Glycosphingolipids Research Articles

Chemoenzymatic Synthesis of Globo-series Glycosphingolipids and Evaluation of Their Immunosuppressive Activities.

Glycosphingolipids (GSLs) play essential roles in many important biological processes, making them attractive synthetic targets. In this paper, a viable chemoenzymatic method is described for the synthesis of globo-series GSLs, namely, Gb4, Gb5, SSEA-4, and Globo H. The strategy uses a chemically synthesized lactoside acceptor equipped with a partial ceramide structure that is uniquely extended by glycosyltransferases in a highly efficient one-pot multiple enzyme (OPME) procedure. A direct and quantitative conversion of Gb4 sphingosine to Globo H sphingosine is achieved by performing two-sequential OPME glycosylations. A reduction and N-acylation protocol allows facile incorporation of various fatty acids into the lipid portions of the GSLs. The chemically well-defined lipid-modified Globo H-GSLs displayed some differences in their immunosuppressive activities, which may benefit the structural modifications of Globo H ceramides in finding new types of immunosuppressive agents. The strategy outlined in this work should be applicable to the rapid access to other complex GSLs.

Enterohemorrhagic Escherichia coli and a Fresh View on Shiga Toxin-Binding Glycosphingolipids of Primary Human Kidney and Colon Epithelial Cells and Their Toxin Susceptibility.

Enterohemorrhagic Escherichia coli (EHEC) are the human pathogenic subset of Shiga toxin (Stx)-producing E. coli (STEC). EHEC are responsible for severe colon infections associated with life-threatening extraintestinal complications such as the hemolytic-uremic syndrome (HUS) and neurological disturbances. Endothelial cells in various human organs are renowned targets of Stx, whereas the role of epithelial cells of colon and kidneys in the infection process has been and is still a matter of debate. This review shortly addresses the clinical impact of EHEC infections, novel aspects of vesicular package of Stx in the intestine and the blood stream as well as Stx-mediated extraintestinal complications and therapeutic options. Here follows a compilation of the Stx-binding glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) and their various lipoforms present in primary human kidney and colon epithelial cells and their distribution in lipid raft-analog membrane preparations. The last issues are the high and extremely low susceptibility of primary renal and colonic epithelial cells, respectively, suggesting a large resilience of the intestinal epithelium against the human-pathogenic Stx1a- and Stx2a-subtypes due to the low content of the high-affinity Stx-receptor Gb3Cer in colon epithelial cells. The review closes with a brief outlook on future challenges of Stx research.

Functions and applications of glycolipid-hydrolyzing microbial glycosidases.

Glycolipids are important components of cell membranes in several organisms. The major glycolipids in mammals are glycosphingolipids (GSLs), which are composed of ceramides. In mammals, GSLs are degraded stepwise from the non-reducing end of the oligosaccharides via exo-type glycosidases. However, endoglycoceramidase (EGCase), an endo-type glycosidase found in actinomycetes, is a unique enzyme that directly acts on the glycosidic linkage between oligosaccharides and ceramides to generate intact oligosaccharides and ceramides. Three molecular species of EGCase, namely EGCase I, EGCase II, and endogalactosylceramidase, have been identified based on their substrate specificity. EGCrP1 and EGCrP2, which are homologs of EGCase in pathogenic fungi, were identified as the first fungal glucosylceramide- and sterylglucoside-hydrolyzing glycosidases, respectively. These enzymes are promising targets for antifungal drugs against pathogenic fungi. This review describes the functions and properties of these microbial glycolipid-degrading enzymes, the molecular basis of their differential substrate specificity, and their applications.

