Glycosphingolipids and cell death

Sphingolipids have been implicated in various cellular processes including growth, cell-cell or ligand-receptor interactions, and differentiation. In addition to their importance as reservoirs of metabolites with important signaling properties, sphingolipids also help provide structural order to plasma membrane lipids and proteins within the bilayer. Glycosylated sphingolipids, and sphingomyelin in particular, are involved in the formation of lipid rafts. Although it is well accepted that ceramide, the backbone of all sphingolipids, plays a critical role in apoptosis, less is known about the biological functions of glycosphingolipids. This review summarizes current knowledge of the involvement of glycosphingolipids in cell death and in other pathological processes and diseases.     

人正常子宫肌肉的鞘糖脂组成及其在不同生理功能阶段的变化

本文测定了新生儿、生育期、更年期和足月妊娠人子宫肌肉的神经节苷脂(Gg)与中性鞘糖脂(N-GSL)的含量,比较了两种鞘糖脂的HPTLC谱。新生儿期Gg的总含量(以脂结合唾液酸LBSA量表示)最高,每克湿重组织约45.2μg,足月妊娠子宫肌肉中的含量最低,为10.4μg,生育期为32.8μg、更年期为39.5μg。N—GSL的含量却以足月妊娠子宫肌肉中最多,达99.4μg。按HPTLC谱分析子宫肌肉中Gg的主要组分为GD_3和GM_3,在子宫发育成熟与妊娠时,肌肉组织中这两种组分的含量变化明显:生育期样品的GD_3由新生儿的25.4％增加到56.6％(按占LBSA总量的百分比计算),GM_3则由33.2％降至16.9％。此外,GM_1和GD_(1a)也明显减少。N—GSL在生育期CMH、CDH和CTH的含量(按占含糖基量的百分比计算)成倍增加,而含五糖基以上的组分则仅为新生儿子宫的1/5。足月妊娠与新生儿子宫肌肉的两类鞘糖脂的HPTLC谱类似,但前者GT1b占19.4％,明显高于新生儿样品(6.1％)。     

Glycosphingolipids in membrane architecture

As part of a program to investigate the behavior and interactions of glycolipids in biological membranes we have synthesized spin-labeled derivatives of 2 families of carbohydrate-bearing ceramides (glycosphingolipids): simple neutral glycolipids and gangliosides. Galactosyl ceramide has been synthesized with the spin label at 3 different positions on the fatty acid chain. It has been studied in bilayers of various different lipids and lipid mixtures and compared to the corresponding phospholipid spin labels. Considerable similarity has been found between the behavior of galactosyl ceramide and phosphatidylcholine. These similarities include a negligible flip-flop rate, a flexibility gradient in the acyl chains, and exclusion from phosphatidylserine domains in the face of a Ca²⁺-induced lateral phase separation. Evidence for dramatic clustering of simple neutral glycolipids has not been found. Glycosphingolipids do seem to have the capacity to increase rigidity in fluid lipid bilayers. A general procedure has been developed for covalent attachment of a nitroxide spin label to the headgroup region of complex glycolipids such as gangliosides. Studies of beef brain gangliosides labeled in this manner and incorporated into bilayers of phosphatidylcholine indicate that the headgroup oligosaccharides are in rapid, random motion as opposed to being in any way immobilized. This headgroup mobility depends very little on the fluidity or rigidity of the bilayer. However, headgroup mobility decreases, perhaps as a result of cooperative headgroup interactions, with increasing bilayer concentration of unlabeled ganglioside.     

糖鞘脂分析技术及相关疾病研究进展

糖鞘脂是一类广泛分布在动物细胞膜表面的糖脂类物质,它在调控细胞识别、黏附、增殖以及凋亡等方面均有重要的生物学作用.本综述主要讨论了在现代分析技术范畴中,糖鞘脂的鉴定及其糖链结构的分离与解析方面的研究进展和糖鞘脂在癌症等疾病发生发展中所起的生物学功能,以及糖鞘脂作为疾病治疗靶标的可能性.随着现代仪器技术,尤其是质谱技术和色谱-质谱联用技术的发展,糖鞘脂的分离与检测也进入了高速发展的时代.目前,使用质谱技术在肝癌、结直肠癌、乳腺癌等恶性肿瘤的组织样本中均发现了不同种类糖鞘脂不同程度的异常表达.其中,岩藻糖基化的糖鞘脂上调表达在众多癌症糖鞘脂检测中尤为突出,故岩藻糖基化的糖鞘脂可能会成为一类癌症的早期诊断标志物.近年来,随着对糖鞘脂理解的不断深入,糖鞘脂在诸多疾病,如癌症血管生成过程中的功能研究成为了热点之一.例如,从肿瘤细胞表面脱落的大多数糖鞘脂在肿瘤微环境中主要起到了促进血管生成的作用,而与此相反的是,另一种结构简单的神经节苷脂GM3却起到了抑制血管生成的作用.本综述汇集了对上述现象在分子水平上的不同解读以及利用此现象对癌症靶向治疗的研究与探索,并对基于抑制糖鞘脂合成的靶向治疗的发展前景进行了分析展望.     

