 3-7 fold increased concentration of the ganglio-tetraosyl gangliosides GM1, GD1a, GT1a, GD1b, and GT1b. Thus, RA-induced neuronal differentiation of SH-SY5Y cells is accompanied by ganglioside changes similar to those observed during embryonic neuronal differentiation.     

Photochemical labeling of human erythrocyte membrane proteins with radioiodinated 4-azidosalicylic acid derivatives of G(M3), G(D3), G(M1), and FucG(M1) gangliosides

Photoreactive gangliosides of high specific radioactivity may prove useful for studies on glycosphingolipid functions. We prepared 4-azidosalicylic acid (ASA) acylated derivatives of GM3, GD3, GM1, and FucGM1 gangliosides (gangliosides-ASA). Gangliosides-ASA were characterized by their TLC mobility, UV spectra, carbohydrate composition, and digestion with leech endoceramidase. After radioiodination to about 200 Ci/mmole gangliosides-ASA were used for photochemical labeling of human erythrocytes. Radioiodinated gangliosides-ASA were incorporated into erythrocytes in a time and concentration dependent manner, the kinetics and extent of incorporation being similar for all the gangliosides-ASA used. Radioiodinated gangliosides-ASA incorporated into erythrocytes were resistant to trypsin digestion while treatment with 1% BSA removed about 90% of the label. Incubation with cholera toxin protected radioiodinated GM1-ASA and, to a lesser extent, FucGM1-ASA but not GM3-ASA and GD3-ASA, against removal with BSA. After photolysis about 40-50% of radioactivity was firmly bound to erythrocyte lipids and proteins. The ratio of lipid- to protein-bound radioactivity ranged from 2.2:1 to 3.2:1. Photolabeled proteins were analyzed by SDS/PAGE followed by autoradiography. Band 3 was the most extensively photolabeled protein with all the radioiodinated gangliosides-ASA used. DIDS, an inhibitor of band 3 protein activity, caused reduction in photolabeling of this protein by about 20%.     

Complex gangliosides affect GD3 accessibility to antibody in developing neuronal cells

Ganglioside expression of embryonic chick retina cells developed in vitro was analyzed by indirect immunofluorescence. Immature neurons were GD3 positive cells and the labeling was chiefly distributed all over their cell membrane. Mature neurons became GD3 negative and expressed complex gangliosides of the a- and b-pathways; nevertheless, the content of GD3 accounted for approximately 40% of the total gangliosides in these cells. Neuraminidase hydrolysis pointed out that GD3 was located in membrane of differentiated cells. The frequency of cells with the GD3 immunostain localized in restricted area of membrane of undifferentiated neurons increased significantly after adding a mixture of bovine brain gangliosides (largely complex gangliosides). Antibody binding to immobilized GD3 showed a dose-dependent inhibition by adding a mixture of bovine brain gangliosides, GM1, GD1a or asialo-GM1. Glycosphingolipids with shorter oligosaccharide chains, as cerebrosides or sulfatides, did not affect this binding. These results suggest that, concomitant with the accretion of content of complex gangliosides, a rearrangement in the membrane would occur, which progressively masks GD3 to its antibody. This rearrangement might affect putative ganglioside functions involved in neuronal differentiation.     

Borrelia burgdorferi shows specificity of binding to glycosphingolipids

Live but not fixed or heat-killed Borrelia burgdorferi bound to galactocerebroside, lactosylceramide, and ceramide trihexoside. In addition, this organism bound to the disialoganglioside GD1a and the trisialoganglioside GT1b but not to gangliosides GM1, GD1b, GM2, and GM3 and not to asialo GM1. This adhesion pattern confirmed earlier findings of binding to galactocerebroside and places this organism within a prokaryotic group which binds to lactosylceramide. The binding to GD1a and GT1b, both of which carry terminal as well as multiple sialic acids, indicates that B. burgdorferi can show specificity of binding within a group of acidic gangliosides. Adhesion could not be inhibited by several concentrations of sugars and sialic acid, indicating more complex binding requirements than for terminal carbohydrates alone. Low-passage strains adhered to the four substrates in greater numbers than strains in culture for long periods of time. OspB mutants in general bound better or at least equally well to several of the glycosphingolipids, and preincubation of substrates with soluble recombinant and affinity-purified Osp did not inhibitor or weakly inhibited the binding of the organisms. These findings suggest that outer surface lipoproteins A and B are not directly involved in adhesion to glycosphingolipids.     

