Series Glycosphingolipids Research Articles

Fucosylation of glycoproteins and glycolipids: opposing roles in cholera intoxication.

Cholera toxin (CT) is the etiological agent of cholera. Here we report that multiple classes of fucosylated glycoconjugates function in CT binding and intoxication of intestinal epithelial cells. In Colo205 cells, knockout (KO) of B3GNT5, which encodes an enzyme required for synthesis of lacto and neolacto series glycosphingolipids (GSLs), reduces CT binding but sensitizes cells to intoxication. Overexpressing B3GNT5 to generate more fucosylated GSLs confers protection against intoxication, indicating that fucosylated GSLs act as decoy receptors for CT. KO of B3GALT5 causes increased production of fucosylated O-linked and N-linked glycoproteins and leads to increased CT binding and intoxication. KO of B3GNT5 in B3GALT5-KO cells eliminates production of fucosylated GSLs but increases intoxication, identifying fucosylated glycoproteins as functional receptors for CT. These findings provide insight into the molecular determinants regulating CT sensitivity of host cells.

Dissociation of globotriaosylceramide and impaired endothelial function in α-galactosidase-A deficient EA.hy926 cells

Fabry disease, a rare, X-linked lysosomal storage disease, arises from deficiency of the lysosomal hydrolase, α-galactosidase A (GLA) which disrupts the catabolism of globo- series glycosphingolipids (GSLs). One potential link between GLA deficiency and vascular dysfunction may be changes in endothelial nitric oxide synthase (eNOS) function. GLA-deficient EA.hy926 cells were obtained by siRNA knockdown of GLA expression and by mutation of GLA with CRISPR/Cas9 gene editing to investigate the effects of GLA deficiency on eNOS. As previously observed with siRNA knockdown of GLA, globotriaosylceramide (Gb3) accumulated in EA.hy926 cells. In contrast, Gb3 did not accumulate in CRISPR/Cas9 gene edited GLA-deficient cells, but instead, globotetraosylceramide (Gb4). However, in both the siRNA and CRISPR/Cas9 models globotriaosylsphingosine (lyso-Gb3) was elevated. As was previously observed with siRNA knockdown of GLA expression, CRISPR/Cas9 GLA-deficient cells had lower eNOS activity. Restoring GLA activity in GLA-deficient cells with exogenous GLA treatment improved eNOS activity. In contrast, treating cells with the glucosylceramide synthase inhibitor, eliglustat, decreased NOS activity. These results suggest that eNOS uncoupling is due to GLA deficiency, and not necessarily due to elevated Gb3 per se. It was observed that lyso-Gb3 inhibits eNOS activity.

Impaired mitophagy causes brown adipose tissue activation and cachexia in Sanfilippo syndrome type A mice

Fabry disease, a rare, X-linked lysosomal storage disease, arises from deficiency of the lysosomal hydrolase, α-galactosidase A (GLA) which disrupts the catabolism of globo- series glycosphingolipids (GSLs). One potential link between GLA deficiency and vascular dysfunction may be changes in endothelial nitric oxide synthase (eNOS) function. GLA-deficient EA.hy926 cells were obtained by siRNA knockdown of GLA expression and by mutation of GLA with CRISPR/Cas9 gene editing to investigate the effects of GLA deficiency on eNOS. As previously observed with siRNA knockdown of GLA, globotriaosylceramide (Gb3) accumulated in EA.hy926 cells. In contrast, Gb3 did not accumulate in CRISPR/Cas9 gene edited GLA-deficient cells, but instead, globotetraosylceramide (Gb4). However, in both the siRNA and CRISPR/Cas9 models globotriaosylsphingosine (lyso-Gb3) was elevated. As was previously observed with siRNA knockdown of GLA expression, CRISPR/Cas9 GLA-deficient cells had lower eNOS activity. Restoring GLA activity in GLA-deficient cells with exogenous GLA treatment improved eNOS activity. In contrast, treating cells with the glucosylceramide synthase inhibitor, eliglustat, decreased NOS activity. These results suggest that eNOS uncoupling is due to GLA deficiency, and not necessarily due to elevated Gb3 per se. It was observed that lyso-Gb3 inhibits eNOS activity.

