Complex Gangliosides Research Articles

A Modular Chemoenzymatic Synthesis of Disialosyl Globopentaosylceramide (DSGb5Cer) Glycan.

The total synthesis of the oligosaccharide moiety of disialosyl globopentaosylceramide (DSGb5 Cer), a dominant ganglioside isolated from malignant renal cell carcinoma tissues, is reported. The synthetic strategy relies on a chemical α(2,6)-sialylation at the internal GalNAc unit of a Gb5 pentasaccharide backbone that furnishes a Neu5Acα(2,6)GalNAc-linked hexasaccharide, suitable for an enzymatic α(2,3)-sialylation of the terminal Gal residue to construct a heptasaccharide glycan. Convergent access to this key α(2,6)-sialylated hexasaccharide was also achieved through a [3+3] glycosylation building upon a Galβ(1,3)[Neu5Acα(2,6)]GalNAc-based trisaccharide donor and a Gb3 acceptor. The synthetic DSGb5 glycan bearing a 6-azidohexyl aglycon at the reducing end could undergo further regioselective functionalization. This approach represents a viable chemoenzymatic method for accessing complex ganglioside glycans and should be useful for the synthesis and biological investigation of DSGb5 derivatives.

S1P-lyase deficiency uncouples ganglioside formation – Potential contribution to tumorigenic capacity

Sphingosine-1-phosphate (S1P) is not only a catabolic intermediate of all sphingolipids but also an evolutionary conserved bioactive lipid with critical functions in cell survival, differentiation, and migration as well as in immunity and angiogenesis. S1P-lyase (SGPL1) irreversibly cleaves S1P in the final step of sphingolipid catabolism. As sphingoid bases and their 1-phosphates are not only metabolic intermediates but also highly bioactive lipids that modulate a wide range of physiological processes, it would be predicted that their elevation might induce adjustments in other facets of sphingolipid metabolism and/or alter cell behavior. We actually found in a previous study that in terminally differentiated neurons SGPL1 deficiency increases sphingolipid formation via recycling at the expense of de novo synthesis. We now investigated whether and how SGPL1 deficiency affects the metabolism of (glyco)sphingolipids in mouse embryonic fibroblasts (MEFs). According to our previous experiments in neurons, we found a strong accumulation of S1P in SGPL1-deficient MEFs. Surprisingly, a completely different situation arose as we analyzed sphingolipid metabolism in this non-differentiated cell type. The production of biosynthetic precursors of complex glycosphingolipids including ceramide, glucosylceramide and also ganglioside GM3 via de novo synthesis and recycling pathway was substantially increased whereas the amount of more complex gangliosides dropped significantly.

Gangliosides in the differentiation process of primary neurons: the specific role of GM1-oligosaccharide

It has been recently reported by our group that GM1-oligosaccharide added to neuroblastoma cells or administered to mouse experimental model mimics the neurotrophic and neuroprotective properties of GM1 ganglioside. In addition to this, differently from GM1, GM1-oligosaccharide is not taken up by the cells, remaining solubilized into the extracellular environment interacting with cell surface proteins. Those characteristics make GM1-oligosaccharide a good tool to study the properties of the endogenous GM1, avoiding to interfere with the ganglioside natural metabolic pathway. In this study, we show that GM1-oligosaccharide administered to mice cerebellar granule neurons by interacting with cell surface induces TrkA-MAP kinase pathway activation enhancing neuron clustering, arborization and networking. Accordingly, in the presence of GM1-oligosaccharide, neurons show a higher phosphorylation rate of FAK and Src proteins, the intracellular key regulators of neuronal motility. Moreover, treated cells express increased level of specific neuronal markers, suggesting an advanced stage of maturation compared to controls. In parallel, we found that in the presence of GM1-oligosaccharide, neurons accelerate the expression of complex gangliosides and reduce the level of the simplest ones, displaying the typical ganglioside pattern of mature neurons. Our data confirms the specific role of GM1 in neuronal differentiation and maturation, determined by its oligosaccharide portion. GM1-oligosacchairide interaction with cell surface receptors triggers the activation of intracellular biochemical pathways responsible for neuronal migration, dendrites emission and axon growth.

