Lysosomal Glycosphingolipid Storage Diseases.

Glycosphingolipids are cell-type-specific components of the outer leaflet of mammalian plasma membranes. Gangliosides, sialic acid-containing glycosphingolipids, are especially enriched on neuronal surfaces. As amphi-philic molecules, they comprise a hydrophilic oligosaccharide chain attached to a hydrophobic membrane anchor, ceramide. Whereas glycosphingolipid formation is catalyzed by membrane-bound enzymes along the secretory pathway, degradation takes place at the surface of intralysosomal vesicles of late endosomes and lysosomes catalyzed in a stepwise fashion by soluble hydrolases and assisted by small lipid-binding glycoproteins. Inherited defects of lysosomal hydrolases or lipid-binding proteins cause the accumulation of undegradable material in lysosomal storage diseases (GM1 and GM2 gangliosidosis; Fabry, Gaucher, and Krabbe diseases; and metachromatic leukodystrophy). The catabolic processes are strongly modified by the lipid composition of the substrate-carrying membranes, and the pathological accumulation of primary storage compounds can trigger an accumulation of secondary storage compounds (e.g., small glycosphingolipids and cholesterol in Niemann-Pick disease).

Glucosylceramide flippases contribute to cellular glucosylceramide homeostasis.

Lipid transport is an essential cellular process with importance to human health, disease development, and therapeutic strategies. Type IV P-type ATPases (P4-ATPases) have been identified as membrane lipid flippases by utilizing nitrobenzoxadiazole (NBD)-labeled lipids as substrates. Among the 14 human type IV P-type ATPases, ATP10D was shown to flip NBD-glucosylceramide (GlcCer) across the plasma membrane. Here, we found that conversion of incorporated GlcCer (d18:1/12:0) to other sphingolipids is accelerated in cells exogenously expressing ATP10D but not its ATPase-deficient mutant. These findings suggest that 1) ATP10D flips unmodified GlcCer as well as NBD-GlcCer at the plasma membrane and 2) ATP10D can translocate extracellular GlcCer, which is subsequently converted to other metabolites. Notably, exogenous expression of ATP10D led to the reduction in cellular hexosylceramide levels. Moreover, the expression of GlcCer flippases, including ATP10D, also reduced cellular hexosylceramide levels in fibroblasts derived from patients with Gaucher disease, which is a lysosomal storage disorder with excess GlcCer accumulation. Our study highlights the contribution of ATP10D to the regulation of cellular GlcCer levels and maintaining lipid homeostasis.

A novel family of sugar-specific phosphodiesterases that remove zwitterionic modifications of GlcNAc.

The zwitterions phosphorylcholine (PC) and phosphoethanolamine (PE) are often found esterified to certain sugars in polysaccharides and glycoconjugates in a wide range of biological species. One such modification involves PC attachment to the 6-carbon of N-acetylglucosamine (GlcNAc-6-PC) in N-glycans and glycosphingolipids (GSLs) of parasitic nematodes, a modification that helps the parasite evade host immunity. Knowledge of enzymes involved in the synthesis and degradation of PC and PE modifications is limited. More detailed studies on such enzymes would contribute to a better understanding of the function of PC modifications and have potential application in the structural analysis of zwitterion-modified glycans. In this study, we used functional metagenomic screening to identify phosphodiesterases encoded in a human fecal DNA fosmid library that remove PC from GlcNAc-6-PC. A novel bacterial phosphodiesterase was identified and biochemically characterized. This enzyme (termed GlcNAc-PDase) shows remarkable substrate preference for GlcNAc-6-PC and GlcNAc-6-PE, with little or no activity on other zwitterion-modified hexoses. The identified GlcNAc-PDase protein sequence is a member of the large endonuclease/exonuclease/phosphatase superfamily where it defines a distinct subfamily of related sequences of previously unknown function, mostly from Clostridium bacteria species. Finally, we demonstrate use of GlcNAc-PDase to confirm the presence of GlcNAc-6-PC in N-glycans and GSLs of the parasitic nematode Brugia malayi in a glycoanalytical workflow.

High expression of B3GALT5 suppresses the galectin-4-mediated peritoneal dissemination of poorly differentiated gastric cancer cells.