Sulfated glucuronyl glycolipids and gangliosides in the optic nerve of humans

Pathologically delayed visual evoked potentials may be present in patients with neuropathy associated with IgM M-proteinemia, which is directed against myelin-associated glycoprotein and sulfated glucuronyl glycolipids (SGGLs), but there are no reports of these antigens in the optic nerve. We recently examined human optic nerve and occipital lobe tissues for the occurrence of SGGLs using the technique of immunostaining on thin-layer chromatographic plates and found them in the optic nerve, but not the occipital lobe. SGGLs in the optic nerve may represent target antigens for CNS involvement by the M-protein in patients with neuropathy. We also studied the ganglioside composition of the optic nerve and found it different from that of the brain. Human optic nerve is characterized by an abundance of the b-series gangliosides, including GD1b, GT1b, and GQ1b. GD1a, which is usually a major component of brain gangliosides, is only a minor species of the optic nerve ganglioside fraction.     

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.     

Long-term ethanol consumption selectively impairs ganglioside pathway in rat brain

Gangliosides are sialo-glycosphingolipids that play important roles in the interaction of cells with their environment and are thus involved in the regulation of many cellular events. Sialic acid residues are important for the conformation of a glycomolecule, their structural stability and their functions. Although decreased brain ganglioside sialic acid has been previously reported as a result of chronic ethanol treatment in rats, no reports are available on the sialylation of specific gangliosides and/or the mechanism leading to depletion of their sialic acid residues. Therefore, in this investigation, we have examined the effects of chronic ethanol treatment on (1) incorporation of [4,5-3H]N-acetylmannosamine (ManNAc) into specific rat brain gangliosides, GD3, GD1a, GT1a, and GT1b; and (2) enzymatic activities of brain sialyltransferase and sialidase at specific subcellular levels. The experiments were done in male Wistar rats pair-fed with either ethanol or control liquid diets for a period of 8 weeks. The rats were intracerebroventricularly injected with labeled ManNAc (30 microCi/rat) and killed after 90 min. Radioactivity was determined in respective ganglioside bands separated on a thin layer chromatography system. Specific activities of sialyltransferase and sialidase were assessed using GM3 and GD3 as substrates, respectively. The results showed significant decreases of 57.7% (p < 0.001) and 68.9% (p < 0.001), respectively, in the labeled ManNAc incorporation into GD3 and GD1a fractions in rats of the ethanol group, compared with rats of the control group. No significant changes were noted in the incorporation of labeled ManNAc into GT1a or GT1b ganglioside fractions between the ethanol and control groups. Concomitantly, compared with control rats, a decrease of 18.9% (p < 0.05), 20.6% (p < 0.05), and 15.8% (p < 0.001) was found in the sialyltransferase activity, respectively, at the whole brain, and brain Golgi and synaptosomal levels. However, dramatic increases of 32.4% (p < 0.05), 105% (p < 0.001), and 150% (p < 0.001) in sialidase activity were found, respectively, at the whole brain and brain cytosol and synaptosomal fractions of rat treated chronically with ethanol. Thus, we conclude that the deleterious actions of ethanol on the sialylation of rat brain gangliosides is specific, and the reduced sialic acid label found in GD3 and GD1a in this study is mainly due to increased activity of brain sialidase. Furthermore, the study reaffirms our tenet that, regardless of whether it is the liver or the brain, glycosylation cascade is one of the main target of the deleterious attacks of ethanol.     