Neobiosynthesis of Glycosphingolipids by Plasma Membrane-associated Glycosyltransferases

Gangliosides, complex glycosphingolipids containing sialic acids, are synthesized in the endoplasmic reticulum and in the Golgi complex. These neobiosynthesized gangliosides move via vesicular transport to the plasma membrane, becoming components of the external leaflet. Gangliosides can undergo endocytosis followed by recycling to the cell surface or sorting to the Golgi complex or lysosomes for remodeling and catabolism. Recently, glycosphingolipid catabolic enzymes (glycohydrolases) have been found to be associated with the plasma membrane, where they display activity on the membrane components. In this work, we demonstrated that ecto-ganglioside glycosyltransferases may catalyze ganglioside synthesis outside the Golgi compartment, particularly at the cell surface. Specifically, we report the first direct evidence of expression and activity of CMP-NeuAc:GM3 sialyltransferase (Sial-T2) at the cell surface of epithelial and melanoma cells, with membrane-integrated ecto-Sial-T2 being able to sialylate endogenously synthesized GM3 ganglioside as well as exogenously incorporated substrate. Interestingly, we also showed that ecto-Sial-T2 was able to synthesize GD3 ganglioside at the cell surface using the endogenously synthesized cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) available at the extracellular milieu. In addition, the expression of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase (GalNAc-T) was also detected at the cell surface of epithelial cells, whose catalytic activity was only observed after feeding the cells with exogenous GM3 substrate. Thus, the relative interplay between the plasma membrane-associated glycosyltransferase and glycohydrolase activities, even when acting on a common substrate, emerges as a potential level of regulation of the local glycosphingolipid composition in response to different external and internal stimuli.     

Novel Neogala-Series Glycosphingolipids with a Terminal Glucose Residue from the Fungus Mariannaea elegans

To study the role of osteopontin, we did cell adhesion and ALP assays of rat bone marrow osteoblastic cells (RBMO) on collagen Type I and osteopontin surfaces. The RBMO proved to adhere much more strongly to the osteopontin and to have higher ALP activity on the osteopontin, which suggests that pre-osteoblasts differentiate into osteoblasts that form bone by recognizing osteopontins.     

GRASP55 regulates intra‐Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis‐trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI‐based retrograde transport vesicles, thus concentrating them in the trans‐Golgi. In genome‐edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis‐Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.     

Immunoglobulin G-class mouse monoclonal antibodies to major brain gangliosides

Mice genetically engineered to lack complex gangliosides are improved hosts for raising antibodies against those gangliosides. We report the generation and characterization of nine immunoglobulin G (IgG)-class monoclonal antibodies (mAbs) raised against the four major brain gangliosides in mammals. These include (designated as ganglioside specificity-IgG subclass) two anti-GM1 mAbs (GM1-1, GM1-2b), three anti-GD1a mAbs (GD1a-1, GD1a-2a, GD1a-2b), one anti-GD1b mAb (GD1b-1), and three anti-GT1b mAbs (GT1b-1, GT1b-2a, GT1b-2b). Each mAb demonstrated high specificity, with little or no cross-reactivity with other major brain gangliosides. Enzyme-linked immunosorbent assay (ELISA) screening against 14 closely related synthetic and purified gangliosides confirmed the high specificity, with no significant cross-reactivity except that of the anti-GD1a mAbs for the closely related minor ganglioside GT1a alpha. All of the mAbs were useful for ELISA, TLC immunooverlay, and immunocytochemistry. Neural cells from wild-type rats and mice were immunostained to differing levels with the anti-ganglioside antibodies, whereas neural cells from mice engineered to lack complex gangliosides (lacking the ganglioside-specific biosynthetic enzyme UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase) remained unstained, demonstrating that most of the mAbs react only with gangliosides and not with related structures on glycoproteins. These mAbs may provide useful tools for delineation of the expression and function of the major brain gangliosides and for probing the pathology of anti-ganglioside autoimmune diseases.     