DIPG-43. Glucosylceramide synthase inhibitors induce ceramide accumulation and sensitise H3K27 Mutant Diffuse midline Glioma to irradiation

Abstract BACKGROUND AND AIMS: Glycosphingolipids (GSL) are amphipathic lipids particularly abundant in the brain where their amount and expression patterns change drastically during the embryonic to postnatal stages and during tumorigenesis. The biosynthesis of GSL begins with the formation of glucosylceramide from ceramide, a step catalysed by the glucosylceramide synthase (UGCG). UGCG can be inhibited by eliglustat, which is used for treating children with Gaucher‘s disease. We have previously shown that the GSL composition is deregulated in H3K27M mutant diffuse midline glioma (H3K27M mut) and that eliglustat inhibits cell proliferation. Here we analysed the mechanism of action of eliglustat in H3K27M mut and its effect on irradiation. METHODS: The concentration of different components of the sphingolipid metabolism (ceramide, ceramide-1-Phosphate (CIP), sphingomyelin, Sphingosine and Sphingosine-1-Phosphate (S1P)) was assessed by mass spectrometry in the H3K27M mut cell line SF8628, before and after treatment with eliglustat. The combination of eliglustat with ionizing radiation was analysed by clonogenic assay. RESULTS: The treatment of H3K27M mut cells with eliglustat resulted in a significant increase in the concentration of ceramide, Sphingosine, C1P, but not S1P. The increase was concentration and time dependent and was not observed after longer incubation. Eliglustat treatment reduced the colony formation ability after irradiation. CONCLUSIONS: Ceramide is a known mediator of apoptosis involved in the molecular mechanisms underlying cellular response to irradiation. Increased endogenous ceramide levels, induced by blocking the synthesis of GSL, may sensitize H3K27M mut cells to irradiation. However, ceramide can be converted in C1P, a potent inhibitor of apoptosis and inducer of cell survival. Thus, the time and concentration dependent shift to ceramide and C1P requires further investigation in order to achieve an appropriate balance between the levels of these two metabolites and identify the optimal therapeutic window for combination with irradiation and potentially chemotherapy

The Role of Adipocyte‐derived Extracellular Vesicles in Diabetes‐associated Endothelial Dysfunction

The development of diabetes (T2D) is strongly associated with obesity, and both are well‐established risk factors for cardiovascular disease. Endothelial dysfunction is an early event in developing vascular complications in obese diabetic (OB‐T2D) adults. Therefore, our long‐term goal is to identify endothelial dysfunction mechanisms in this population, mainly those related to the multifaceted crosstalk between dysfunctional adipocytes and endothelial cells. Our recent findings revealed that ex vivocultured human adipose tissues (AT) release extracellular vesicles (EVs) that are efficiently captured by endothelial cells. These EVs are lipid‐enclosed structures that carry bioactive cargos and are considered vital mediators of intercellular communication. Adipocyte‐derived EVs (adiposomes) mediate signaling between cell types within the AT and circulate in the blood to communicate signals to remote cells and tissues. The purpose of this study is to investigate the role of adiposomes in communicating the unhealthy milieu of dysfunctional AT to endothelial cells in OB‐T2D. Altered lipid metabolism and enhanced ceramide synthesis in OB‐T2D induce adiposome production. Under hyperglycemia, ceramides could be glycosylated into glycosphingolipids (GSLs), selectively packed in adiposomes to be cleared away from adipocytes. We hypothesize that the AT from OB‐T2D patients creates excess GSL‐rich adiposomes, which are incorporated into endothelial cells causing alterations in endothelial cell properties and function. To test this hypothesis, AT samples were collected from OB‐T2D and non‐OB subjects (n=10, each). Nanoparticle analysis and confocal microscopy were used to analyze these adiposomes, and mass spectrometry and Western blotting techniques were used to examine their content. Adiposome uptake, lipid fusion, and changes in endothelial membrane structure and signaling pathways were also assessed following adiposome incubation with microvascular endothelial cells. Our data showed that in comparison to non‐obese controls, adiposomes isolated from OB‐T2D adipose tissues contained higher levels of GSLs. When added to cultured endothelial cells, GSL‐rich adiposomes tended to accumulate in an endothelial structure known as caveolae. These caveolae cover more than 50% of the endothelial cell surface and serve to concentrate signal transduction molecules such as endothelial nitric oxide synthase (eNOS) and others involved in angiogenesis, transcytosis, and permeability. According to our findings, the phosphorylation of the key caveolar protein caveolin‐1 by GSL‐rich adiposomes caused caveolar fission from the endothelial cell surface. This effect was mediated via an activation of Src kinase, as it was inhibited by the Src kinase inhibitor, PP2. These molecular events were accompanied by reductions in endothelial nitric oxide synthase (eNOS) activation and nitric oxide production, disturbances in endothelial gap junction, and increased permeability. Accordingly, we conclude that dysfunctional AT in OB‐T2D patients produce GSL‐loaded adiposomes that fuse with endothelial cells and activate the Src kinase pathway resulting in caveolar fission and endothelial cell dysfunction.