Effects of glucosylceramide synthase inhibitor and ganglioside GQ1b on synchronous oscillations of intracellular Ca2+ in cultured cortical neurons

To evaluate the role of endogenous gangliosides in synapse formation, the glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), was used to deplete glycosphingolipids (GSLs) of cultured rat cerebral cortical neurons. Synapse formation between the neurons was estimated by the synchronous oscillation of synaptic activity monitored by fura-2 calcium imaging. Treatment with D-PDMP resulted in dose- and time-dependent decreases in the frequency of synchronous oscillations. When a series of gangliosides (GM3, GM1, GD3, GD1b, GT1b, and GQ1b) was supplemented to the GSL depleted cells, only GQ1b was able to normalize the decreased frequency by D-PDMP. These results suggest that de novo synthesis of a particular molecular species of the gangliosides, GQ1b, is essential for synapse formation and synaptic activity.     

Ganglioside GD1alpha functions in the adhesion of metastatic tumor cells to endothelial cells of the target tissue

We studied the role of glycosphingolipids expressed on the cell surfaces of a metastatic tumor cell line. Glycosphingolipid compositions of the low-metastatic murine lymphosarcoma cell line RAW117-P and its sub-line, RAW117-H10, which shows higher metastatic potential for the liver than P cells, were compared. Both types of cells had LacCer, Gg3Cer, and Gg4Cer as the major neutral glycosphingolipids and GM1b and GD1alpha as the gangliosides. There are differences in glycosphingolipid contents, the neutral glycosphingolipid contents in the parental cells being 1.5-fold higher than that in the variant ones. In contrast, the level of GD1alpha in H10 cells was twice as much as that in the P cells; however, the expression of other gangliosides was down-regulated. On the basis of the results of glycosphingolipid analysis, we investigated the functional role of GD1alpha in H10 cells in the adhesion of the tumor cells to the target tissue by using hepatic sinusoidal endothelial (HSE) cells. GD1alpha and GM1b inhibited the adhesion when HSE cells were incubated prior to coculture with the tumor cells. This inhibitory effect by GD1alpha and GM1b was observed within 30 min after addition of H10 cells to HSE cells and was dose dependent. GD1alpha showed a higher inhibitory effect on the adhesion than GM1b, whereas other glycosphingolipids showed no inhibitory effect. Anti-GD1alpha monoclonal antibody also inhibited the adhesion between the H10 and HSE cells. When cultured without fetal bovine serum for 30 min in a various glycosphingolipids-coated dish for bacterial culture, HSE cells adhered to the area coated with GD1alpha but not to areas coated with other glycosphingolipids. HSE cell adhesion depended on the amount of GD1alpha coated on the plate. These data indicate that GD1alpha functions as an adhesion molecule in the process of metastasis of H10 cells.     

Does hysteroscopy improve upon the sensitivity of dilatation and curettage in the diagnosis of endometrial hyperplasia or carcinoma?

We report the occurrence of a relapsing, severe predominantly motor neuropathy in a 75-year-old man with an IGM-K M-protein binding to gangliosides GM2, GM3, GM4, GD1a, GT1b and LM1. Motor nerve conduction velocities were slowed with conduction block. A superficial peroneal nerve biopsy specimen revealed segmental demyelination and remyelination. The patient improved after repeated plasma exchanges, and the antibody titer decreased in association with clinical recovery. This IgM M-protein has a unique, previously unreported binding specificity for terminal NeuAcalpha2-3Galbeta- moiety in common to all gangliosides bound by the antibody except GM2. M-proteins with this affinity may be involved in the pathogenesis of this and other cases of motor-dominant demyelinating neuropathy.     

Effects of gonadotropin and testosterone treatments on prostate volume and serum prostate specific antigen levels in male hypogonadism

Various monosialo- and disialo-gangliosides and their derivatives were examined by delayed ion extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF MS) in the reflector mode with alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid used as the matrix. Native gangliosides were generally found to give good spectra in the negative ion mode. 2,5-Dihydroxybenzoic acid was a better matrix for gangliosides than alpha-cyano-4-hydroxycinnamic acid, because this matrix seemed to minimize loss of sialic acid and carbon dioxide of gangliosides. About 1 pmol of ganglioside was able to be detected with this matrix. When "A-series" gangliosides such as GD1a and GalNAc-GD1a gave undesirable extra peaks probably due to loss of sialic acid besides molecule-related ion peaks, the methyl-esterification of the gangliosides at the carboxyl groups of sialic acids was found to be necessary to obtain good DE MALDI-TOF mass spectra in the positive ion mode. In contrast, "B-series" gangliosides such as GD1b, GD2, and GD3 gave rise to major dehydrated molecule-related ion [M-H2O-H]- peaks in the negative ion mode without the pretreatment of methyl-esterification. The DE MALDI-TOF mass spectrometric analysis enabled us to distinguish between GD1a and GD1b, which have the same molecular weight. It was also found that not only a purified sample, but also a mixed sample of various gangliosides was amenable to the identification of them by DE MALDI-TOF MS.     