Distinct Cerebellar Glycosphingolipid Phenotypes in Wistar and Lewis Rats

Three strains of laboratory rats (Lewis, Wistar and Sprague–Dawley) commonly used for the study of movement disorders differ in gait traits. Sialic acid containing glycosphingolipids, called gangliosides, are abundant in the nervous system, where they affect numerous neurochemical events. Cerebellum plays the key role in the integration of body movements. This study investigated glycosphingolipid phenotypes with the hypothesis that they differ in cerebella of the three rat strains. In addition, forebrain and brain stem glycosphingolipid phenotypes were determined. Total glycosphingolipid fractions (neutral and acidic) were analysed by high performance thin-layer chromatography (HPTLC). Complex gangliosides were detected with HPTLC immunostaining by using cholera toxin B subunit after the neuraminidase pretreatment. The most significant differences were found in the cerebellum glycosphingolipid content. Lewis rats showed three fold higher monohexaosylceramides (galactosylceramide + sulfatide)/gangliosides ratio compared to Wistar rats. On the other hand, the cerebellum of Wistar rats contained an increased content of complex gangliosides GD1b, GT1b and GQ1b, fourfold, twofold and tenfold, respectively, compared to Lewis rats. This study shows that Wistar and Lewis rat strains have pronounced differences in glycosphingolipid content and the composition of cerebellum.

Halogen-directed chemical sialylation: pseudo-stereodivergent access to marine ganglioside epitopes.

Sialic acids are conspicuous structural components of the complex gangliosides that regulate cellular processes. Their importance in molecular recognition manifests itself in drug design (e.g. Tamiflu®) and continues to stimulate the development of effective chemical sialylation strategies to complement chemoenzymatic technologies. Stereodivergent approaches that enable the α- or β-anomer to be generated at will are particularly powerful to attenuate hydrogen bond networks and interrogate function. Herein, we demonstrate that site-selective halogenation (F and Br) at C3 of the N-glycolyl units common to marine Neu2,6Glu epitopes enables pseudo-stereodivergent sialylation. α-Selective sialylation results from fluorination, whereas traceless bromine-guided sialylation generates the β-adduct. This concept is validated in the synthesis of HLG-1 and Hp-s1 analogues.

Reduced sphingolipid hydrolase activities, substrate accumulation and ganglioside decline in Parkinson\u2019s disease

BackgroundHaploinsufficiency in the Gaucher disease GBA gene, which encodes the lysosomal glucocerebrosidase GBA, and ageing represent major risk factors for developing Parkinson’s disease (PD). Recently, more than fifty other lysosomal storage disorder gene variants have been identified in PD, implicating lysosomal dysfunction more broadly as a key risk factor for PD. Despite the evidence of multiple lysosomal genetic risks, it remains unclear how sphingolipid hydrolase activities, other than GBA, are altered with ageing or in PD. Moreover, it is not fully known if levels of glycosphingolipid substrates for these enzymes change in vulnerable brain regions of PD. Finally, little is known about the levels of complex gangliosides in substantia nigra which may play a significant role in ageing and PD.MethodsTo study sphingolipid hydrolase activities and glycosphingolipid expression in ageing and in PD, two independent cohorts of human substantia nigra tissues were obtained. Fluorescent 4-methylumbelliferone assays were used to determine multiple enzyme activities. The lysosomal GBA and non-lysosomal GBA2 activities were distinguished using the inhibitor NB-DGJ. Sensitive and quantitative normal-phase HPLC was performed to study glycosphingolipid levels. In addition, glycosphingolipid levels in cerebrospinal fluid and serum were analysed as possible biomarkers for PD.ResultsThe present study demonstrates, in two independent cohorts of human post-mortem substantia nigra, that sporadic PD is associated with deficiencies in multiple lysosomal hydrolases (e.g. α-galactosidase and β-hexosaminidase), in addition to reduced GBA and GBA2 activities and concomitant glycosphingolipid substrate accumulation. Furthermore, the data show significant reductions in levels of complex gangliosides (e.g. GM1a) in substantia nigra, CSF and serum in ageing, PD, and REM sleep behaviour disorder, which is a strong predictor of PD.ConclusionsThese findings conclusively demonstrate reductions in GBA activity in the parkinsonian midbrain, and for the first time, reductions in the activity of several other sphingolipid hydrolases. Furthermore, significant reductions were seen in complex gangliosides in PD and ageing. The diminished activities of these lysosomal hydrolases, the glycosphingolipid substrate accumulation, and the reduced levels of complex gangliosides are likely major contributors to the primary development of the pathology seen in PD and related disorders with age.