Peritoneal metastasis frequently accompanies metastatic and/or recurrent gastric cancer, leading to a poor prognosis owing to a lack of effective treatment. Hence, there is a pressing need to enhance our understanding of the mechanisms and molecules driving peritoneal metastasis. In a previous study, galectin-4 inhibition impeded peritoneal metastasis in a murine model. This study examined the glycan profiles of cell surface proteins and glycosphingolipids (GSLs) in cells with varying tumorigenic potentials to understand the intricate mechanisms underlying galectin-4-mediated regulation, particularly glycosylation. Detailed mass spectrometry analysis showed that galectin-4 knockout cells exhibit increased expression of lacto-series GSLs with ß1,3-linked galactose while showing no significant alterations in neolacto-series GSLs. We conducted real-time polymerase chain reaction (PCR) analysis to identify candidate glycosyltransferases that synthesize increased levels of GSLs. Subsequently, we introduced the candidate B3GALT5 gene and selected the clones with high expression levels. B3GALT5 gene-expressing clones showed GSL glycan profiles like those of knockout cells and significantly reduced tumorigenic ability in mouse models. These clones exhibited diminished proliferative capacity and showed reduced expression of galectin-4 and activated AKT. Moreover, co-localization of galectin-4 with flotillin-2 (a raft marker) decreased in B3GALT5-expressing cells, implicating GSLs in galectin-4 localization to lipid rafts. D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (a GSL synthase inhibitor) also affected galectin-4 localization in rafts, suggesting the involvement of GSL microdomains. We discovered that B3GALT5 plays a crucial role in regulating peritoneal metastasis of malignant gastric cancer cells by suppressing cell proliferation and modulating lipid rafts and galectin-4 via mechanisms that are yet to be elucidated.

Human genetic defects of sphingolipid synthesis.

Sphingolipids are ubiquitous lipids, present in the membranes of all cell types, the stratum corneum and the circulating lipoproteins. Autosomal recessive as well as dominant diseases due to disturbed sphingolipid biosynthesis have been identified, including defects in the synthesis of ceramides, sphingomyelins and glycosphingolipids. In many instances, these gene variants result in the loss of catalytic function of the mutated enzymes. Additional gene defects implicate the subcellular localization of the sphingolipid-synthesizing enzyme, the regulation of its activity, or even the function of a sphingolipid-transporter protein. The resulting metabolic alterations lead to two major, non-exclusive types of clinical manifestations: a neurological disease, more or less rapidly progressive, associated or not with intellectual disability, and an ichthyotic-type skin disorder. These phenotypes highlight the critical importance of sphingolipids in brain and skin development and homeostasis. The present article reviews the clinical symptoms, genetic and biochemical alterations, pathophysiological mechanisms and therapeutic options of this relatively novel group of metabolic diseases.

Depletion of b-series ganglioside prevents limb length discrepancy after growth plate injury.

INTRODUCTION: Growth plate damage in long bones often results in progressive skeletal growth imbalance and deformity, leading to significant physical problems. Gangliosides, key glycosphingolipids in cartilage, are notably abundant in articular cartilage and regulate chondrocyte homeostasis. This suggests their significant roles in regulating growth plate cartilage repair. METHODS: Chondrocytes from 3 to 5 day-old C57BL/6 mice underwent glycoblotting and mass spectrometry. Based on the results of the glycoblotting analysis, we employed GD3 synthase knockout mice (GD3-/-), which lack b-series gangliosides. In 3-week-old mice, physeal injuries were induced in the left tibiae, with right tibiae sham operated. Tibiae were analyzed at 5 weeks postoperatively for length and micro-CT for growth plate height and bone volume at injury sites. Tibial shortening ratio and bone mineral density were measured by micro-CT. RESULTS: Glycoblotting analysis indicated that b-series gangliosides were the most prevalent in physeal chondrocytes among ganglioside series. At 3 weeks, GD3-/- exhibited reduced tibial shortening (14.7 ± 0.2 mm) compared to WT (15.0 ± 0.1 mm, P = 0.03). By 5 weeks, the tibial lengths in GD3-/- (16.0 ± 0.4 mm) closely aligned with sham-operated lengths (P = 0.70). Micro-CT showed delayed physeal bridge formation in GD3-/-, with bone volume measuring 168.9 ± 5.8 HU at 3 weeks (WT: 180.2 ± 3.2 HU, P = 0.09), but normalizing by 5 weeks. CONCLUSION: This study highlights that GD3 synthase knockout mice inhibit physeal bridge formation after growth plate injury, proposing a new non-invasive approach for treating skeletal growth disorders.