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.     

Differential cell- and immuno-biological properties of murine B16-F1 and F10 melanomas: oncogene c-fos expression, sensitivity to LAK cells and/or IL-2, and components of gangliosides

Differential cell- and immuno-biological properties of two murine melanoma B16 variants, B16-F1 and F10, were investigated. Studies focused on the expression of proto-oncogene c-fos, sensitivities to LAK cells and/or IL-2, and modulation of the expression of ganglioside components after treatment with IL-2. Proto-oncogene c-fos was found to be highly expressed in F10 lines by an in situ hybridization technique and also in F10 lung metastatic nests by immunofluorescent staining with anti-c-fos antibody. F1 melanomas were more sensitive to local injection of IL-2. F10 melanomas hardly responded to IL-2 treatment, but successive injections of a combination of LAK cells and IL-2 did cause prolongation of survival rates, even of F10 melanoma-burdened mice. A major component of gangliosides of both F1 and F10 melanomas was GM3. Production of GM3 in F10 melanomas treated with IL-2 for 4 days increased, and, if the treatment was continued for 7 days, minor components of gangliosides, such as GM2, GM1, and GD1a, appeared only in F1 melanomas, while the increase of production of GM3 disappeared in both melanomas. These experimental results may provide clues for additional mechanisms which allow these two murine melanoma variants to show different implantation and metastasis rates.     

Ganglioside synthesis during the development of neuronal polarity. Major changes occur during axonogenesis and axon elongation, but not during dendrite growth or synaptogenesis

Changes in the levels and types of gangliosides occur during neuronal differentiation and development, but no studies have correlated these changes with defined events in neuronal morphogenesis. Here, we have analyzed the relationship between ganglioside synthesis and the development of axons and dendrites in polarized neurons, using hippocampal neurons cultured in such a way that axons and dendrites are generated by a defined sequence of events and in which there is virtually no contamination by glial cells. Neurons were labeled with [4,5-3H]dihydrosphingosine, which was rapidly incorporated into cells and metabolized to 3H-labeled glycosphingolipids. The rate of 3H-labeled glycosphingolipid synthesis was directly proportional to the initial rate of [4,5-3H]dihydrosphingosine uptake and was linear versus time for up to 9 h of incubation. The major changes in 3H-labeled ganglioside synthesis occurred during the period of axonogenesis and rapid axon growth. During axonogenesis, there was a significant increase in the synthesis of complex gangliosides (i.e. GM1, GD1a, GD1b, and GT1b) with a corresponding reduction in the synthesis of glucosylceramide and ganglioside GD3. During the stage of rapid axon growth, the ratio of a- to b-series gangliosides increased significantly. However, during dendritogenesis, dendrite growth, and synaptogenesis, there was little change in ganglioside synthesis, with a small and gradual increase in the ratio of a- to b-series gangliosides and an increase in the synthesis of gangliosides GD1a and GT1b. These results indicate that despite major changes in neuronal morphology and functionality as neurons mature, changes in ganglioside synthesis are restricted to early stages of neuronal development, namely axonogenesis and rapid axon elongation.     