Human caudate nucleus exhibits a highly complex ganglioside pattern as revealed by high-resolution multistage Orbitrap MS

Caudate nucleus (CN) is a specialized part of the dorsal striatum of each brain hemisphere involved in numerous cognitive processes. Caudate dysfunctions are associated with Alzheimer’s, Parkinson’s and Huntington’s disease, autism, and even schizophrenia. Most of the studies upon CN and related diseases were conducted using neuroimaging techniques, which, in some instances have reached contradictory conclusions. This state-of-the-art technique triggered the development of methods able to provide information at the molecular level. In this context, here we have implemented high resolution (HR) mass spectrometry (MS) and multistage MS (MSn), for mapping and structural analysis of gangliosides in human CN. Due to the high resolution and mass accuracy (average value: 1.5 ppm), 100 distinct species, of which 90 were di-, tri-, tetra-, and pentasialylated and 17 fucosylated and acetylated and, for the first time, species modified by CH3COO−, were reliably identified in the native CN ganglioside extract. Additionally, two structurally-relevant species, GD2 (d18:1/18:0) and GD2 (d18:1/16:2), were characterized with MS2–MS3 by collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD). The set of data collected by high-resolution mass spectrometry (HR-MS) revealed a much higher complexity of the CN ganglioside pattern than ever reported, and the species associated to this brain region, potentially implicated in many of its functions.

Visual Function in Mice Lacking GM3 Synthase

ABSTRACTPurpose: Most complex gangliosides in vertebrates are formed from ganglioside GM3. GM3 deficiency in humans can result in epilepsy and visual impairment. To investigate whether a deficiency of GM3 is involved in visual function, ST3GAL5−/− mice with mutations in the ST3GAL5 gene-coded GM3 synthase were employed.Materials and Methods: Sixty mice were employed in this study. The glycosphingolipids of mice retinas were analyzed through high performance thin layer chromatography. The morphology of the optic nerves and retinas were evaluated by hematoxylin and eosin staining and immunohistochemical analysis using an anti-glial fibrillary acidic protein (GFAP) antibody. An electroretinogram (ERG) was applied on the eyes of 4, 9, 12, and 14-month-old mice. Also, visual evoked potential (VEP) was applied on 13-month-old mice.Results: The GM3 in the retinas was detected in ST3GAL5+/+ mice but not ST3GAL5−/− mice. Also, GM1b and GD1α expressions and lactosylceramide accumulation were found in the ST3GAL5−/− mouse retinas. There was no significant difference in GFAP expression in the retinas or optic discs between ST3GAL5+/+ and ST3GAL5−/− mice. Furthermore, the outcome of ERG and VEP analysis showed no disparity between the two strains in 13 and 14-month-old mice.Conclusion: In the eye, neither histopathological abnormalities nor abnormal functions of the retina were found in GM3-deficient mice. Differing from the situation in patients with GM3 deficiency, the lack of GM3 in mice did not lead to optic nerve atrophy.

Identification of 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) as main O-acetylated sialic acid species of GD2 in breast cancer cells.

