Expression of GD2 and GD3 gangliosides in human embryonic neural stem cells

NSCs (neural stem cells) are undifferentiated neural cells endowed with a high potential for proliferation and a capacity for self-renewal with retention of multipotency to differentiate into neurons and glial cells. It has been recently reported that GD3, a b-series ganglioside, is a marker molecule for identifying and isolating mouse NSCs. However, the expression of gangliosides in human NSCs is largely unknown. In the present study, we analysed the expression of gangliosides, GD2 and GD3, in human NSCs that were isolated from human brains at gestational week 17 in the form of neurospheres, which are floating clonal aggregates formed by NSCs in vitro. Employing immunocytochemistry, we found that human NSCs were strongly reactive to anti-GD2 antibody and relatively weakly reactive to anti-GD3 antibody. Treatment of these cells with an organic solvent such as 100% methanol, which selectively removes glycolipids from plasma membrane, abolished the immunoreactivity with those antibodies, indicating that the reactivity was due to GD2 and GD3, but not to GD2-/GD3-like glycoproteins or proteoglycans. The immunoreactivity of human NSCs to antibody against SSEA-1 (stage-specific embryonic antigen-1), a well-known carbohydrate antigen of NSCs, was not decreased by the treatment with 100% methanol, indicating that SSEA-1 is mainly carried by glycoproteins and/or proteoglycans in human NSCs. Our study suggests that GD2 and GD3 can be marker gangliosides for identifying human NSCs.     

GD3 ganglioside and apoptosis

Lipid and glycolipid mediators are important messengers of the adaptive responses to stress, including apoptosis. In mammalian cells, the intracellular accumulation of ganglioside GD3, an acidic glycosphingolipid, contributes to mitochondrial damage, a crucial event during the apoptopic program. GD3 is a minor ganglioside in most normal tissues. Its expression increases during development and in pathological conditions such as cancer and neurodegenerative disorders. Intriguingly, GD3 can mediate additional biological events such as cell proliferation and differentiation. These diverse and opposing effects indicate that tightly regulated mechanisms, including 9-O-acetylation, control GD3 function, by affecting intracellular levels, localization and structure of GD3, and eventually dictate biological outcomes and cell fate decisions.     

Formation of Ganglioside GD 1b-Lactone in Rat Brain from Intracisternally Administered GD 1b

The presence of ganglioside GD1b, in lactone form GD1b-L, was ascertained in rat brain. The possible formation of GD1b-L from GD1b in brain was explored by the intracisternal injection of GD1b, 3H-labelled at the level of the terminal galactose. This was followed by recognition of the radioactive gangliosides formed at different times (1, 3, and 7 days) after injection. Whereas at 0 time after injection the only radioactive ganglioside was GD1b, after 1, 3, and 7 days other radioactive gangliosides were also found, thus indicating GD1b penetration into the brain tissue, followed by metabolic processing. Besides GD1b, the following radioactive gangliosides were recognized: GM1 and GM2, derived from GD1b degradation; GT1b, formed by the direct sialylation of GD1b; and GD1b-L, produced by metabolic lactonization. The radioactivity carried by GD1b-L was maximal 3 days after injection; its time course was different from that of the other gangliosides, suggesting that the process of lactonization is separate from that of both degradation and glycosylation. Under the same experimental conditions, some radioactive gangliosides also appeared in the liver, although in much smaller amounts than in brain. Radioactive GD1b-L could not be detected in liver, thus indicating that metabolic lactonization is a tissue- or organ-specific process.     

Ganglioside GD3 and GD3-lactone mediated regulation of the intermolecular organization in mixed monolayers with dipalmitoylphosphatidylcholine