Quantitative analysis of bacterial toxin affinity and specificity for glycolipid receptors by surface plasmon resonance

The primary virulence factors of many pathogenic bacteria are secreted protein toxins which bind to glycolipid receptors on host cell surfaces. The binding specificities of three such toxins for different glycolipids, mainly from the ganglioside series, were determined by surface plasmon resonance (SPR) using a liposome capture method. Unlike microtiter plate and thin layer chromatography overlay assays, the SPR/liposome methodology allows for real time analysis of toxin binding under conditions that mimic the natural cell surface venue of these interactions and without any requirement for labeling of toxin or receptor. Compared to conventional assays, the liposome technique showed more restricted oligosaccharide specificities for toxin binding. Cholera toxin demonstrated an absolute requirement for terminal galactose and internal sialic acid residues (as in GM1) with tolerance for substitution with a second internal sialic acid (as in GD1b). Escherichia coli heat-labile enterotoxin bound to GM1 and tolerated removal or extension of the internal sialic acid residue (as in asialo-GM1 and GD1b, respectively) but not substitution of the terminal galactose of GM1. Tetanus toxin showed a requirement for two internal sialic acid residues as in GD1b. Extension of terminal galactose with a single sialic acid was tolerated to some extent. The SPR analyses also yielded rate and affinity constants which are not attainable by conventional assays. Complex binding profiles were observed in that the association and dissociation rate constants varied with toxin:receptor ratios. The sub-nanomolar affinities of cholera toxin and heat-labile enterotoxin for liposome-anchored gangliosides were attributable largely to very slow dissociation rate constants. The SPR/liposome technology should have general applicability in the study of glycolipid-protein interactions and in the evaluation of reagents designed to interfere with these interactions.     

The effects of reactive centre loop length upon serpin polymerisation

Our rapid method of microwave-mediated saponification for preparing lysoglycosphingolipids from their parent glycosphingolipids was also able to prepare lysogangliosides or modified lysogangliosides, which were identified by delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometric (DE MALDI-TOF MS) analysis. When GM3, GM2, and GM1 isolated from adult human brain gangliosides were subjected to the saponification, GM3 was found to give rise to only lyso-GM3 containing de-N-acetylneuraminic acid (de-N-acetyl lyso-GM3), whereas the GM2 produced both lyso-GM2 and the de-N-acetyl compound, and GM1 also gave both lyso-GM1 and the de-N-acetyl compound. In the saponification of GM1 and GDla, isolated from rat brain gangliosides, GM1 similarly produced both lyso-GM1 and the de-N-acetyl compound, but GDla was found to give rise to both dehydrated de-N-monoacetyl and dehydrated de-N-diacetyl lyso-GDla. However, the saponification of the GM1 fraction isolated from porcine brain gangliosides gave rise not only to both lyso-GM1 and the de-N-acetyl compound, but also unexpectedly to both lyso-fucosyl GM1 and its de-N-acetyl compound. The untreated GM1 fraction was examined by TLC and DE MALDI-TOF mass spectrometry, and proved to contain fucosyl-GM1. The DE MALDI-TOF MS analysis of the prepared lyso-gangliosides showed that their long chain bases consisted of d18:1 and d20:1 sphingosines in various ratios reflecting those of the different mammalian brain gangliosides.     

Reactivity of neuroborreliosis patients (Lyme disease) to cardiolipin and gangliosides

A subset of patients (50%) with neuroborreliosis (Lyme disease) showed IgG reactivity to cardiolipin in solid phase ELISA. In addition, a subset of patients with neuroborreliosis (29%) and syphilis (59%) had IgM reactivity to gangliosides with a Gal(beta 1-3) GalNac terminal sequence (GM1, GD1b, and asialo GM1). Anti-ganglioside IgM antibodies were significantly more frequent in these two groups of patients compared to patients with cutaneous and articular Lyme disease, primary antiphospholipid syndrome, systemic lupus erythematosus and normal controls. Correlative evidence and adsorption experiments indicated that antibodies to cardiolipin had separate specificities from those directed against the gangliosides. IgM antibodies to Gal(beta 1-3) GalNac gangliosides appeared to have similar specificities since these were positively correlated and inhibitable by cross adsorption assays. Given the clinical associations of patients with neuroborreliosis and syphilis with IgM reactivity to gangliosides sharing the Gal(beta 1-3) GalNac terminus, we suggest that these antibodies could represent a response to injury in neurological disease or a cross reactive event caused by spirochetes.