Mainly restricted to the nervous system in healthy adults, complex gangliosides such as GD3 and GD2 have been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. Interestingly, O-acetylated forms of GD2, not expressed in human peripheral nerve fibers, are highly expressed in GD2+ tumor cells. Very little information is known regarding the expression of O-acetylated disialogangliosides in breast cancer (BC) cell lines. Here, we analyzed the expression of GD2, GD3 and their O-acetylated forms O-acetyl-GD2 (OAcGD2) and O-acetyl-GD3 (OAcGD3) in BC cells. We used Hs 578T and SUM159PT cell lines, as well as cell clones over-expressing GD3 synthase derived from MDA-MB-231 and MCF-7. Using flow cytometry and immunocytochemistry/confocal microscopy, we report that BC cells express b-series gangliosides GD3 and GD2, as well as significant amounts of OAcGD2. However, OAcGD3 expression was not detected in these cells. O-acetylation of gangliosides isolated from BC cells was examined by LC-MS analysis of sialic acid DMB-derivatives. We report that the main acetylated form of sialic acid expressed in BC gangliosides is 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). These results highlight a close interrelationship between Neu5,9Ac2 and OAcGD2 expression, and suggest that OAcGD2 is synthetized from GD2 and not from OAcGD3 in BC cells.

Host membrane glycosphingolipids and lipid microdomains facilitate Histoplasma capsulatum internalisation by macrophages.

Recognition and internalisation of intracellular pathogens by host cells is a multifactorial process, involving both stable and transient interactions. The plasticity of the host cell plasma membrane is fundamental in this infectious process. Here, the participation of macrophage lipid microdomains during adhesion and internalisation of the fungal pathogen Histoplasma capsulatum (Hc) was investigated. An increase in membrane lateral organisation, which is a characteristic of lipid microdomains, was observed during the first steps of Hc-macrophage interaction. Cholesterol enrichment in macrophage membranes around Hc contact regions and reduced levels of Hc-macrophage association after cholesterol removal also suggested the participation of lipid microdomains during Hc-macrophage interaction. Using optical tweezers to study cell-to-cell interactions, we showed that cholesterol depletion increased the time required for Hc adhesion. Additionally, fungal internalisation was significantly reduced under these conditions. Moreover, macrophages treated with the ceramide-glucosyltransferase inhibitor (P4r) and macrophages with altered ganglioside synthesis (from B4galnt1-/- mice) showed a deficient ability to interact with Hc. Coincubation of oligo-GM1 and treatment with Cholera toxin Subunit B, which recognises the ganglioside GM1, also reduced Hc association. Although purified GM1 did not alter Hc binding, treatment with P4 significantly increased the time required for Hc binding to macrophages. The content of CD18 was displaced from lipid microdomains in B4galnt1-/- macrophages. In addition, macrophages with reduced CD18 expression (CD18low ) were associated with Hc at levels similar to wild-type cells. Finally, CD11b and CD18 colocalised with GM1 during Hc-macrophage interaction. Our results indicate that lipid rafts and particularly complex gangliosides that reside in lipid rafts stabilise Hc-macrophage adhesion and mediate efficient internalisation during histoplasmosis.

Loss of Enzyme Activity in Mutated B4GALNT1 Gene Products in Patients with Hereditary Spastic Paraplegia Results in Relatively Mild Neurological Disorders: Similarity with Phenotypes of B4galnt1 Knockout Mice

B4GALNT1 is an enzyme essential for the synthesis of complex gangliosides, whose absence leads to progressive neurodegeneration with aging in mice. Recently, eleven cases of hereditary spastic paraplegia with mutation in the coding region of B4GALNT1 were reported. However, changes in the enzymatic activity of their products have never been studied. We have constructed expression vectors for individual mutant cDNAs, and examined their activities by cell-free in vitro enzyme assays, and flow cytometry of cells transfected with their expression vectors. Among them, almost all mutant genes showed the complete loss of B4GALNT1 activity in both the in vitro enzyme assays and flow cytometry. Two mutants exceptionally showed weak activity. One of them, M4, had a mutation at amino acid 228 with a premature termination codon. Interestingly, the intensity of fluorescence of GM2 measured by flow cytometry was equivalent between the WT and M4 mutant, although the positive cell population was relatively small in M4. Western immunoblotting of cell lysates from transfectants with cDNA plasmids revealed 67-kDa bands except those containing premature termination codons or frame-shift mutation. Taken together with the clinical findings of patients, loss of enzyme activity may be responsible for the clinical features of hereditary spastic paraplegia, whereas the intensity of neurological disorders was relatively milder than expected. These clinical features of patients including those with male hypogonadism are very similar to the abnormal phenotypes detected in B4galnt1-deficient mice.