Sphingolipids: Less Enigmatic but Still Many Questions about the Role(s) of Ceramide in the Synthesis/Function of the Ganglioside Class of Glycosphingolipids.

While much has been learned about sphingolipids, originally named for their sphinx-like enigmatic properties, there are still many unanswered questions about the possible effect(s) of the composition of ceramide on the synthesis and/or behavior of a glycosphingolipid (GSL). Over time, studies of their ceramide component, the sphingoid base containing the lipid moiety of GSLs, were frequently distinct from those performed to ascertain the roles of the carbohydrate moieties. Due to the number of classes of GSLs that can be derived from ceramide, this review focuses on the possible role(s) of ceramide in the synthesis/function of just one GSL class, derived from glucosylceramide (Glc-Cer), namely sialylated ganglio derivatives, initially characterized and named gangliosides (GGs) due to their presence in ganglion cells. While much is known about their synthesis and function, much is still being learned. For example, it is only within the last 15-20 years or so that the mechanism by which the fatty acyl component of ceramide affected its transport to different sites in the Golgi, where it is used for the synthesis of Glu- or galactosyl-Cer (Gal-Cer) and more complex GSLs, was defined. Still to be fully addressed are questions such as (1) whether ceramide composition affects the transport of partially glycosylated GSLs to sites where their carbohydrate chain can be elongated or affects the activity of glycosyl transferases catalyzing that elongation; (2) what controls the differences seen in the ceramide composition of GGs that have identical carbohydrate compositions but vary in that of their ceramide and vice versa; (3) how alterations in ceramide composition affect the function of membrane GGs; and (4) how this knowledge might be applied to the development of therapies for treating diseases that correlate with abnormal expression of GGs. The availability of an updatable data bank of complete structures for individual classes of GSLs found in normal tissues as well as those associated with disease would facilitate research in this area.

Characterization of novel nonacid glycosphingolipids as biomarkers of human gastric adenocarcinoma.

Changes in glycosphingolipid structures have been shown to occur during the development of several types of human cancers, generating cancer-specific carbohydrate structures that could be used as biomarkers for diagnosis and therapeutic targeting. In this study, we characterized nonacid glycosphingolipids isolated from a human gastric adenocarcinoma by mass spectrometry, enzymatic hydrolysis, and by binding with a battery of carbohydrate-recognizing ligands. We show that the majority of the complex nonacid glycosphingolipids had type 2 (Galß4GlcNAc) core chains (neolactotetraosylceramide, the Lex, H type 2, x2, and the P1 pentaosylceramides, and the Ley, A type 2, and neolacto hexaosylceramides). We also found glycosphingolipids with type 1 (Galß3GlcNAc) core (lactotetraosylceramide and the H type 1 pentaosylceramide) and globo (GalαGal) core chains (globotriaosylceramide and globotetraosylceramide). Interestingly, we characterized two complex glycosphingolipids as a P1 heptaosylceramide (Galα4Galß4GlcNAcß3Galß4GlcNAcß3Gal ß4Glcß1Cer) and a branched P1 decaosylceramide (Galα4Gal ß4GlcNAcß3(Galα4Galß4GlcNAcß6)Galß4GlcNAcß3Galß4Glc ß1Cer). These are novel glycosphingolipid structures and the first reported cases of complex glycosphingolipids larger than pentaosylceramide carrying the P1 trisaccharide. We propose that these P1 glycosphingolipids may represent potential biomarkers for the early diagnosis of gastric cancer.

A sensitive method for determining UDP-glucose: ceramide glucosyltransferase (UGCG) activity in biological samples using deuterated glucosylceramide as acceptor substrate.