Disease severity scoring system for acid sphingomyelinase deficiency: Severity score domains and components

Fabry disease, a rare, X-linked lysosomal storage disease, arises from deficiency of the lysosomal hydrolase, α-galactosidase A (GLA) which disrupts the catabolism of globo- series glycosphingolipids (GSLs). One potential link between GLA deficiency and vascular dysfunction may be changes in endothelial nitric oxide synthase (eNOS) function. GLA-deficient EA.hy926 cells were obtained by siRNA knockdown of GLA expression and by mutation of GLA with CRISPR/Cas9 gene editing to investigate the effects of GLA deficiency on eNOS. As previously observed with siRNA knockdown of GLA, globotriaosylceramide (Gb3) accumulated in EA.hy926 cells. In contrast, Gb3 did not accumulate in CRISPR/Cas9 gene edited GLA-deficient cells, but instead, globotetraosylceramide (Gb4). However, in both the siRNA and CRISPR/Cas9 models globotriaosylsphingosine (lyso-Gb3) was elevated. As was previously observed with siRNA knockdown of GLA expression, CRISPR/Cas9 GLA-deficient cells had lower eNOS activity. Restoring GLA activity in GLA-deficient cells with exogenous GLA treatment improved eNOS activity. In contrast, treating cells with the glucosylceramide synthase inhibitor, eliglustat, decreased NOS activity. These results suggest that eNOS uncoupling is due to GLA deficiency, and not necessarily due to elevated Gb3 per se. It was observed that lyso-Gb3 inhibits eNOS activity.

Altered (neo-) lacto series glycolipid biosynthesis impairs \u03b12-6 sialylation on N-glycoproteins in ovarian cancer cells

The (neo-) lacto series glycosphingolipids (nsGSLs) comprise of glycan epitopes that are present as blood group antigens, act as primary receptors for human pathogens and are also increasingly associated with malignant diseases. Beta-1, 3-N-acetyl-glucosaminyl-transferase 5 (B3GNT5) is suggested as the key glycosyltransferase for the biosynthesis of nsGSLs. In this study, we investigated the impact of CRISPR-Cas9 -mediated gene disruption of B3GNT5 (∆B3GNT5) on the expression of glycosphingolipids and N-glycoproteins by utilizing immunostaining and glycomics-based PGC-UHPLC-ESI-QTOF-MS/MS profiling. ∆B3GNT5 cells lost nsGSL expression coinciding with reduction of α2-6 sialylation on N-glycoproteins. In contrast, disruption of B4GALNT1, a glycosyltransferase for ganglio series GSLs did not affect α2-6 sialylation on N-glycoproteins. We further profiled all known α2-6 sialyltransferase-encoding genes and showed that the loss of α2-6 sialylation is due to silencing of ST6GAL1 expression in ∆B3GNT5 cells. These results demonstrate that nsGSLs are part of a complex network affecting N-glycosylation in ovarian cancer cells.

Structure-based rationale for differential recognition of lacto- and neolacto- series glycosphingolipids by the N-terminal domain of human galectin-8.

Glycosphingolipids are ubiquitous cell surface molecules undertaking fundamental cellular processes. Lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) are the representative core structures for lacto- and neolacto-series glycosphingolipids. These glycolipids are the carriers to the blood group antigen and human natural killer antigens mainly found on blood cells, and are also principal components in human milk, contributing to infant health. The β-galactoside recognising galectins mediate various cellular functions of these glycosphingolipids. We report crystallographic structures of the galectin-8 N-terminal domain (galectin-8N) in complex with LNT and LNnT. We reveal the first example in which the non-reducing end of LNT binds to the primary binding site of a galectin, and provide a structure-based rationale for the significant ten-fold difference in binding affinities of galectin-8N toward LNT compared to LNnT, such a magnitude of difference not being observed for any other galectin. In addition, the LNnT complex showed that the unique Arg59 has ability to adopt a new orientation, and comparison of glycerol- and lactose-bound galectin-8N structures reveals a minimum atomic framework for ligand recognition. Overall, these results enhance our understanding of glycosphingolipids interactions with galectin-8N, and highlight a structure-based rationale for its significantly different affinity for components of biologically relevant glycosphingolipids.