Glial Sulfatides and Neuronal Complex Gangliosides Are Functionally Interdependent in Maintaining Myelinating Axon Integrity.

Sulfatides and gangliosides are raft-associated glycolipids essential for maintaining myelinated nerve integrity. Mice deficient in sulfatide (cerebroside sulfotransferase knock-out, CST−/−) or complex gangliosides (β-1,4-N-acetylegalactosaminyltransferase1 knock-out, GalNAc-T−/−) display prominent disorganization of proteins at the node of Ranvier (NoR) in early life and age-dependent neurodegeneration. Loss of neuronal rather than glial complex gangliosides underpins the GalNAc-T−/− phenotype, as shown by neuron- or glial-specific rescue, whereas sulfatide is principally expressed and functional in glial membranes. The similarities in NoR phenotype of CST−/−, GalNAc-T−/−, and axo–glial protein-deficient mice suggests that these glycolipids stabilize membrane proteins including neurofascin155 (NF155) and myelin-associated glycoprotein (MAG) at axo–glial junctions. To assess the functional interactions between sulfatide and gangliosides, CST−/− and GalNAc-T−/− genotypes were interbred. CST−/−× GalNAc-T−/− mice develop normally to postnatal day 10 (P10), but all die between P20 and P25, coinciding with peak myelination. Ultrastructural, immunohistological, and biochemical analysis of either sex revealed widespread axonal degeneration and disruption to the axo–glial junction at the NoR. In addition to sulfatide-dependent loss of NF155, CST−/− × GalNAc-T−/− mice exhibited a major reduction in MAG protein levels in CNS myelin compared with WT and single-lipid-deficient mice. The CST−/− × GalNAc-T−/− phenotype was fully restored to that of CST−/− mice by neuron-specific expression of complex gangliosides, but not by their glial-specific expression nor by the global expression of a-series gangliosides. These data indicate that sulfatide and complex b-series gangliosides on the glial and neuronal membranes, respectively, act in concert to promote NF155 and MAG in maintaining the stable axo–glial interactions essential for normal nerve function.SIGNIFICANCE STATEMENT Sulfatides and complex gangliosides are membrane glycolipids with important roles in maintaining nervous system integrity. Node of Ranvier maintenance in particular requires stable compartmentalization of multiple membrane proteins. The axo–glial adhesion molecules neurofascin155 (NF155) and myelin-associated glycoprotein (MAG) require membrane microdomains containing either sulfatides or complex gangliosides to localize and function effectively. The cooperative roles of these microdomains and associated proteins are unknown. Here, we show vital interdependent roles for sulfatides and complex gangliosides because double (but not single) deficiency causes a rapidly lethal phenotype at an early age. These findings suggest that sulfatides and complex gangliosides on opposing axo–glial membranes are responsible for essential tethering of the axo–glial junction proteins NF155 and MAG, which interact to maintain the nodal complex.

Chloroquine Restores Ganglioside Homeostasis and Improves Pathological and Behavioral Outcomes Post-stroke in the Rat.

Perturbations of ganglioside homeostasis have been observed following stroke whereby toxic simple gangliosides GM2 and GM3 accumulate, while protective complex species GM1 and GD1 are reduced. Thus, there is a need for therapeutic interventions which can prevent ganglioside dysregulation after stroke. A pharmacological intervention using chloroquine was selected for its transient lysosomotropic properties which disrupt the activity of catabolic ganglioside enzymes. Chloroquine was administered both in vitro (0.1μM), to primary cortical neurons exposed to GM3 toxicity, and in vivo (45mg/kgi.p.), to 3-month-old male Wistar rats that underwent a severe stroke injury. Chloroquine was administered for seven consecutive days beginning 3days prior to the stroke injury. Gangliosides were examined using MALDI imaging mass spectrometry at 3 and 21days after the injury, and motor deficits were examined using the ladder task. Chloroquine treatment prevented ganglioside dysregulation 3days post-stroke and partially prevented complex ganglioside depletion 21days post-stroke. Exogenous GM3 was found to be toxic to primary cortical neurons which was protected by chloroquine treatment. Motor deficits were prevented in the forelimbs of stroke-injured rats with chloroquine treatment and was associated with decreased inflammation, neurodegeneration, and an increase in cell survival at the site of injury. Chloroquine administration prevents ganglioside dysregulation acutely, protects against GM3 toxicity in neurons, and is associated with long-term functional and pathological improvements after stroke in the rat. Therefore, targeting lipid dysregulation using lysosomotropic agents such as chloroquine may represent a novel therapeutic avenue for stroke injuries.