Glucosylceramide synthase (UGCG) is a key enzyme in the biosynthesis of glycosphingolipids and its activity is related to the resistance to anticancer drugs and is involved in the derangement of metabolism in various diseases. Moreover, UGCG acts as a major controller of the balanced levels of individual brain sphingolipids that may trigger neurodegeneration in Gaucher disease and in Parkinson disease associated to pathogenic variants in the glucocerebrosidase-encoding gene GBA. We have developed an effective method for determining UGCG activity in vitro using deuterated ceramide as an acceptor, and quantitation of the formed deuterated glucosylceramide by liquid chromatography coupled with tandem mass spectrometry. The method enabled us to determine the kinetic parameters of UGGC and the effect of the inhibitor GZ667161 on the enzyme activity expressed in model cells, as well as to measure UGCG specific activity in human fibroblasts using a simple crude cell homogenate. This novel approach may be useful in determining the actual UGCG activity levels in patient cells and tissues of animal models of diseases, and to study novel drugs targeting glycosphingolipid metabolism.

Sulfatide-selectin signaling in the spinal cord induces mechanical allodynia.

Sulfatide is a sulfated glycosphingolipid that is present abundantly in myelin sheaths of the brain and spinal cord. It is synthesized by a cerebroside sulfotransferase encoded by Gal3st1, which catalyzes the transfer of sulfate from 3'-phosphoadenylylsulfate to galactosylceramide. We previously reported that Gal3st1 gene expression in the spinal cord is up-regulated 1 day after intraplantar injection of complete Freund's adjuvant (CFA), indicating that sulfatide is involved in inflammatory pain. In the present study, we found that intrathecal injection of sulfatide led to mechanical allodynia. Sulfatide caused levels of glial fibrillary acidic protein (GFAP) and nitric oxide in the spinal cord to increase. Mechanical allodynia induced by intrathecal injection of sulfatide was blocked by nitric oxide synthase inhibitors and by suppression of astrocyte activation by L-α-aminoadipate. These results suggest that sulfatide-induced mechanical allodynia involved glial activation and nitric oxide production. Blocking selectin, a sulfatide-binding protein, with bimosiamose attenuated sulfatide-induced allodynia and ameliorated CFA-induced mechanical allodynia during inflammatory pain. Finally, elevated levels of sulfatide concentration in the spinal cord were observed during CFA-induced inflammatory pain. The elevated sulfatide levels enhanced selectin activation in the spinal cord, resulting in mechanical allodynia. Our data suggest that sulfatide-selectin interaction plays a key role in inflammatory pain.

Cell type- and region-specific translatomes in an MPTP mouse model of Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, characterized by the progressive loss of nigrostriatal dopaminergic neurons (DANs), involving the dysregulation of both neurons and glial cells. Cell type- and region-specific gene expression profiles can provide an effective source for revealing the mechanisms of PD. In this study, we adopted the RiboTag approach to obtain cell type (DAN, microglia, astrocytes)- and brain region (substantia nigra, caudate-putamen)-specific translatomes at an early stage in an MPTP-induced mouse model of PD. Through DAN-specific translatome analysis, the glycosphingolipid biosynthetic process was identified as a significantly downregulated pathway in the MPTP-treated mice. ST8Sia6, a key downregulated gene related to glycosphingolipid biosynthesis, was confirmed to be downregulated in nigral DANs from postmortem brains of patients with PD. Specific expression of ST8Sia6 in DANs exerts anti-inflammatory and neuroprotective effects in MPTP-treated mice. Through cell type (microglia vs. astrocyte) and brain region (substantia nigra vs. caudate-putamen) comparisons, nigral microglia showed the most intense immune responses. Microglia and astrocytes in the substantia nigra showed similar levels of activation in interferon-related pathways and interferon gamma (IFNG) was identified as the top upstream regulator in both cell types. This work highlights that the glycosphingolipid metabolism pathway in the DAN is involved in neuroinflammation and neurodegeneration in an MPTP mouse model of PD and provides a new data source for elucidating the pathogenesis of PD.

Venglustat, an orally administered glucosylceramide synthase inhibitor: Assessment over 3 years in adult males with classic Fabry disease in an open-label phase 2 study and its extension study.