The interactions of dpPC with ganglioside GD3 and two lactones, GD3LacI or GD3LacII, in lipid monolayers occur with reduced, unaltered, or increased molecular area and surface potential/molecule, respectively. dpPC is fully miscible with GD3 and GD3LacI but films with GD3LacII show immiscibility above 75 mol% lactone. At low proportions of GD3 in mixtures with dpPC, GD3 undergoes condensation and depolarization; dpPC is depolarized and its molecular area is reduced above 50 mol% GD3. GD3LacI forms ideally mixed films with dpPC. Mixtures of dpPC with GD3LacII at mole fractions below 0.3 show increased mean molecular area and surface potential/molecule mostly due to lactone alterations. Between mole fractions of 0.3 and 0.75 the surface parameters of dpPC are altered, and above these proportions both lipids are immiscible. Defined variations of molecular properties induced by ganglioside lactonization are selectively transduced to changes of the intermolecular organization and surface electrostatics in mixed interfaces with dpPC. Thus, changes in the relative proportions of a ganglioside and its lactone forms may act as sensitive biotransducers for membrane-mediated cellular functions, without the need for metabolically altering the concentration of gangliosides.     

神经节苷脂GD3与肿瘤的血管生成作用(英文)

 血管生成作用 (angiogenesis)是实体瘤 (solidtumor)生长和扩散的必要条件 .实体瘤的微血管密度与肿瘤的恶性程度成正相关 ,而且也与病人的预后密切相关 .因此 ,对抗血管生成作用是一种很有吸引力的肿瘤疗法 .神经节苷脂GD3在多种类型的肿瘤中超常表达 .一般认为 ,神经节苷脂GD3有增强肿瘤本身及邻近组织中的血管生成作用 ,从而促进肿瘤的演进和转移 .最近的研究工作为这一假设提供了有力的实验证据 .应用GD3合酶的反意DNA转染肿瘤细胞从而抑制细胞中的GD3合酶的表达 ,极大地降低了细胞的内源GD3含量 .进一步的研究证明 ,抑制肿瘤细胞的GD3合成明显地降低了该肿瘤细胞的血管内皮生长因子 (VEGF)的水平 ,并使血管生成作用降至最小限度 .这些实验说明GD3在肿瘤的血管生成中具有重要的作用 .此外 ,GD3作为肿瘤的一种相关抗原 ,它与血管生成因子的协同效应将在未来的联合基因疗法中起到重要的作用     

Disialogangliosides GD2 and GD3 are involved in the attachment of human melanoma and neuroblastoma cells to extracellular matrix proteins

Human melanoma cells express relatively large amounts of the disialogangliosides GD3 and GD2 on their surface whereas neuroblastoma cells express GD2 as a major ganglioside. Monoclonal antibodies (Mabs) directed specifically to the carbohydrate moiety of GD3 and GD2 inhibit melanoma and neuroblastoma cell attachment to various substrate adhesive proteins, e.g. collagen, vitronectin, laminin, fibronectin, and a heptapeptide, glycyl-L-arginyl-glycyl-L-aspartyl-L-seryl-L-prolyl- L-cysteine, which constitutes the cell attachment site of fibronectin. Cells that are preattached to a fibronectin substrate can also be induced to detach and round up in the presence of purified anti- ganglioside Mab. Moreover, when melanoma cells that contain both GD2 and GD3 are incubated with Mabs directed to both of these molecules an additive inhibition is observed. The specificity of this inhibition is demonstrated since Mabs of various isotypes directed to either protein or carbohydrate epitopes on a number of other major melanoma or neuroblastoma cell surface antigens have no effect on cell attachment. A study of the kinetics involved in this inhibition indicates that significant effects occur during the first 5 min of cell attachment, suggesting an important role for GD2 and GD3 in the initial events of cell-substrate interactions. The role of gangliosides in cell attachment apparently does not directly involve a strong interaction with fibronectin since we could not observe any binding of radiolabeled fibronectin or fragments of the molecule known to contain the cell attachment site to melanoma gangliosides separated on thin-layer chromatograms. An alternative explanation would be that gangliosides may play a role in the electrostatic requirements for cell-substrate interactions. In this regard, controlled periodate oxidation of terminal, unsubstituted sialic acid residues on the cell surface not only specifically destroys the antigenic epitopes on GD2 and GD3 recognized by specific Mabs but also inhibits melanoma cell and neuroblastoma cell attachment. In fact, the periodate-induced ganglioside oxidation and the inhibition of cell attachment are equally dose dependent. These data suggest that cell-substratum interactions may depend in part on the electrostatic environment provided by terminal sialic acid residues of cell surface gangliosides and possibly other anionic glycoconjugates.     