Abstract 1110: Ganglioside GM2 mediated tumor growth, progression, and metastasis involves YAP-dependent transcriptional program

Abstract Purpose: Recent report from our laboratory showed a novel role of tumor derived ganglioside, GM2 in mediating tumor cell migration as well invasion. In the present study we report a novel role of GM2 in mediating anchorage independent growth (AIG) and epithelial-mesenchymal transition (EMT) leading to tumor growth, progression and metastasis, through a HIPPO-YAP dependent transcriptional program. Experimental procedures : Permanent GM2-synthase knockout (KO) cells were generated using targeted genome editing tool, TALEN. TALEN was used to generate Renca-vGM2-syn KO cells (from a GM2-over-expressing variant of a mouse kidney cancer cell line, Renca-v). Renca-vWT as well as Renca-vGM2-syn KO cells were used to define the role of GM2 in AIG, EMT and tumorigenesis both in vitro and in vivo. DNA microarray analysis was used to elucidate the mechanism behind GM2-mediated AIG and EMT. To define involvement of YAP in GM2-mediated EMT, real time PCR was used to validate YAP-target gene expression in GM2-synthase KO or silenced (siRNA) cells as well as in cells treated with exogenous GM2, in the presence or absence of specific inhibitors of the Hippo-YAP signaling pathway. CRISPR-Cas9 mediated YAP/TAZ-double KO (HelaYAP/TAZ KO) cells were used to conclusively prove the involvement of YAP/TAZ in GM2-mediated EMT. Results : Renca-vGM2-syn KO cells show reduced AIG, higher cell adhesion possibly through increased anoikis sensitivity, indicating a critical role of GM2-synthase and complex gangliosides in GM2-mediated EMT. Furthermore, Renca-vGM2-syn KO cell line shows reduced tumor growth potential, significant reduction in experimental lung metastasis with increased median survival in a syngeneic mice tumor model. Gene expression profiling using DNA microarray with GM2-synthase silenced cells reveal significant modulation of YAP target genes, suggesting a critical role of the Hippo-YAP signaling axis. Molecular insight reveals that GM2-synthase knockout or siRNA mediated knockdown results in significant downregulation of YAP activated genes namely Ctgf, Cyr 61, Pdgf-c, Lox, etc. and upregulation of YAP repressed gene Ddit4, while exogenous addition of GM2 in GM2 deficient cells (Renca, MCF-7 and Hela) show opposite results. Verteporfin mediated disruption of YAP transcriptional program abrogated GM2 mediated modulation of YAP target genes. Finally, significant decrease in YAP-target gene expression in YAP/TAZ-double KO cells, but not in either YAP or TAZ (paralogue of YAP) single KOs confirm the definitive involvement of YAP/TAZ in GM2-mediated EMT. Conclusion : Our findings confirm a novel role of GM2 in triggering EMT by targeting YAP, ultimately leading to increased tumorigenic potential and metastatic activity in tumor cells. Citation Format: Barun Mahata, Abhisek Sarkar, Elora Khamrui, Sohini Chakraborty, Zhumur Ghosh, Kaushik Biswas. Ganglioside GM2 mediated tumor growth, progression, and metastasis involves YAP-dependent transcriptional program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1110.

TNF differentially regulates ganglioside biosynthesis and expression in breast cancer cell lines.