Venglustat inhibits the enzymatic conversion of ceramide to glucosylceramide, reducing available substrate for the synthesis of more complex glycosphingolipids. It offers a potential new approach to the treatment of patients with Fabry disease (α-Gal A deficiency), in whom progressive accumulation of such glycosphingolipids, including globotriaosylceramide (GL-3), in the lysosomes of a wide range of cell types often leads to vital organ complications in adulthood. An international, open-label, single-arm, Phase 2a uncontrolled 26-week clinical study (NCT02228460) and a 130-week extension study (NCT02489344) were conducted to assess the safety, pharmacodynamics, pharmacokinetics, and exploratory efficacy of 15 mg once daily oral venglustat in treatment-naïve adult male patients with classic Fabry disease. Of 11 patients (18-37 years old) who initially enrolled, nine completed the 26-week study and seven completed the extension study. A total of 169 treatment-emergent adverse events (TEAEs) were reported by nine patients, the majority being mild (73%) and unrelated to the study drug (70%). Nine serious TEAEs (serious adverse events) and 11 severe TEAEs, including a self-harm event, were reported. No deaths or treatment-related life-threatening adverse events were reported. Skin GL-3 scores in superficial skin capillary endothelium (SSCE), estimated by light microscopy, were unchanged from baseline at Week 26 in five patients, decreased in three patients, and increased in one patient. There was no significant change in GL-3 scores or significant shift in grouped GL-3 scores. Five of six patients had reductions from baseline in GL-3 score at the end of the extension study. At Weeks 26 and 156 the mean (standard deviation) changes from baseline in the fraction of the volume of SSCE cytoplasm occupied by GL-3 inclusions, measured by electron microscopy unbiased stereology, were - 0.06 (0.03) (p = 0.0010) and - 0.12 (0.04) (p = 0.0008), respectively. Venglustat treatment reduced markers in the synthetic and degradative pathway of major glycosphingolipids; proximal markers reduced rapidly and more distal markers (plasma GL-3 and globotriaosylsphingosine) reduced progressively. There were no biochemical or histological indications of progression of Fabry disease over 3 years of follow-up. These findings confirm target engagement and the pharmacodynamic effects of venglustat in adult males with classic Fabry disease. However, further clinical evaluation in larger studies is needed to determine efficacy and safety.

Porcine intestinal glycosphingolipids recognized by Brachyspira hyodysenteriae.

Swine dysentery caused by Brachyspira hyodysenteriae is a disease present worldwide with an important economic impact on the farming business, resulting in an increased use of antibiotics. In the present study, we investigated the binding of B. hyodysenteriae to glycosphingolipids from porcine small intestinal epithelium in order to determine the glycosphingolipids involved in B. hyodysenteriae adhesion. Specific interactions between B. hyodysenteriae and two non-acid glycosphingolipids were obtained. These binding-active glycosphingolipids, were characterized by mass spectrometry as lactotetraosylceramide (Galß3GlcNAcß3Galß4Glcß1Cer) and the B5 glycosphingolipid (Galα3Galß4GlcNAcß3Galß4Glcß1Cer). Comparative binding studies using structurally related reference glycosphingolipids showed that B. hyodysenteriae binding to lactotetraosylceramide required an unsubstituted terminal Galß3GlcNAc sequence, while for binding to the B5 pentaosylceramide the terminal Galα3Galß4GlcNAc sequence is the minimum element recognized by the bacteria. Binding of Griffonia simplicifolia IB4 lectin to pig colon tissue sections from healthy control pig and B. hyodysenteriae infected pigs showed that in the healthy pigs the Galα3Gal epitope was mainly present in the lamina propria. In contrast, in four out of five pigs with swine dysentery there was an increased expression of Galα3Gal in the goblet cells and in the colonic crypts, where B. hyodysenteriae also was present. The one pig that had recovered by the time of necropsy had the Galα3Gal epitope only in the lamina propria. These data are consistent with a model where a transient increase in the carbohydrate sequence recognized by the bacteria occur in colonic mucins during B. hyodysenteriae infection, suggesting that the mucins may act as decoys contributing to clearance of the infection. These findings may lead to novel strategies for treatment of B. hyodysenteriae induced swine dysentery.

Process Engineering and Glycosyltransferase Improvement for Short Route Chemoenzymatic Total Synthesis of GM1 Gangliosides.