Autophagy and cell death

Autophagy exerts dual functions in cancer, acting as both a tumor suppressor, for example, by preventing the accumulation of damaged proteins and organelles, and as a tumor promoter that supports tumor growth. Many anticancer therapies engage autophagy as part of a cellular response. However, the question of whether or not autophagic activity in cells undergoing cell death is the cause of death or whether it is actually an attempt to support survival in response to cellular stress conditions has been discussed with great controversy.     

Changes of the ganglioside pattern and content in human fibroblasts by high density cell population subculture progression

In this study we show that the ganglioside content and pattern of human skin fibroblasts change along the process of cell subculture progression by varying the cell density.GM3, GD3 and GD1a were components of the total cell ganglioside mixtures extracted from cells, but GD1a was in all the extracts a minor component or very scant. Other gangliosides present in traces were not characterised. The fibroblast ganglioside content of 52 pools of cells obtained from 5 different cell lines cultured at variable cell density ranged from 2.0 to 13.1 nmoles per mg of cell protein. The molar ratio between GM3 and GD3 varied from 418 to 0.6 in the ganglioside mixtures, as determined by densitometric quantitative analysis after thin layer chromatographic separation.Both the ganglioside content and the GM3/GD3 molar ratio were constant along several passages of subculture progression performed by plating cells collected at confluence. Instead, when the subculture progression was performed by plating cells collected at a few days after reaching confluence, a progressive increase of the ganglioside content was observed. GD3 increased proportionally more than GM3 so that a progressive decrease of the ratio between GM3 and GD3 was observed. In some experiments, GD3 was very scant at the beginning of the progression, while it was near 30% after 5 passages under these conditions. The progressive increase of GD3 along the high density cell population subculture progression was associated to a moderate increase of the mRNA GD3 synthase. Published in 2003.     

Gene-linked shift in ganglioside distribution influences growth and vascularity in a mouse astrocytoma

Brain tumor growth and progression is dependent upon vascularity, and is associated with altered ganglioside composition and distribution. In this study, we examined the influence of gangliosides on growth and vascularity in a malignant mouse astrocytoma, CT-2A. Ganglioside distribution was altered in CT-2A tumor cells using an antisense construct to beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T), a key enzyme that uses the simple ganglioside GM3 as a substrate for the synthesis of the more complex gangliosides, GM2, GM1 and GD1a. GalNAc-T gene expression was significantly lower in CT-2A cells stably transfected with the antisense GalNAc-T plasmid, pcDNA3.1/TNG (CT-2A/TNG) than in either non-transfected CT-2A or mock-transfected (CT-2A/V) control tumor cells. GM3 was elevated from 16% to 58% of the total ganglioside distribution, whereas GM1 and GD1a were reduced from 17% and 49% to 10% and 17%, respectively, in CT-2A/TNG tumor cells. Growth, vascularity (blood vessel density and Matrigel assay) and vascular endothelial growth factor (VEGF) expression was significantly less in CT-2A/TNG tumors than in control CT-2A brain tumors. In addition, the expression of VEGF, hypoxia-inducible factor 1alpha (HIF-1alpha) and neuropilin-1 (NP-1) was significantly lower in CT-2A/TNG tumor cells than in control CT-2A tumor cells. These data suggest that gene-linked changes in ganglioside composition influence the growth and angiogenic properties of the CT-2A astrocytoma.     

Role of Glycosphingolipids in HIV-1 Entry: Requirement of Globotriosylceramide (Gb3) in CD4/CXCR4-dependent Fusion

We have recently shown that addition of human erythrocyte glycosphingolipids (GSL) to non-human CD4⁺ or GSL-depleted human CD4⁺ cells rendered those cells susceptible to gp120-gp41-mediated cell fusion (Puri et al., BBRC, 1998). One GSL fraction (Fraction 3) isolated from human erythrocyte GSL mixture exhibited the highest recovery of fusion following incorporation into CD4⁺ non-human and GSL-depleted HeLa-CD4 cells (HeLa-CD4/GSL^–). Structural analysis of Fraction 3 showed that this GSL had identical head group as the known GSL, Gal(14)Gal(1 4)Glc-Ceramide (Gb3) (Puri et al., PNAS, 1998). Here we report that presence of Gb3 in CD4⁺/CXCR4⁺ cells but not CD4⁺/CXCR4^– cells allows fusion with HIV-1_Lai-envelope glycoprotein expressing cells (TF228). Therefore, Gb3 functions in conjunction with HIV-1 co-receptor, CXCR4 to promote fusion. We propose that Gb3 functions by recruiting CD4 and/or CXCR4 at the fusion site through structurally specific interactions.     