Gangliosides are glycosphingolipids concentrated in glycolipid-enriched membrane microdomains. Mainly restricted to the nervous system in healthy adult, complex gangliosides such as GD3 and GD2 have been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. GD3 synthase (GD3S), the key enzyme that controls the biosynthesis of complex gangliosides, was shown to be over-expressed in Estrogen Receptor (ER)-negative breast cancer tumors, and associated with a decreased overall survival of patients. We previously demonstrated that GD3S expression in ER-negative breast cancer cells induced a proliferative phenotype and an increased tumor growth. In addition, our results clearly indicate that Tumor Necrosis Factor (TNF) induced GD3S over-expression in breast cancer cells via NFκB pathway. In this study, we analyzed the effect of TNF on ganglioside biosynthesis and expression in breast cancer cells from different molecular subtypes. We showed that TNF up-regulated the expression of GD3S in MCF-7 and Hs578T cells, whereas no change was observed for MDA-MB-231. We also showed that TNF induced an increased expression of complex gangliosides at the cell surface of a small proportion of MCF-7 cells. These results demonstrate that TNF differentially regulates gangliosides expression in breast cancer cell lines and establish a possible link between inflammation at the tumor site environment, expression of complex gangliosides and tumor development.

Glycosphingolipid levels and glucocerebrosidase activity are altered in normal aging of the mouse brain.

Aging is the predominant risk factor for both genetic and sporadic Parkinson’s disease (PD). The majority of PD cases are nonfamilial, and the connection between aging and PD-associated genes is not well understood. Haploinsufficiency of the GBA gene, leading to a reduction in glucocerebrosidase (GCase) activity, is one of the most common genetic risk factors for PD. Furthermore, GCase activity is also reduced in brain regions of sporadic PD patients, with a corresponding accumulation of its glycosphingolipid (GSL) substrates. Recent findings in PD patients and aging control cases, and in human PD patient induced pluripotent stem cell neurons, have shown an age-dependent reduction in GCase activity and an elevation of some GSLs. We therefore asked whether aging-induced changes to both lysosomal and nonlysosomal GCase activity and GSL homeostasis in the brain could also be reflected in other nonhuman mammalian systems. Increases in brain polyubiquitin and the lysosomal-associated membrane protein, LAMP2A, were found in 24-month-old wild-type mice compared to 1.5-month-old mice. A lipidomic analysis was performed on brains of wild-type mice of different strains between 1.5 and 24 months of age. Aging created GSL changes that are reminiscent of sporadic PD. Levels of glucosylceramide, glucosylsphingosine, lactosylceramide, and GM1a were elevated in the brain of aged mice, and levels of complex gangliosides, GD1a, GD1b, and GT1b, were reduced with age. Parallel biochemical analyses revealed a change in lipid metabolism probably mediated by lysosomal hydrolases, with reduced GCase and increased neuraminidase activity. Based on these data, we hypothesize that perturbation of GSL metabolism in the aging brain may precede or may be part of abnormal protein handling and may accelerate PD pathophysiological processes in vulnerable neurons in PD and other age-related neurodegenerative disorders.

Membrane-lipid homeostasis in a prodromal rat model of Alzheimer's disease: Characteristic profiles in ganglioside distributions during aging detected using MALDI imaging mass spectrometry

BackgroundAccumulation of simple gangliosides GM2 and GM3, and gangliosides with longer long-chain bases (d20:1) have been linked to toxicity and the pathogenesis of Alzheimer's disease (AD). Conversely, complex gangliosides, such as GM1, have been shown to be neuroprotective. Recent evidence using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) has demonstrated that a-series gangliosides are differentially altered during normal aging, yet it remains unclear how simple species are shifting relative to complex gangliosides in the prodromal stages of AD. MethodsGanglioside profiles in wild-type (Wt) and transgenic APP21 Fischer rats were detected and quantified using MALDI-IMS at P0 (birth), 3, 12, and 20 months of age and each species quantified to allow for individual species comparisons. ResultsTg APP21 rats were found to have a decreased level of complex gangliosides in a number of brain regions as compared to Wt rats and showed higher levels of simple gangliosides. A unique pattern of expression was observed in the white matter as compared to gray matter regions, with an age-dependent decrease in GD1 d18:1 species observed and significantly elevated levels of GM3 in Tg APP21 rats. ConclusionsThese results are indicative of a pathological shift in ganglioside homeostasis during aging that is exacerbated in Tg APP21 rats. General significanceGanglioside dysregulation may occur in the prodromal stages of neurodegenerative diseases like AD.