High Diversity of Glycosphingolipid Glycans of Colorectal Cancer Cell Lines Reflects the Cellular Differentiation Phenotype.

Colorectal cancer (CRC)–associated changes of protein glycosylation have been widely studied. In contrast, the expression of glycosphingolipid (GSL) patterns in CRC has, hitherto, remained largely unexplored. Even though GSLs are major carriers of cell surface carbohydrates, they are understudied due to their complexity and analytical challenges. In this study, we provide an in-depth analysis of GSL glycans of 22 CRC cell lines using porous graphitized carbon nano–liquid chromatography coupled with electrospray ionization–mass spectrometry. Our data revealed that the GSL expression varies among different cell line classifications, with undifferentiated cell lines showing high expression of blood group A, B, and H antigens while for colon-like cell lines the most prominent GSL glycans contained (sialyl)-LewisA/X and LewisB/Y antigens. Moreover, the GSL expression correlated with relevant glycosyltransferases that are involved in their biosynthesis as well as with transcription factors (TFs) implicated in colon differentiation. Additionally, correlations between certain glycosyltransferases and TFs at mRNA expression level were found, such as FUT3, which correlated with CDX1, ETS2, HNF1A, HNF4A, MECOM, and MYB. These TFs are upregulated in colon-like cell lines pointing to their potential role in regulating fucosylation during colon differentiation. In conclusion, our study reveals novel layers of potential GSL glycans regulation relevant for future research in colon differentiation and CRC.

Vesicular Ganglioside GM1 From Breast Tumor Cells Stimulated Epithelial-to-Mesenchymal Transition of Recipient MCF-10A Cells.

Small extracellular vesicles (sEVs) are a type of membrane structure secreted by cells, which are involved in physiological and pathological processes by participating in intercellular communication. Glycosphingolipids (GSLs) are enriched in sEV and can be delivered to recipient cells. In this study, we found that overexpression of B3GALT4, the glycosyltransferase responsible for ganglioside GM1 synthesis, can induce the epithelial–mesenchymal transition (EMT) process in MCF-10A cells. Moreover, GM1 was verified to be presented on sEV from breast cancer cells. Overexpression of B3GALT4 resulted in elevated vesicular GM1 levels and increased sEV secretion in breast cancer cells. Proteomic analysis revealed that eleven sEV secretion-related proteins were differentially expressed, which might contribute to the altered sEV secretion. Of the identified proteins, 15 oncogenic differentially expressed proteins were documented to be presented in sEV. With the treatment of GM1-enriched sEV from breast cancer cells, the EMT process was induced in recipient non-tumorigenic epithelial MCF-10A cells. Our findings demonstrated that GM1-enriched sEVs derived from breast cancer cells induced the EMT process of recipient cells, which might provide essential information on the biological function of vesicular GM1.

Update on Glycosphingolipids Abundance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer. Low numbers of HCC patients being suitable for liver resection or transplantation and multidrug resistance development during pharmacotherapy leads to high death rates for HCC patients. Understanding the molecular mechanisms of HCC etiology may contribute to the development of novel therapeutic strategies for prevention and treatment of HCC. UDP-glucose ceramide glycosyltransferase (UGCG), a key enzyme in glycosphingolipid metabolism, generates glucosylceramide (GlcCer), which is the precursor for all glycosphingolipids (GSLs). Since UGCG gene expression is altered in 0.8% of HCC tumors, GSLs may play a role in cellular processes in liver cancer cells. Here, we discuss the current literature about GSLs and their abundance in normal liver cells, Gaucher disease and HCC. Furthermore, we review the involvement of UGCG/GlcCer in multidrug resistance development, globosides as a potential prognostic marker for HCC, gangliosides as a potential liver cancer stem cell marker, and the role of sulfatides in tumor metastasis. Only a limited number of molecular mechanisms executed by GSLs in HCC are known, which we summarize here briefly. Overall, the role GSLs play in HCC progression and their ability to serve as biomarkers or prognostic indicators for HCC, requires further investigation.