猴腺病毒GD株的分离与鉴定

目的 体外分离出猴腺病毒(simian adenovirus,SAdV)毒株,并对其进行鉴定,为SAdV的生物学特性、致病机制和诊断试剂等方面的研究提供基础.方法 采用PCR法对猕猴粪便样本进行SAdV筛查,将PCR检测为阳性的样本处理后接种BSC-1细胞,盲传3代后用PCR扩增测序并负染色电镜观察.结果 从广东地区食蟹猴粪便样本中成功分离出一株SAdV,细胞病变明显,PCR检测呈阳性,电镜下可观察到典型腺病毒样形态的病毒粒子,命名为GD株.经测序鉴定,发现GD株属HAdV-G亚属,与SAdV-1进化发生距离最近.结论 GD株的分离成功,使猴腺病毒基因转移载体的构建成为可能,有助于猴腺病毒替代载体的发展.     

Potentiation of interleukin-2 production and its binding by monoclonal antibodies to the gangliosides GD3 and GD2

Summary Previous studies have shown that monoclonal antibodies (mAbs) against certain gangliosides, which induced remissions in patients with melanoma, also potentiated the response of lymphocytes to a variety of stimuli, including lectins, interleukin-2 (IL-2) and antigens. The present studies have investigated the mechanism of these effects on lymphocytes. Although the mAbs potentiated phytohemagglutinin (PHA)-induced IL-2 production at high concentrations of mAbs and of PHA, this did not appear to explain their potentiation of the proliferative responses of lymphocytes. Hence, although IL-2 production was minimal or absent from the CD8⁺ subset the latter showed the highest degree of augmentation. Furthermore, addition of IL-2 to PHA-stimulated cultures did not produce similar augmentation of mitogenic responses to that produced by the mAb to GD3 or GD2. The augmented and normal mitogenic responses were, however, dependent on IL-2, as shown by their inhibition with mAbs against IL-2. The antiganglioside mAbs did not have significant effects on IL-2 receptor expression measured by mAbs to Tac. However, the mAbs appeared to increase the affinity of binding of radiolabelled IL-2 to IL-2 receptor and increased internalization of the latter. These results suggest that the effects of the mAbs on IL-2 production may be distinguished from their effects on the proliferative responses of T cells and that the latter were associated with changes in affinity and internalization of ¹²⁵I-IL-2. Whether the latter is a direct cause of the increased proliferative response remains unknown. The ability of mAbs to GD2 and GD3 to increase IL-2 production and to enhance IL-2-dependent proliferative responses suggests the may have valuable clinical roles as immunopotentiating agents.     

Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a

We have synthesized several ganglio-oligosaccharide structures using glycosyltransferases from Campylobacter jejuni. The enzymes, alpha-(2-->3/8)-sialyltransferase (Cst-II), beta-(1-->4)-N-acetylgalactosaminyltransferase (CgtA), and beta-(1-->3)-galactosyltransferase (CgtB), were produced in large-scale fermentation from Escherichia coli and further characterized based on their acceptor specificities. 2-Azidoethyl-glycosides corresponding to the oligosaccharides of GD3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GM2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GD2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), and GM1 (beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) were synthesized in high yields (gram-scale). In addition, a mammalian alpha-(2-->3)-sialyltransferase (ST3Gal I) was used to sialylate GM1 and generate GD1a (alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) oligosaccharide. We also cloned and expressed a rat UDP-N-acetylglucosamine-4'epimerase (GalNAcE) in E. coli AD202 cells for cost saving in situ conversion of less expensive UDP-GlcNAc to UDP-GalNAc.     

