Ganglioside Pattern Research Articles

Capillary Electrophoresis-Laser Induced Fluorescence Method Development and Validation for Quantification of Nine Gangliosides-Application to Analysis of Cell Lines of CNS Origin.

Gangliosides are sialic acid-containing glycosphingolipids that play an essential role in many biological and pathophysiological processes. They are present in high amounts in the central nervous system and their abnormal metabolism or expression has been observed in many diseases. We have developed and validated a sensitive capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the separation and quantification of oligosaccharides digested from nine gangliosides of high biological relevance. APTS was used for the labeling of the glycans. Reverse polarity CE was performed for the separation of the labeled glycans bearing negative charges. The optimized background electrolyte is a 15 mM lithium acetate buffer with pH of 5 containing 5% w/v linear polyacrylamide, which allows for the separation of all nine gangliosides. Validation parameters including linearity, precision, and accuracy were evaluated. LOQ and LOD were in the nM range, comparable to those of LC-MS techniques. The method was used to identify and quantify the ganglioside pattern of glioblastoma and neuroblastoma cell lines. The presented method is a valuable tool for further investigations aiming at understanding the role of gangliosides in various neurological diseases or CNS tumors.

Gangliosides as Therapeutic Targets for Neurodegenerative Diseases.

Gangliosides, sialic acid-containing glycosphingolipids, are abundant in cell membranes and primarily involved in controlling cell signaling and cell communication. The altered ganglioside pattern has been demonstrated in several neurodegenerative diseases, characterized during early-onset or infancy, emphasizing the significance of gangliosides in the brain. Enzymes required for the biosynthesis of gangliosides are linked to several devastating neurological disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegia (HSP). In this review, we summarized not only the critical roles of biosynthetic enzymes and their inhibitors in ganglioside metabolism but also the efficacy of treatment strategies of ganglioside to address their significance in those diseases.

Advances in Mass Spectrometry of Gangliosides Expressed in Brain Cancers.

Gangliosides are highly abundant in the human brain where they are involved in major biological events. In brain cancers, alterations of ganglioside pattern occur, some of which being correlated with neoplastic transformation, while others with tumor proliferation. Of all techniques, mass spectrometry (MS) has proven to be one of the most effective in gangliosidomics, due to its ability to characterize heterogeneous mixtures and discover species with biomarker value. This review highlights the most significant achievements of MS in the analysis of gangliosides in human brain cancers. The first part presents the latest state of MS development in the discovery of ganglioside markers in primary brain tumors, with a particular emphasis on the ion mobility separation (IMS) MS and its contribution to the elucidation of the gangliosidome associated with aggressive tumors. The second part is focused on MS of gangliosides in brain metastases, highlighting the ability of matrix-assisted laser desorption/ionization (MALDI)-MS, microfluidics-MS and tandem MS to decipher and structurally characterize species involved in the metastatic process. In the end, several conclusions and perspectives are presented, among which the need for development of reliable software and a user-friendly structural database as a search platform in brain tumor diagnostics.

Ganglioside-focused Glycan Array Reveals Abnormal Anti-GD1b Auto-antibody in Plasma of Preclinical Huntington’s Disease

Huntington's disease (HD) is a progressive and devastating neurodegenerative disease marked by inheritable CAG nucleotide expansion. For offspring of HD patients carrying abnormal CAG expansion, biomarkers that predict disease onset are crucially important but still lacking. Alteration of brain ganglioside patterns has been observed in the pathology of patients carrying HD. Here, by using a novel and sensitive ganglioside-focused glycan array, we examined the potential of anti-glycan auto-antibodies for HD. In this study, we collected plasma from 97 participants including 42 control (NC), 16 pre-manifest HD (pre-HD), and 39 HD cases and measured the anti-glycan auto-antibodies by a novel ganglioside-focused glycan array. The association between plasma anti-glycan auto-antibodies and disease progression was analyzed using univariate and multivariate logistic regression. The disease-predictive capacity of anti-glycan auto-antibodies was further investigated by receiver operating characteristic (ROC) analysis. We found that anti-glycan auto-antibodies were generally higher in the pre-HD group when compared to the NC and HD groups. Specifically, anti-GD1b auto-antibody demonstrated the potential for distinguishing between pre-HD and control groups. Moreover, in combination with age and the number of CAG repeat, the level of anti-GD1b antibody showed excellent predictability with an area under the ROC curve (AUC) of 0.95 to discriminate between pre-HD carriers and HD patients. With glycan array technology, this study demonstrated abnormal auto-antibody responses that showed temporal changes from pre-HD to HD.