Glycosphingolipids in human embryonic stem cells and breast cancer stem cells, and potential cancer therapy strategies based on their structures and functions.

Expression profiles of glycosphingolipids (GSLs) in human embryonic stem cell (hESC) lines and their differentiated embryoid body (EB) outgrowth cells, consisting of three germ layers, were surveyed systematically. Several globo- and lacto-series GSLs were identified in undifferentiated hESCs and during differentiation of hESCs to EB outgrowth cells, and core structure switching of these GSLs to gangliosides was observed. Such switching was attributable to altered expression of key glycosyltransferases (GTs) in GSL biosynthetic pathways, reflecting the unique stage-specific transitions and mechanisms characteristic of the differentiation process. Lineage-specific differentiation of hESCs was associated with further GSL alterations. During differentiation of undifferentiated hESCs to neural progenitor cells, core structure switching from globo- and lacto-series to primarily gangliosides (particularly GD3) was again observed. During differentiation to endodermal cells, alterations of GSL profiles were distinct from those in differentiation to EB outgrowth or neural progenitor cells, with high expression of Gb4Cer and low expression of stage-specific embryonic antigen (SSEA)-3, -4, or GD3 in endodermal cells. Again, such profile changes resulted from alterations of key GTs in GSL biosynthetic pathways. Novel glycan structures identified on hESCs and their differentiated counterparts presumably play functional roles in hESCs and related cancer or cancer stem cells, and will be useful as surface biomarkers. We also examined GSL expression profiles in breast cancer stem cells (CSCs), using a model of epithelial-mesenchymal transition (EMT)-induced human breast CSCs. We found that GD2 and GD3, together with their common upstream GTs, GD3 synthase (GD3S) and GD2/GM2 synthase, maintained stem cell phenotype in breast CSCs. Subsequent studies showed that GD3 was associated with epidermal growth factor receptor (EGFR), and activated EGFR signaling in breast CSCs and breast cancer cell lines. GD3S knockdown enhanced cytotoxicity of gefitinib (an EGFR kinase inhibitor) in resistant MDA-MB468 cells, both in vitro and in vivo. Our findings indicate that GD3S contributes to gefitinib resistance in EGFR-positive breast cancer cells, and is a potentially useful therapeutic target in drug-resistant breast cancers.

Structure and function of glycosphingolipids on small extracellular vesicles.

Extracellular vesicles (EVs) are membrane-delineated particles secreted by most types of cells under both normal and pathophysiological conditions. EVs are believed to mediate intercellular communication by serving as carriers of different bioactive ingredients, including proteins, nucleic acids and lipids. Glycoconjugates are complex molecules consisting of covalently linked carbohydrate with proteins or lipids. These glycoconjugates play essential roles in the sorting of vesicular protein and the uptake of small extracellular vesicles (30–100 nm, sEVs) into recipient cells. Glycosphingolipids (GSLs), one subtype of glycolipids, which are ubiquitous membrane components in almost all living organisms, are also commonly distributed on sEVs. However, the study of functional roles of GSLs on sEVs are far behind than other functional cargos. The purpose of this review is to highlight the importance of GSLs on sEVs. Initially, we described classification and structure of GSLs. Then, we briefly introduced the essential functions of GSLs, which are able to interact with functional membrane proteins, such as growth factor receptors, integrins and tetraspanins, to modulate cell growth, adhesion and cell motility. In addition, we discussed analytical methods for studying GSLs on sEVs. Finally, we focused on the function of GSLs on sEVs, including regulating the aggregation of extracellular α-synuclein (α-syn) or extracellular amyloid-β (Aβ) and influencing tumor cell malignancy.