Large-scale synthesis of GM1, an important ganglioside in mammalian cells especially those in the nervous system, is needed to explore its therapeutic potential. Biocatalytic production is a promising platform for such a purpose. We report herein the development of process engineering and glycosyltransferase improvement strategies to advance chemoenzymatic total synthesis of GM1. Firstly, a new short route was developed for chemical synthesis of lactosylsphingosine from the commercially available Garner's aldehyde. Secondly, two glycosyltransferases including Campylobacter jejuni ß1-4GalNAcT (CjCgtA) and ß1-3-galactosyltransferase (CjCgtB) were improved on their soluble expression in E. coli and enzyme stability by fusing with an N-terminal maltose binding protein (MBP). Thirdly, the process for enzymatic synthesis of GM1 sphingosines from lactosylsphingosine was engineered by developing a multistep one-pot multienzyme (MSOPME) strategy without isolating intermediate glycosphingosines and by adding a detergent, sodium cholate, to the later enzymatic glycosylation steps. Installation of a desired fatty acyl chain to GM1 glycosphingosines led to the formation of target GM1 gangliosides. The combination of glycosyltransferase improvement with chemical and enzymatic process engineering represents a significant advance in obtaining GM1 gangliosides containing different sialic acid forms by total chemoenzymatic synthesis in a short route and with high efficiency.

A Biomarker Study in Patients with GBA1-Parkinson's Disease and Healthy Controls.

BACKGROUND: Molecules related to glucocerebrosidase (GCase) are potential biomarkers for development of compounds targeting GBA1-associated Parkinson's disease (GBA-PD). OBJECTIVES: Assessing variability of various glycosphingolipids (GSLs) in plasma, peripheral blood mononuclear cells (PBMCs), and cerebrospinal fluid (CSF) across GBA-PD, idiopathic PD (iPD), and healthy volunteers (HVs). METHODS: Data from five studies were combined. Variability was assessed of glucosylceramide (various isoforms), lactosylceramide (various isoforms), glucosylsphingosine, galactosylsphingosine, GCase activity (using fluorescent 4-methylumbeliferryl-ß-glucoside), and GCase protein (using enzyme-linked immunosorbent assay) in plasma, PBMCs, and CSF if available, in GBA-PD, iPD, and HVs. GSLs in leukocyte subtypes were compared in HVs. Principal component analysis was used to explore global patterns in GSLs, clinical characteristics (Movement Disorder Society - Unified Parkinson's Disease Rating Scale Part 3 [MDS-UPDRS-3], Mini-Mental State Examination [MMSE], GBA1 mutation type), and participant status (GBA-PD, iPD, HVs). RESULTS: Within-subject between-day variability ranged from 5.8% to 44.5% and was generally lower in plasma than in PBMCs. Extracellular glucosylceramide levels (plasma) were slightly higher in GBA-PD compared with both iPD and HVs, while intracellular levels were comparable. GSLs in the different matrices (plasma, PBMCs, CSF) did not correlate. Both lactosylceramide and glucosylsphingosine were more abundant in granulocytes compared with monocytes and lymphocytes. Absolute levels of GSL isoforms differed greatly. GBA1 mutation types could not be differentiated based on GSL data. CONCLUSIONS: Glucosylceramide can stably be measured over days in both plasma and PBMCs and may be used as a biomarker in clinical trials targeting GBA-PD. Glucosylsphingosine and lactosylceramide are stable in plasma but are strongly affected by leukocyte subtypes in PBMCs. GBA-PD could be differentiated from iPD and HVs, primarily based on glucosylceramide levels in plasma. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

A concise chemoenzymatic total synthesis of neutral Globo-series glycosphingolipids Globo A and Globo B, and Forssman and para-Forssman antigens.