Decoding Distinct Ganglioside Patterns of Native and Differentiated Mesenchymal Stem Cells by a Novel Glycolipidomics Profiling Strategy.

Gangliosides are an indispensable glycolipid class concentrated on cell surfaces with a critical role in stem cell differentiation. Nonetheless, owing to the lack of suitable methods for scalable analysis covering the full scope of ganglioside molecular diversity, their mechanistic properties in signaling and differentiation remain undiscovered to a large extent. This work introduces a sensitive and comprehensive ganglioside assay based on liquid chromatography, high-resolution mass spectrometry, and multistage fragmentation. Complemented by an open-source data evaluation workflow, we provide automated in-depth lipid species-level and molecular species-level annotation based on decision rule sets for all major ganglioside classes. Compared to conventional state-of-the-art methods, the presented ganglioside assay offers (1) increased sensitivity, (2) superior structural elucidation, and (3) the possibility to detect novel ganglioside species. A major reason for the highly improved sensitivity is the optimized spectral readout based on the unique capability of two parallelizable mass analyzers for multistage fragmentation. We demonstrated the high-throughput universal capability of our novel analytical strategy by identifying 254 ganglioside species. As a proof of concept, 137 unique gangliosides were annotated in native and differentiated human mesenchymal stem cells including 78 potential cell-state-specific markers and 38 previously unreported gangliosides. A general increase of the ganglioside numbers upon differentiation was observed as well as cell-state-specific clustering based on the ganglioside species patterns. The combination of the developed glycolipidomics assay with the extended automated annotation tool enables comprehensive in-depth ganglioside characterization as shown on biological samples of interest. Our results suggest ganglioside patterns as a promising quality control tool for stem cells and their differentiation products. Additionally, we believe that our analytical workflow paves the way for probing glycolipid-based biochemical processes shedding light on the enigmatic processes of gangliosides and glycolipids in general.

Gangliosidome of a Human Hippocampus in Temporal Lobe Epilepsy Resolved by High-Resolution Tandem Mass Spectrometry.

In this study, we developed a high-resolution tandem mass spectrometry (HR MS) approach to assess presumed changes in gangliosidome of a human hippocampus affected by temporal lobe epilepsy (TLE) in comparison with a normal hippocampus. Gangliosides, membrane glycolipids, are particularly diverse and abundant in the human brain, and participate in ion transport and modulation of neuronal excitability. Changes in structural ganglioside pattern potentially linked to TLE molecular pathogenesis have not been explored in detail. Aiming to characterize TLE-specific gangliosidome, we analyzed the native gangliosides purified from a human hippocampal tissue sample affected by TLE and a control hippocampus using HR MS. Marked differences of ganglioside expression were shown in TLE vs. control, particularly with respect to the sialylation degree of components, discovered as a characteristic feature of TLE. Another major finding is the occurrence of tetrasialofucogangliosides in TLE and species modified by either O-acetylation or CH3COO−. Structural analysis by higher-energy collisional dissociation (HCD) MS/MS gave rise to fragmentation patterns implying that the GQ1b (d18:1/18:0) isomer is specifically associated with TLE. Further investigation in a larger sample is needed in order to confirm the discovery of ganglioside structures specifically expressed in human TLE and to provide information on the probable role of gangliosides in the molecular events underlying seizures.

MO635: Early Diabetic Kidney Disease in Type 2 Diabetes Mellitus Patients is Associated with A Particular Ganglioside Profile, Identified by High-Resolution Tandem Mass Spectrometry:A Pilot Study