Role of Globotriaosylceramide in Physiology and Pathology

At first glance, the biological function of globoside (Gb) clusters appears to be that of glycosphingolipid (GSL) receptors for bacterial toxins that mediate host-pathogen interaction. Indeed, certain bacterial toxin families have been evolutionarily arranged so that they can enter eukaryotic cells through GSL receptors. A closer look reveals this molecular arrangement allocated on a variety of eukaryotic cell membranes, with its role revolving around physiological regulation and pathological processes. What makes Gb such a ubiquitous functional arrangement? Perhaps its peculiarity is underpinned by the molecular structure itself, the nature of Gb-bound ligands, or the intracellular trafficking unleashed by those ligands. Moreover, Gb biological conspicuousness may not lie on intrinsic properties or on its enzymatic synthesis/degradation pathways. The present review traverses these biological aspects, focusing mainly on globotriaosylceramide (Gb3), a GSL molecule present in cell membranes of distinct cell types, and proposes a wrap-up discussion with a phylogenetic view and the physiological and pathological functional alternatives.

Glycosphingolipid-Glycan Signatures of Acute Myeloid Leukemia Cell Lines Reflect Hematopoietic Differentiation.

Aberrant expression of certain glycosphingolipids (GSLs) is associated with the differentiation of acute myeloid leukemia (AML) cells. However, the expression patterns of GSLs in AML are still poorly explored because of their complexity, the presence of multiple isomeric structures, and tedious analytical procedures. In this study, we performed an in-depth GSL glycan analysis of 19 AML cell lines using porous graphitized carbon liquid chromatography-mass spectrometry revealing strikingly different GSL glycan profiles between the various AML cell lines. The cell lines of the M6 subtype showed a high expression of gangliosides with α2,3-sialylation and Neu5Gc, while the M2 and M5 subtypes were characterized by high expression of (neo)lacto-series glycans and Lewis A/X antigens. Integrated analysis of glycomics and available transcriptomics data revealed the association of GSL glycan abundances with the transcriptomics expression of certain glycosyltransferases (GTs) and transcription factors (TFs). In addition, correlations were found between specific GTs and TFs. Our data reveal TFs GATA2, GATA1, and RUNX1 as candidate inducers of the expression of gangliosides and sialylation via regulation of the GTs ST3GAL2 and ST8SIA1. In conclusion, we show that GSL glycan expression levels are associated with hematopoietic AML classifications and TF and GT gene expression. Further research is needed to dissect the regulation of GSL expression and its role in hematopoiesis and associated malignancies.

Gangliosides in T cell development and function of mice.

The molecular diversity of glycosphingolipids (GSLs) that arouse during thecourseof evolution clearly plays an essential role in maintenance of biological homeostasis. Why is such a wide variety of GSLs necessary, and what gave rise to the expression mechanisms that are selective and specific to individual cells, tissues, or organs? What is the biological significance of these mechanisms? The same questions apply to GSLs involved in T cell development and activation.Primary CD4+ T cells and CD8+ T cells preferentially express differing ganglioside series: a-series and o-series, respectively. Conversely, a-series and o-series ganglioside deficiency results respectively in CD4+ and CD8+ T cell dysfunction. Dynamic changes in ganglioside expression occur during T cell development in thymus. Ganglioside GM3 synthase deficiency, which results in lack of a-series gangliosides, ameliorated CD4+ T cell-mediated airway hypersensitivity in a mouse model of allergic asthma. In this review, we summarize findings from these and many studies to illustrate the key roles of gangliosides in T cell differentiation and function.

Analysis and Comparison of Mouse and Human Brain Gangliosides via Two-Stage Matching of MS/MS Spectra.

Glycosphingolipids (GSLs), including gangliosides, are essential components of the cell membrane. Because of their vital biological functions, a facile method for the analysis and comparison of GSLs in biological issues is desired. To this end, a new method for GSL analysis was developed based on two-stage matching of the carbohydrate and glycolipid product ions of experimental and reference MS/MS spectra of GSLs. The applicability of this method to the analysis of gangliosides in biological tissues was verified using human plasma and mouse brains spiked with standards. The method was then used to characterize endogenous gangliosides in mouse and human brains. It was shown that each endogenous ganglioside species had varied lipid forms and that mouse and human brains had different compositions of ganglioside species and lipid forms. Moreover, a 36-carbon ceramide is found to represent the major lipid form for mouse brain gangliosides, while the major lipid form for most human brain gangliosides is a 38-carbon ceramide. This study has verified that the two-stage MS/MS spectral matching method could be used to study gangliosides or GSLs and their lipid forms in complex biological samples, thereby having a broad application.