Glycosphingolipids of Human Peripheral Nervous System Myelins Isolated from Cauda Equina

Abstract: Compositions of neutral and sulfated glucuronyl glycosphingolipids purified from human motor and sensory nerves and myelins were studied. Higher neutral glycosphingolipids (fraction B), which were separated from GazlCer (fraction A), were analyzed by TLC and TLC-immunostaining. Both nerve myelins contained paragloboside (nLc₄Cer) and nLc₆Cer dominantly as major higher glycosphingolipids and very little globoside (Gb₄Cer), whereas both nerves contained Gb₄Cer and nLc₄Cer. Besides these major glycosphingolipids, a neutral glycolipid containing asialoGMI (Gg₄Cer) epitope and other minor components such as ceramide trihexoside and ceramide dihexoside were detected in both nerves and their myelins. Furthermore, sulfated glucuronyl nLc₄Cer and n Lc₆Cer, which were monoclonal antibody HNK-1 reactive glycolipids, were detected in both nerves and myelins.     

Sialic Acid Linkage in Glycosphingolipids Is a Molecular Correlate for Trafficking and Delivery of Extracellular Cargo

Gangliosides, glycosphingolipids containing sialic acid moieties, are well known mediators of transmembrane signaling and endocytosis at the plasma membrane. However, little is known about their precise regulatory role at the cell periphery for intracellular sorting of extracellular cargo. Here we inspected published scientific literature for two types of cargoes, namely bacterial toxins and viruses, regarding their usage of gangliosides. We derived a rather simple yet surprisingly consistent framework to classify 20 viruses from 12 different families and five type AB bacterial toxins into two broad categories. We propose that gangliosides with terminally attached sialic acids classify cargo for uptake and trafficking early in the endocytic pathway while gangliosides with internally attached sialic acids associate with uptake and trafficking of cargo late in the endocytic system. Our study provides a testable hypothesis for future investigations into a wide range of trafficking events. It could be utilized as a framework for other intracellular pathogens where lipids are known to be involved in recognition and trafficking. For instance, predictions can be put forward and evaluated based on ganglioside binding patterns and intracellular trafficking routes. Finally, incorporation of our classifier into large scale systems‐biology studies could help reveal related molecular determinants in subcellular sorting .     

Functional cloning of genes that suppress oxidative stress-induced cell death: TCTP prevents hydrogen peroxide-induced cell death

We used retroviral-mediated expression cloning to identify cDNAs that inhibit cell death induced by oxidative stress. To isolate the genes, we introduced a murine embryonic retroviral cDNA library into NIH/3T3 cells, and selected for cells resistant to hydrogen peroxide. The surviving cells were cloned, and the integrated cDNAs were rescued by polymerase chain reaction. Several of the isolated cDNAs are known to be involved in modulating the redox state of cells. Other cDNAs encode proteins known to suppress apoptosis caused by reasons other than oxidative stress. These included polyadenylate-binding protein, cytosolic 1 (Pabpc1) and translationally controlled tumor protein (TCTP).     

Neutral Glycosphingolipids and Ceramide Composition of Ethylnitrosourea-Induced Rat Neural Tumors: Accumulation of Ceramide in Tumors

Abstract: Experimental rat neural tumors in offspring were induced transplacentally by a single injection of a chemical carcinogen, ethylnitrosourea, 20 mg/kg body weight, in the tail vein of the mother. The neutral glycosphingolipid, sulfatide, and ceramide composition of the tumors and the normal tissues from which the tumors originated is described. The content of nonhydroxy fatty acid (NFA) and hydroxy fatty acid (HFA) containing ceramide in all the neural tumors so far examined was significantly increased compared with the corresponding normal neural tissue. Some 8 to 18 mol% of total neutral glycolipids was as ceramide in neurinomas, oligodendrogliomas, and menin-giomas. Lactosylceramide in normal neural tissues was about 1 mol% of the total neutral glycosphingolipids. In various neural tumors lactosylceramide increased up to 8 mol%. NFA- and HFA-containing cerebrosides constitute 94–100% of the neutral glycosphingolipids in normal neural tissues. In various neural tumors the mol percent of cerebrosides was significantly reduced. A high performance liquid chromatographic method was modified to analyze simultaneously ceramides, cerebrosides, and higher neutral glycosphingolipids.