Abstract BACKGROUND AND AIMS Metabolic biomarkers are of high importance for the identification of early renal involvement in the course of type 2 diabetes mellitus (DM). Gangliosides, which are complex cell type-specific glycosphingolipids, consist of a sialylated oligosaccharide chain attached to a ceramide of different composition with respect to the sphingoid base and fatty acid residues [1]. Gangliosides play a major role in the pathogenesis of diabetic kidney disease (DKD). The aim of the study was to assess the pattern of urinary gangliosides in patients with type 2 DM. METHOD The urine gangliosidome of 30 type 2 DM patients (10 normo-, 10 micro-, and 10 macroalbuminuric) has been investigated in a cross-sectional pilot study, by a comparative assay with 10 healthy controls. Following the optimization of urine ganglioside extraction procedure [2, 3], we have determined the composition of native ganglioside mixtures from the 24-h collected urine samples using a modern bioanalytical platform based on nanoelectrospray ionization (nanoESI) high-resolution mass spectrometry (HR MS) on an Orbitrap instrument. RESULTS HR MS screening and fragmentation analysis by tandem MS revealed that: (i) the urinary gangliosidome of type 2 DM patients contains a significantly higher number of distinct species differing in either their glycan or ceramide structure than the controls; (ii) the ganglioside urinary level was significantly increased in normoalbuminuric patients compared with healthy control subjects; (iii) the gangliosidome of macroalbuminuric patients is characterized by an elevated overall sialic acid content than normo- and microalbuminuric diabetics and more complex structures, including fucosylated, O-GalNAc- and CH3COO--modified compounds; (iv) the degree of sialylation of species presented correlations with the level of albuminuria and renal function; (v) further detailed structural analysis of a type GQ1(d18:1/18:0) species (identified only in macroalbuminuric patients) demonstrated that the present isomer is of type D (all four syalic acids are linked to the inner galactose). CONCLUSION HR MS by tandem MS methods, using an Orbitrap instrument, are a reliable tool for the identification of a particular ganglioside profile in the urine of type 2 DM patients. The sialylated species and those altered by peripheral attachments to the glycan core might be considered useful indicators of early DKD.

High-resolution mass spectrometry reveals a complex ganglioside pattern and novel polysialylated structures associated with the human motor cortex.

We have developed here a superior methodology based on high-resolution mass spectrometry for screening and fragmentation analysis of gangliosides extracted and purified from the human motor cortex . The experiments, conducted on a nanoelectrospray Orbitrap mass spectroscope in the negative ion mode, allowed the discrimination in the native mixture extracted from human motor cortex of no less than 83 different gangliosides, which represents the highest number of structures identified so far in this brain region. The spectral data, acquired in high-resolution mass spectrometry mode with a remarkable sensitivity and an average mass accuracy of 4.48 ppm, also show that the gangliosidome of motor cortex is generally characterized by species exhibiting a much higher degree of sialylation than previously known. Motor cortex was found dominated by complex structures with a sialylation degree ≥3, exhibiting long saccharide chains, in the G1 class. Fucogangliosides and species with the glycan chain elongated by either O-acetylation and/or acetate anion attachments were also detected; the later modification was for the first time discovered in this brain region. Of major significance is the identification of hepta and octasialylated species of GS1 and GO1 type, which are among the structures with the longest oligosaccharide chain discovered so far in the human brain. In the last stage of research, tandem mass spectrometry performed by higher energy collision dissociation provided structural data documenting the occurrence of GT1b (d18:1/20:0) isomer in the human motor cortex.

High resolution mass spectrometry provides novel insights into the ganglioside pattern of brain cavernous hemangioma

In this study we have optimized nanoelectrospray ionization (nanoESI) high resolution mass spectrometry (HR MS) performed on Orbitrap instrument in the negative ion mode for the determination of the composition and structure of gangliosides extracted from human brain cavernous hemangioma.The optimized HR MS platform, allowed the discrimination of 62 ions, corresponding to 52 different ganglioside species, which represents roughly twice the number of species existing in the current inventory of human brain hemangioma-associated gangliosides. The experiments revealed a ganglioside pattern dominated by GD-type of structures as well as an elevated incidence of species characterized by a low degree of sialylation and short glycan chains, including asialo GA1 (d18:1/18:0), which offer a new perspective upon the ganglioside composition in this benign tumor. Many of the structures are characteristic for this type of tumor only and are to be considered in further investigations for their potential use in early brain hemangioma diagnosis based on molecular markers. The detailed fragmentation analysis performed by collision-induced dissociation (CID) tandem MS provided information of structural elements related to the glycan core and ceramide moiety, which confirmed the molecular configuration of GD3 (d18:1/24:1) and GD3 (d18:1/24:2) species with potential biomarker role.