Glucosylceramide in T cells regulates the pathology of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disease in the colon characterized by excessive activation of T cells. Glycosphingolipids (GSLs) are composed of lipid rafts in cellular membranes, and their content is linked to immune cell function. In the present study, we investigated the involvement of GSLs in IBD. Microarray data showed that in IBD patients, the expression of only UDP-glucose ceramide glucosyltransferase (UGCG) decreased among the GSLs synthases. Ad libitum access to dextran sulfate sodium (DSS) resulted in decreased UGCG and glucosylceramide (GlcCer) content in mesenteric lymph nodes and T cells from the spleen. Furthermore, the knockdown of Ugcg in T cells exacerbated the pathogenesis of colitis, which was accompanied by a decrease in Treg levels. Treatment with GlcCer nanoparticles prevented DSS-induced colitis. These results suggested that GlcCer in T cells is involved in the pathogenesis of IBD. Furthermore, GlcCer nanoparticles are a potential efficacious therapeutic target for IBD patients.

Glycosphingolipid Levels in Urine Extracellular Vesicles Enhance Prediction of Therapeutic Response in Lupus Nephritis.

The development of nephritis increases the risk of morbidity and mortality in systemic lupus erythematosus (SLE) patients. While standard induction therapies, such as mycophenolate mofetil (MMF) induce clinical remission (i.e., complete response) in approximately 50% of SLE patients with nephritis, many patients fail to respond. Therapeutic response is often not assessed until 6–12 months after beginning treatment. Those patients that fail to respond to treatment continue to accumulate organ damage, thus, there is a critical need to predict which patients will fail therapy before beginning treatment, allowing physicians to optimize therapy. Our previous studies demonstrated elevated urine, but not serum, glycosphingolipids (GSLs) in SLE patients with nephritis compared to SLE patients without nephritis, suggesting the urine GSLs were derived from the kidney. In this study, we measured the GSLs hexosylceramide and lactosylceramide in extracellular vesicles isolated from longitudinal urine samples of LN patients that were treated with MMF for 12 months. GSL levels were significantly elevated in the baseline samples (prior to treatment) of non-responders compared to complete responders. While a few other proteins measured in the whole urine were higher in non-responders at baseline, only GSLs demonstrated a significant ability to discriminate treatment response in lupus nephritis patients.

Oligosaccharide Presentation Modulates the Molecular Recognition of Glycolipids by Galectins on Membrane Surfaces.

Galectins are a family of glycan binding proteins that stand out for the wide range of biological phenomena in which they are involved. Most galectin functions are associated with their glycan binding capacities, which are generally well characterized at the oligosaccharide level, but not at the glycoprotein or glycolipid level. Glycolipids form the part of cell membranes where they can act as galectin cellular receptors. In this scenario, glycan presentation as well as the membrane chemical and structural features are expected to have a strong impact in these molecular association processes. Herein, liposomes were used as membrane mimicking scaffolds for the presentation of glycosphingolipids (GSLs) and to investigate their interaction with Galectin-3 and the N-domain of Galectin-8 (Gal8N). The binding towards GM3 and GM1 and their non-silaylated GSLs was compared to the binding to the free glycans, devoid of lipid. The analysis was carried out using a combination of NMR methods, membrane perturbation studies, and molecular modeling. Our results showed a different tendency of the two galectins in their binding capacities towards the glycans, depending on whether they were free oligosaccharides or as part of GSL inserted into a lipid bilayer, highlighting the significance of GSL glycan presentation on membranes in lectin binding.

Structural characterization and analysis of different epimers of neutral glycosphingolipid LcGg4 by ion mobility spectrometry-mass spectrometry.