Altered Expression of Ganglioside Metabolizing Enzymes Results in GM3 Ganglioside Accumulation in Cerebellar Cells of a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis.

Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene. Most JNCL patients exhibit a 1.02 kb genomic deletion removing exons 7 and 8 of this gene, which results in a truncated CLN3 protein carrying an aberrant C-terminus. A genetically accurate mouse model (Cln3Δex7/8 mice) for this deletion has been generated. Using cerebellar precursor cell lines generated from wildtype and Cln3Δex7/8 mice, we have here analyzed the consequences of the CLN3 deletion on levels of cellular gangliosides, particularly GM3, GM2, GM1a and GD1a. The levels of GM1a and GD1a were found to be significantly reduced by both biochemical and cytochemical methods. However, quantitative high-performance liquid chromatography analysis revealed a highly significant increase in GM3, suggesting a metabolic blockade in the conversion of GM3 to more complex gangliosides. Quantitative real-time PCR analysis revealed a significant reduction in the transcripts of the interconverting enzymes, especially of β-1,4-N-acetyl-galactosaminyl transferase 1 (GM2 synthase), which is the enzyme converting GM3 to GM2. Thus, our data suggest that the complex a-series gangliosides are reduced in Cln3Δex7/8 mouse cerebellar precursor cells due to impaired transcription of the genes responsible for their synthesis.

Isobaric Labeling of Intact Gangliosides toward Multiplexed LC-MS/MS-Based Quantitative Analysis.

Gangliosides are sialic acid-containing glycosphingolipids recognized to play essential role in biological processes. Both the glycan and lipid structures influence their biological function and thus necessitate their determination as intact molecular species. To our knowledge, no multiplexed method for intact gangliosides currently exists. In this paper, we aimed to demonstrate an approach for isobaric labeling of intact gangliosides. Specifically, we carried out the rapid, chemoselective oxidation of sialic acid side chain in common ganglioside core structures using NaIO4 followed by ligation with a carbonyl-reactive isobaric tandem mass tag (TMT) reagent and subsequent RPLC-MS/MS analysis. Attachment of the isobaric label was observed to improve the ionization efficiency of complex gangliosides using electrospray ionization. Fragmentation of the resulting [M + 2H]2+ ions of TMT-labeled gangliosides provided information-rich spectra containing fragments from the glycan, lipid, and TMT reporter ions. This facile approach enabled simultaneous quantification of up to six samples as well as identification of glycan and lipid compositions in a single injection. As a proof-of-concept, using porcine brain total ganglioside extracts pooled at known ratios, we obtained overall sample-to-sample precision of <12% RSD and mean error of <10%. This showcased the great promise and feasibility of this strategy for high-throughput analysis of intact gangliosides in biological extracts.

Biology of GM3 Ganglioside.

Since the successful molecular cloning in 1998 of GM3 synthase (GM3S, ST3GAL5), the enzyme responsible for initiating biosynthesis of all complex gangliosides, the efforts of our research group have been focused on clarifying the physiological and pathological implications of gangliosides, particularly GM3. We have identified isoforms of GM3S proteins having distinctive lengths of N-terminal cytoplasmic tails, and found that these cytoplasmic tails define subcellular localization, stability, and in vivo activity of GM3S isoforms. Our studies of the molecular pathogenesis of type 2 diabetes, focused on interaction between insulin receptor and GM3 in membrane microdomains, led to a novel concept: type 2 diabetes and certain other lifestyle-related diseases are membrane microdomain disorders resulting from aberrant expression of gangliosides. This concept has enhanced our understanding of the pathophysiological roles of GM3 and related gangliosides in various diseases involving chronic inflammation, such as insulin resistance, leptin resistance, and T-cell function and immune disorders (e.g., allergic asthma). We also demonstrated an essential role of GM3 in murine and human auditory systems; a common pathological feature of GM3S deficiency is deafness. This is the first direct link reported between gangliosides and auditory functions.