Dynamic changes in patterns of ganglioside expression characterize maturation of lymphoid lineage cells into effector cells and suggest a close relationship between CD8+ T cells and NK cells

Abstract Gangliosides are glycosphingolipids that play important regulatory roles in cell signaling and differentiation. To investigate their expression patterns during lymphoid cell development, human PBMC were stained with recombinant B subunits of LT-IIa, LT-IIb, or LT-IIc and with select mAb. FACS analysis showed that ganglioside ligands for LT-IIaB, LT-IIbB, and LT-IIcB were increasingly expressed during transition from naïve to memory to effector memory CD4+ T cells, with ganglioside ligands for LT-IIbB exhibiting the greatest increase. A similar trend was observed in CD8+ T cell subsets, except no major changes in the expression of LT-IIaB ligands were detected. Patterns of ganglioside expression in NK cell subsets were remarkably similar to those observed in the CD8+ T cells: The ganglioside ligands for LT-IIbB and LT-IIcB were increasingly expressed during transition from tissue-resident to effector to differentiated/exhausted NK cells, with ganglioside ligands for LT-IIbB exhibiting the greatest increase and ligands for LT-IIaB showing little change across subsets. Finally, expression of LT-IIaB- and LT-IIcB-binding gangliosides increased during transition from naïve to memory to antibody-secreting B cells, with ganglioside ligands for LT-IIaB exhibiting the greatest increase and ligands for LT-IIbB showing little change across subsets. Our results demonstrated the existence of dynamic changes in the patterns of gangliosides expressed throughout lymphoid cell development and differentiation. The distinct patterns suggest the existence of cell signaling differences between and within the distinct lymphoid cell lineages and a close developmental and functional relationship between CD8+ T cells and NK cells.

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.

Physiology of gangliosides and the role of antiganglioside antibodies in human diseases.

Gangliosides are structurally and functionally polymorphic sialic acid containing glycosphingolipids that are widely distributed in the human body. They play important roles in protecting us against immune attacks, yet they can become targets for autoimmunity and act as receptors for microbes, like the influenza viruses, and toxins, such as the cholera toxin. The expression patterns of gangliosides vary in different tissues, during different life periods, as well as in different animals. Antibodies against gangliosides (AGA) can target immune attack e.g., against neuronal cells and neutralize their complement inhibitory activity. AGAs are important especially in acquired demyelinating immune-mediated neuropathies, like Guillain–Barré syndrome (GBS) and its variant, the Miller–Fisher syndrome (MFS). They can emerge in response to different microbial agents and immunological insults. Thereby, they can be involved in a variety of diseases. In addition, antibodies against GM3 were found in the sera of patients vaccinated with Pandemrix®, who developed secondary narcolepsy, strongly supporting the autoimmune etiology of the disease.

Orbitrap mass spectrometry for monitoring the ganglioside pattern in human cerebellum development and aging.

We have developed here a superior approach based on high-resolution (HR) mass spectrometry (MS) for monitoring the changes occurring with development and aging in the composition and structure of cerebellar gangliosidome. The experiments were focused on the comparative screening and structural analysis of gangliosides expressed in fetal and aged cerebellum by Orbitrap MS with nanoelectrospray ionization (nanoESI) in the negative ion mode. The employed ultrahigh-resolution MS platform allowed the discrimination, without the need of previous separation, of 159 ions corresponding to 120 distinct species in the native ganglioside mixtures from fetal and aged cerebellar biopsies, many more than detected before, when MS platforms of lower resolution were employed. A number of gangliosides, in particular polysialylated belonging to GT, GQ, GP, and GS classes, modified by O-fucosylation, O-acetylation, or CH3 COO- were discovered here, for the first time in human cerebellum. These components, found differently expressed in fetal and aged tissues, indicated that the ganglioside profile in cerebellum is development stage- and age-specific. Following the fragmentation analysis by high-energy collision-induced dissociation (HCD) tandem MS (MS/MS), we have also observed that the intimate structure of certain compounds has not changed during the development and aging of the brain, an aspect which could open new directions in the investigation of ganglioside biomarkers in cerebellar tissue.