Ganglioside GD3 biosynthesis in normal and mutant mouse embryos

CMP-sialic acid:GM3 sialyltransferase (GD3 synthase; EC 2.4.99.8) was characterized in a membrane-enriched preparation (P2 pellet) from mouse embryos at embryonic day 12 (E-12). Gangliosides GD3 and GM3 were the major radiolabeled products of the reaction. Optimum GD3 synthase activity was obtained at pH 6.0 using 0.1% detergent Triton CF-54. The Km values for GM3 and CMP-sialic acid were 55 and 80 microM, respectively. The Vmax value was calculated as 622 pmol/mg protein/hr. Ganglioside GD3, as end product, induced a two-step reduction of enzyme activity in the range of concentrations from 0 to 34 microM (40%) and from 150 to 300 microM (65%). The rate of GD3 formation was similar in whole embryos and in embryo head and body regions. GD3 synthase activity in tw1/tw1 mutant mouse embryos, which express defects in neuronal differentiation, was only 40% of that in normal wild-type (+/+) embryos. Enzyme activity in heterozygous (+/twl) embryos was similar to that in +/+ embryos. These findings suggest that the reduced GD3 synthase activity in the mutants might arise as a consequence of failed nervous system development and might reflect a secondary rather than a primary effect of the mutation.     

Ganglioside GD3 in the serum of tumor patients

Gangliosides of normal human blood serum and of blood serum of tumour hosts were studied. It has been shown the first time that ganglioside (NeuAc)2LacCer is present in the blood serum of different tumour hosts, but is absent in normal blood serum. The reasons for the appearance of this ganglioside in blood serum are discussed.     

Structural and immunological characterization of O-acetylated GD2. Evidence that GD2 is an acceptor for ganglioside O-acetyltransferase in human melanoma cells.

We have shown previously that Golgi-enriched vesicles from the human melanoma cell line Melur can transfer [3H]acetate from [acetyl-3H]acetyl-CoA to endogenous GD3 to form [acetyl-3H]O-acetyl-GD3 (Manzi, A. E., Sjoberg, E. R., Diaz, S., and Varki, A. (1990) J. Biol. Chem. 265, 13091-13103). Applying the same approach in the human melanoma cell line M21, label was found in [acetyl-3H]O-acetyl-GD3 and also in a species co-migrating with unsubstituted GD3 on TLC. Both were sialidase-sensitive and alkali-labile, indicating incorporation as [3H]O-acetyl esters on sialic acids. Immunological reactivity, sialidase sensitivity, chromatographic behavior, and the known ganglioside pattern of M21 cells suggested that the slower migrating species might be [acetyl-3H]O-acetyl-GD2. Sialic acids released from this labeled molecule by sialidase showed esterification with [3H]acetate at both C7 and C9 hydroxyls. Lipid extracts from cells metabolically labeled with [3H]galactose showed a corresponding ganglioside, which upon alkali treatment yielded a species migrating with GD2. Analysis of purified ganglioside by high performance thin layer chromatography immuno-overlays, fast atom bombardment-mass spectrometry in positive and negative ion modes, periodate oxidation resistance, linkage analysis by permethylation and gas chromatography-mass spectrometry, and 500 MHz 1H NMR was consistent with the following structure: 9-O Ac-Neu5Ac alpha 2-8Neu5Ac alpha 2-3(GalNAc beta 1-4) Gal beta 1-4Gluc beta 1-1' ceramide Total gangliosides from M21 were analyzed by high performance thin layer chromatography immuno-overlay with monoclonal antibodies D1.1, JONES, 27A, and 8A2, all known to, or suspected of reacting with 9-O-acetylated gangliosides. The first three bound well to 9-O-acetyl-GD3 and a slower migrating 9-O-acetylated ganglioside, which was distinct from 9-O-acetyl-GD2. Antibody 8A2 reacted weakly with purified 9-O-acetyl-GD2 and strongly with two other 9-O-acetylated gangliosides migrating slower than 9-O-acetyl-GD2. Thus, the family of O-acetylated gangliosides in melanoma cells is much more complex than previously appreciated.     

抗神经节苷脂GD2抗体药物的研究现状

神经母细胞瘤(neuroblastoma,NB)是儿童最常见的颅外实体肿瘤,其中高危NB患儿在接受化疗、放疗和手术等联合治疗后仍有约40％因复发而死亡.近年来,随着免疫治疗研究的不断深入,以抗神经节苷脂GD2抗体(anti-ganglioside GD2 antibody,简称抗GD2抗体)药物为基础的免疫治疗已成为高...     