LcGg4, a neutral glycosphingolipid (GSL) and cancer antigen, its epimers GalNAc-LcGg4 and GlcNAc-LcGg4, and three lipid forms of GalNAc-LcGg4 were studied by mass spectrometry (MS). It was found that different forms of GalNAc-LcGg4 carrying homologous (d16:1/18:0) and (d18:1/18:0) lipids were easily separated and identified using liquid chromatography (LC)-MS. In addition, like gangliosides, homologous lipid forms of GalNAc-LcGg4 showed the same fragmentation pattern, except for a uniform shift of their glycolipid product ions by a certain m/z number determined by the varied lipid structure. It was also disclosed that LcGg4 and its epimers GalNAc-LcGg4 and GlcNAc-LcGg4, which are different only in the C4-configuration of their non-reducing end sugar residues, gave the same MS/MS product ions in similar relative intensities, as well as the same LC retention time, suggesting the challenge to differentiate epimeric GSLs by LC-MS. However, ion mobility spectrometry (IMS)-MS was able to efficiently separate and distinguish these epimers. This study has demonstrated the promise of IMS-MS for isomeric GSL characterization and the IMS-MS and LC-MS/MS combination for natural GSL analysis.

Pathological α-syn aggregation is mediated by glycosphingolipid chain length and the physiological state of α-syn in vivo

GBA1 mutations that encode lysosomal β-glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher disease (GD) and are strong risk factors for synucleinopathies, including Parkinson's disease and Lewy body dementia. Only a subset of subjects with GBA1 mutations exhibit neurodegeneration, and the factors that influence neurological phenotypes are unknown. We find that α-synuclein (α-syn) neuropathology induced by GCase depletion depends on neuronal maturity, the physiological state of α-syn, and specific accumulation of long-chain glycosphingolipid (GSL) GCase substrates. Reduced GCase activity does not initiate α-syn aggregation in neonatal mice or immature human midbrain cultures; however, adult mice or mature midbrain cultures that express physiological α-syn oligomers are aggregation prone. Accumulation of long-chain GSLs (≥C22), but not short-chain species, induced α-syn pathology and neurological dysfunction. Selective reduction of long-chain GSLs ameliorated α-syn pathology through lysosomal cathepsins. We identify specific requirements that dictate synuclein pathology in GD models, providing possible explanations for the phenotypic variability in subjects with GCase deficiency.

Assembly of Glycochips with Mammalian GSLs Mimetics toward the On-site Detection of Biological Toxins.

According to our previously proposed scheme, each of three kinds of glycosphingolipid (GSL) derivatives, that is, lactosyl ceramide [Lac–Cer (1)] and gangliosides [GM1-Cer (2) and GT1b-Cer (3)], was installed onto the glass surface modified with Au nanoparticles. In the present study, we tried to apply microwave irradiation to promote their installing reactions. Otherwise, this procedure takes a lot of time as long as a conventional self-assembled monolayer (SAM) technique is applied. Using an advanced microwave reactor capable of adjusting ambient temperatures within a desired range, various GSL glycochips were prepared from the derivatives (1)–(3) under different microwave irradiation conditions. The overall assembling process was programed with an IC controller to finish in 1 h, and the derived GSL glycochips were evaluated in the analysis of three kinds of biological toxins [a Ricinus agglutinin (RCA120), botulinum toxin (BTX), and cholera toxin (CTX)] using a localized surface plasmon resonance (LSPR) biosensor. In the LSPR analysis, most of the irradiated GSL chips showed an enhanced response to the targeting toxin when they were irradiated under optimal temperature conditions. Lac–Cer chips showed the highest response to RCA120 (an agglutinin with β-D-Gal specificity) when the microwave irradiation was conducted at 30–35 °C. Compared to our former Lac–Cer glycochips with the conventional SAM condition, their response was enhanced by 3.6 times. Analogously, GT1b chips gained an approximately 4.1 times enhancement in their response to botulinum type C toxin (BTX/C) when the irradiation was conducted around at 45–60 °C. In the LSPR evaluation of the GM1-Cer glycochips using CTX, an optimal condition also appeared at around 30–35 °C. On the other hand, the microwave irradiation did not lead to a notable increase compared to the former GM1-Cer chips derived with the SAM technique. Judging from these experimental results, the microwave irradiation effectively promotes the installing process for all the three kinds of the GSL derivatives, while the optimal thermal condition becomes different from each other. Many bacterial and botanic proteinous toxins are composed of such carbohydrate binding domains or subunits that can discriminate both the key epitope structure and the dimension of glycoconjugates on the host cell surface. It is assumed that the optimal irradiation and thermal conditions are required to array these semi-synthetic GSL derivatives on the Au nanoparticles in a proper density and geometry for tight adhesion with each of the biological toxins.