B4GALNT1 induces angiogenesis, anchorage independence growth and motility, and promotes tumorigenesis in melanoma by induction of ganglioside GM2/GD2

β-1,4-N-Acetyl-Galactosaminyltransferase 1 (B4GALNT1) encodes the key enzyme B4GALNT1 to generate gangliosides GM2/GD2. GM2/GD2 gangliosides are surface glycolipids mainly found on brain neurons as well as peripheral nerves and skin melanocytes and are reported to exacerbate the malignant potential of melanomas. In order to elucidate the mechanism, we performed functional analyses of B4GALNT1-overexpressing cells. We analyzed ganglioside pattern on four melanoma and two neuroblastoma cell lines by high performance liquid chromatography (HPLC). We overexpressed B4GALNT1 in GM2/GD2-negative human melanoma cell line (SH4) and confirmed production of GM2/GD2 by HPLC. They showed higher anchorage independence growth (AIG) in colony formation assay, and exhibited augmented motility. In vitro, cell proliferation was not affected by GM2/GD2 expression. In vivo, GM2/GD2-positive SH4 clones showed significantly higher tumorigenesis in NOD/Scid/IL2Rγ-null mice, and immunostaining of mouse CD31 revealed that GM2/GD2 induced remarkable angiogenesis. No differences were seen in melanoma stem cell and Epithelial-Mesenchymal Transition markers between GM2/GD2-positive and -negative SH4 cells. We therefore concluded that B4GALNT1, and consequently GM2/GD2, enhanced tumorigenesis via induction of angiogenesis, AIG, and cell motility. RNA-Seq suggested periostin as a potential key factor for angiogenesis and AIG. These findings may lead to development of novel therapy for refractory melanoma.

Cerebrospinal fluid: Profiling and fragmentation of gangliosides by ion mobility mass spectrometry

The proximity of cerebrospinal fluid (CSF) with the brain, its permanent renewal and better availability for research than tissue biopsies, as well as ganglioside (GG) shedding from brain to CSF, impelled lately the development of protocols for the characterization of these glycoconjugates and discovery of central nervous system biomarkers expressed in CSF. Currently, the investigation of CSF gangliosides is focused on concentration measurements of the predominant classes and much less on their profiling and structural analysis.Since we have demonstrated recently the high performance of ion mobility separation mass spectrometry (IMS MS) for compositional and structural determination of human brain GGs, in the present study we have implemented for the first time IMS MS for the exploration of human CSF gangliosidome, in order to generate the first robust mass spectral database of CSF gangliosides. IMS MS separation and screening revealed 113 distinct GG species in CSF, having similar compositions to the species detected in human brain. In comparison with the brain tissue, we have discovered in CSF several components containing fatty acids with odd number of carbon atoms and/or short glycan chains. By tandem MS (MS/MS) we have further analyzed the structure of GD3(d18:1/18:0) and GD2(d18:1/18:0), two glycoforms exhibiting short carbohydrate chains found in CSF, but discovered and characterized previously in brain as well. According to the present results, human CSF and brain show a similar ganglioside pattern, a finding that might be useful in clinical research focused on discovery of ganglioside species associated to neurodegenerative diseases and brain tumors.

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.

Gangliosides profiling in serum of breast cancer patient: GM3 as a potential diagnostic biomarker.

Gangliosides altered during the pathological conditions and particularly in cancers. Here, we aimed to profile the gangliosides in breast cancer serum and propose potential biomarkers. LC-FTMS method was first used to identify all the ganglioside species in serum, then LC-MS/MS-MRM method was employed to quantitate the levels of gangliosides in serum from healthy volunteers and patients with benign breast tumor or breast cancer. 49 ganglioside species were determined, including GM1, GM2, GM3, GD1, GD3 and GT1 species. Compared to healthy volunteers, the levels of GM1, GM2, GM3, GD1 and GD3 displayed a rising trend in breast cancer patients. In particular, as the major glycosphingolipid component, GM3 showed excellent diagnostic accuracy in cancer serum (AUC > 0.9). PCA profile of the GM3 species showed clear distinction between normal and cancer serum. What's more, ROC curve proved great diagnostic accuracy of GM3 between cancer and benign serum. In addition, GM3 was discovered as a diagnostic marker to differentiate luminal B subtype from other subtypes. Furthermore, a positive correlation between GM3 and Ki-67 status of patients was identified. In conclusion, our results introduced the alteration patterns of serum gangliosides in breast cancer and suggested serum GM3 as a potential diagnostic biomarker in breast cancer diagnosis and luminal B subtype distinction.

Gangliosides in Membrane Organization.