Globo A is a neutral Globo-series glycosphingolipid (GSL) that shows natural properties of a cytotoxicity receptor NKp44 binding ligand. The highly complex heptasaccharide glycan structure of Globo A combined with its biological profile provides a unique target for the development of a synthetic method to facilitate its bioactivity studies. Here, a concise chemoenzymatic route to the synthesis of Globo A and its α1,3-galactose-linked congener Globo B is reported. The key to success was the use of a synthetic azido ß-Globo H sphingosine (Globo H-ßSph) as an acceptor substrate and two glycosyl transferases, an α1,3-N-acetylgalactosaminyltransferase from Helicobacter mustelae (BgtA) and a human blood group B α1,3-galactosyltransferase (h1,3GTB), for stereoselective construction of the terminal α1,3-GalNAc and α1,3-Gal linkages, respectively. The azido-Sph lipid sidechain is further elaborated by reduction and a chemoselective N-acylation to complete the total synthesis of the neutral Globo-series GSLs. In addition, the synthesis of Forssman and para-Forssman antigens were prepared. The strategy may be suitable for accessing other complex GSLs and related lipid-modified GSL derivatives.

Vibrio-binding gangliosides in fish intestinal tracts.

It has been clarified that pathogens bind to glycosphingolipid (GSL) receptors in mammals, but there have been very few reports on pathogen-binding GSLs in fish. Vibrios are facultative anaerobic bacteria ubiquitous in marine and brackish environments. They are members of the normal intestinal microflora of healthy fish, but some species can cause a disease called vibriosis in fish and shellfish when the hosts are physiologically or immunologically weakened. The adherence of vibrios to host intestinal tracts is a significant event not only for survival and growth but also in terms of pathogenicity. We show in this mini-review that sialic acid-containing GSLs (gangliosides), GM4 and GM3, are receptors to which vibrios adhere to epithelial cells in the intestinal tract of fish. We also describe the enzymes responsible for synthesizing these Vibrio-binding gangliosides in fish.

Relationship of Sulfatides Physiological Function and Peroxisome Proliferator-Activated Receptor α.

Sulfatides are unique sphingolipids present in the serum and the plasma membrane. Sulfatides exert important functions in a number of systems in the human body, including the nervous, immune, cardiovascular, and coagulation systems.Furthermore, it is closely related to tumor occurrence, development, and metastasis. Peroxisome proliferators-activated receptor α (PPARα) is a class of the nuclear receptor superfamily of transcription factors, which is a potential regulator of sulfatides. This review not only summarizes the current knowledge on the physiological functions of sulfatides in various systems, but also discusses the possible PPARα regulatory mechanisms in sulfatide metabolism and functions. The results of the present analysis provide deep insights and further novel ideas for expanding the research on the physiological function and clinical application of sulfatides.

Genetic knockout of porcine GGTA1 or CMAH/GGTA1 is associated with the emergence of neo-glycans.

BACKGROUND: Pig-derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α-Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. METHODS: The N-glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1-KO and GGTA1/CMAH-KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection. RESULTS: We identified biantennary and core-fucosylated N-glycans terminating with immunogenic α-Gal- and α-Gal-/Neu5Gc-epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH-KO pigs, respectively. Levels of N-glycans terminating with galactose bound in ß(1-4)-linkage to N-acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N-glycans capped with Neu5Gc were increased in GGTA1-KO pigs compared to WT, but were not detected in GGTA1/CMAH-KO pigs. Similarly, the ganglioside Neu5Gc-GM3 was found in WT and GGTA1-KO but not in GGTA1/CMAH-KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. CONCLUSION: Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human-like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic.

Chemoenzymatic Synthesis of N-Acetyl Analogues of 9-O-Acetylated b-Series Gangliosides.

The stable N-acetyl analogues of biologically important 9-O-acetylated b-series gangliosides including 9NAc-GD3, 9NAc-GD2, 9NAc-GD1b, and 9NAc-GT1b were chemoenzymatically synthesized from a GM3 sphingosine. Two chemoenzymatic methods using either 6-azido-6-deoxy-N-acetylmannosamine (ManNAc6N3) as a chemoenzymatic synthon or 6-acetamido-6-deoxy-N-acetylmannosamine (ManNAc6NAc) as an enzymatic precursor for 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc) were developed and compared for the synthesis of 9NAc-GD3. The latter method was found to be more efficient and was used to produce the desired 9-N-acetylated glycosylsphingosines. Furthermore, glycosylsphingosine acylation reaction conditions were improved to obtain target 9-N-acetylated gangliosides in a faster reaction with an easier purification process compared to the previous acylation conditions.