Disialoganglioside GD2 distributes preferentially into substrate- associated microprocesses on human melanoma cells during their attachment to fibronectin

Human melanoma cells (M21) actively attach and spread on a fibronectin substrate. Indirect immunofluorescence assays with specific monoclonal antibodies directed to the disialoganglioside GD2, the major ganglioside expressed on M21 melanoma cells, indicate that during the cell attachment process this molecule redistributes into microprocesses that make direct contact with the fibronectin substrate. Scanning and transmission immunoelectron microscopic studies with anti-GD2 monoclonal antibodies and immuno-gold staining demonstrate that GD2 preferentially localizes into substrate-associated microprocesses that emanate from the plasma membrane of the M21 cells. Staining with monoclonal antibodies directed to other melanoma surface antigens fails to demonstrate a similar distribution pattern on these cells. Direct evidence is provided that GD2 is involved in M21 cell attachment to fibronectin, since treatment of these cells with anti-GD2 monoclonal antibodies causes cell rounding and detachment from a fibronectin substrate. Moreover, scanning electron microscopy demonstrates that this loss of attachment of fibronectin is characterized by a perturbation of the cell attachment-promoting microprocesses that in the presence of these antibodies lose contact with the fibronectin substrate.     

Disruption of GM2/GD2 synthase gene resulted in overt expression of 9-O-acetyl GD3 irrespective of Tis21

GM2/GD2 synthase gene knockout mice lack all complex gangliosides, which are abundantly expressed in the nervous systems of vertebrates. In turn, they have increased precursor structures GM3 and GD3, probably replacing the roles of the depleted complex gangliosides. In this study, we found that 9-O-acetyl GD3 is also highly expressed as one of the major glycosphingolipids accumulating in the nervous tissues of the mutant mice. The identity of the novel component was confirmed by neuraminidase treatment, thin layer chromatography-immunostaining, two-dimensional thin layer chromatography with base treatment, and mass spectrometry. All candidate factors reported to be possible inducer of 9-O- acetylation, such as bitamine D binding protein, acetyl CoA transporter, or O-acetyl ganglioside synthase were not up-regulated. Tis21 which had been reported to be a 9-O-acetylation inducer was partially down-regulated in the null mutants, suggesting that Tis21 is not involved in the induction of 9-O-acetyl-GD3 and that accumulated high amount of GD3 might be the main factor for the dramatic increase of 9-O-acetyl GD3. The ability to acetylate exogenously added GD3 in the normal mouse astrocytes was examined, showing that the wild-type brain might be able to synthesize very low levels of 9-O-acetyl GD3. Increased 9-O-acetyl GD3, in addition to GM3 and GD3, may play an important role in the compensation for deleted complex gangliosides in the mutant mice.     

GD3-replica peptides selected from a phage peptide library induce a GD3 ganglioside antibody response

GD3-replica peptides were obtained from a phage peptide library and an anti-GD3 monoclonal antibody (Mab) (4F6), and anti-GD3 Mabs were generated by immunizing a peptide GD3P4. A Mab, 3D2 was found to recognize GD3 by immunohistochemical approaches. Amino acid analysis of heavy and light chain variable regions of 4F6 and 3D2 showed that the respective chains had the same length, and only a few different amino acid substitutions were found. The present data indicate that the immunogenic GD3P4 is processed in a certain size and exposed on the antigen-presenting cells with a molecular shape quite similar to that of the GD3 epitope in 4F6.     

Gangliosides GM3 and GD3 in human stomach and breast tumors

Gangliosides of human gastric and mammary tumours and of homologous normal tissues were studied by using biochemical methods and specific antisera. It was found that in most cases GM3, GD3 and GM1 are predominant gangliosides, whereas several polar components are minor ones. A comparison of the relative amount of ganglioside fractions revealed that in gastric tumours the per cent content of polar compounds is higher than in intact tissue; however, the absolute content of all gangliosides is markedly increased. A comparative study of the composition of mammary tumour and normal tissue gangliosides demonstrated two types of changes: i) the absolute content of all gangliosides in tumour tissue was increased and, ii) the increase in the content of total gangliosides was paralleled with the appearance of a new fraction (presumably GM4), the decrease of the GD3 content and the disappearance of polar gangliosides. A possible mechanism of this effect is discussed.