Since the structure of GM1 was elucidated 55years ago, researchers have been attracted by the sialylated glycans of gangliosides. Gangliosides head groups, protruding toward the extracellular space, significantly contribute to the cell glycocalyx; and in certain cells, such as neurons, are major determinants of the features of the cell surface. Expression of glycosyltransferases involved in the de novo biosynthesis of gangliosides is tightly regulated along cell differentiation and activation, and is regarded as the main metabolic mechanism responsible for the acquisition of cell-specific ganglioside patterns. The resulting sialooligosaccharides are characterized by a high degree of geometrical complexity and by highly dynamic properties, which seem to be functional for complex interactions with other molecules sitting on the same cellular membrane (cis-interactions) or soluble molecules present in the extracellular environment, or molecules associated with the surface of other cells (trans-interactions). There is no doubt that the multifaceted biological functions of gangliosides are largely dependent on oligosaccharide-mediated molecular interactions. However, gangliosides are amphipathic membrane lipids, and their chemicophysical, aggregational, and, consequently, biological properties are dictated by the properties of the monomers as a whole, which are not merely dependent on the structures of their polar head groups. In this chapter, we would like to focus on the peculiar chemicophysical features of gangliosides (in particular, those of the nervous system), that represent an important driving force determining the organization and properties of cellular membranes, and to emphasize the causal connections between altered ganglioside-dependent membrane organization and relevant pathological conditions.

Electrospray ionization ion mobility mass spectrometry provides novel insights into the pattern and activity of fetal hippocampus gangliosides

Gangliosides (GGs), a particular class of glycosphingolipids ubiquitously found in tissues and body fluids, exhibit the highest expression in the central nervous system, especially in brain. GGs are involved in crucial processes, such as neurogenesis, synaptogenesis, synaptic transmission, cell adhesion, growth and proliferation. For these reasons, efforts are constantly invested into development and refinement of specific methods for GG analysis. We have recently shown that ion mobility separation (IMS) mass spectrometry (MS) has the capability to provide consistent compositional and structural information on GGs at high sensitivity, resolution and mass accuracy. In the present paper, we have implemented IMS MS for the first time in the study of a highly complex native GG mixture extracted and purified from human fetal hippocampus. As compared to previous studies, where no separation techniques prior to MS were applied, IMS MS technique has not just generated valuable novel information on the GG pattern characteristic for hippocampus in early developmental stage, but also provided data related to the GG molecular involvement in the synaptic functions by the discovery of 25 novel structures modified by CH3COO−. The detection and identification in fetal hippocampus of a much larger number of GG species than ever reported before was possible due to the ion mobility separation according to the charge state, the carbohydrate chain length and the degree of sialylation. By applying IMS in conjunction with collision induced dissociation (CID) tandem MS (MS/MS), novel GG species modified by CH3COO− attachment, discovered here for the first time, were sequenced and structurally investigated in details. The present findings, based on IMS MS, provide a more reliable insight into the expression and role of gangliosides in human hippocampus, with a particular emphasis on their cholinergic activity at this level.

Ganglioside Profiling of the Human Retina: Comparison with Other Ocular Structures, Brain and Plasma Reveals Tissue Specificities.

Gangliosides make a wide family of glycosphingolipids, highly heterogeneous in both the ceramide moiety and the oligosaccharide chain. While ubiquitously expressed in mammalian tissues, they are particularly abundant in the brain and the peripheral nervous system. Gangliosides are known to play a crucial role in the development, maintenance and functional integrity of the nervous system. However, the expression and roles of gangliosides in the retina, although often considered as a window on the brain, has been far less studied. We performed an in-depth analysis of gangliosides of the human retina, especially using powerful LC/MS methods. We compared the pattern of ganglioside classes and ceramide molecular species of this tissue with other ocular structures and with brain and plasma in elderly human individuals. About a hundred of ganglioside molecular species among 15 distinct classes were detected illustrating the huge structural diversity of these compounds. The retina exhibited a very diverse ganglioside profile and shared several common features with the brain (prominence of tetraosylgangliosides, abundance of d20:1 long chain base and 18:0 fatty acid…). However, the retina stood out with the specific expression of GD3, GT3 and AcGT3, which further presented a peculiar molecular species distribution. The unique ganglioside pattern we observed in the human retina suggests that these ganglioside species play a specific role in the structure and function of this tissue. This lipidomic study, by highlighting retina specific ganglioside species, opens up novel research directions for a better understanding of the biological role of gangliosides in the retina.