Glucosylceramide synthase inhibitors differentially affect expression of glycosphingolipids.

Glucosylceramide synthase (GCS) catalyzes the first committed step in the biosynthesis of glucosylceramide (GlcCer)-related glycosphingolipids (GSLs). Although inhibitors of GCS, PPMP and PDMP have been widely used to elucidate their biological function and relevance, our comprehensive literature review revealed that the available data are ambiguous. We therefore investigated whether and to what extent GCS inhibitors affect the expression of lactosylceramide (LacCer), neolacto (nLc4 and P1), ganglio (GM1 and GD3) and globo (Gb3 and SSEA3) series GSLs in a panel of human cancer cell lines using flow cytometry, a commonly applied method investigating cell-surface GSLs after GCS inhibition. Their cell-surface GSL expression considerably varied among cell lines and more importantly, sublethal concentrations (IC10) of both inhibitors preferentially and significantly reduced the expression of Gb3 in the cancer cell lines IGROV1, BG1, HT29 and T47D, whereas SSEA3 was only reduced in BG1. Unexpectedly, the neolacto and ganglio series was not affected. LacCer, the precursor of all GlcCer-related GSL, was significantly reduced only in BG1 cells treated with PPMP. Future research questions addressing particular GSLs require careful consideration; our results indicate that the extent to which there is a decrease in the expression of one or more particular GSLs is dependent on the cell line under investigation, the type of GCS inhibitor and exposure duration.

Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells.

BackgroundThe GBGT1 gene encodes the globoside alpha-1,3-N-acetylgalactosaminyltransferase 1. This enzyme catalyzes the last step in the multi-step biosynthesis of the Forssman (Fs) antigen, a pentaglycosyl ceramide of the globo series glycosphingolipids. While differential GBGT1 mRNA expression has been observed in a variety of human tissues being highest in placenta and ovary, the expression of GBGT1 and the genes encoding the glycosyltransferases and glycosidases involved in the biosynthesis of Fs as well as the possible involvement of DNA methylation in transcriptional regulation of GBGT1 expression have not yet been investigated.ResultsRT-qPCR profiling showed high GBGT1 expression in normal ovary surface epithelial (HOSE) cell lines and low GBGT1 expression in all (e.g. A2780, SKOV3) except one (OVCAR3) investigated ovarian cancer cell lines, a finding that was confirmed by Western blot analysis. Hierarchical cluster analysis showed that GBGT1 was even the most variably expressed gene of Fs biosynthesis-relevant glycogenes and among the investigated cell lines, whereas NAGA which encodes the alpha-N-acetylgalactosaminidase hydrolyzing Fs was not differentially expressed. Bisulfite- and COBRA-analysis of the CpG island methylation status in the GBGT1 promoter region demonstrated high or intermediate levels of GBGT1 DNA methylation in all ovarian cancer cell lines (except for OVCAR3) but marginal levels of DNA methylation in the two HOSE cell lines. The extent of DNA methylation inversely correlated with GBGT1 mRNA and protein expression. Bioinformatic analysis of GBGT1 in The Cancer Genome Atlas ovarian cancer dataset demonstrated that this inverse correlation was also found in primary ovarian cancer tissue samples confirming our cell line-based findings. Restoration of GBGT1 mRNA and protein expression in low GBGT1-expressing A2780 cells was achieved by 5-aza-2’-deoxycytidine treatment and these treated cells exhibited increased helix pomatia agglutinin-staining, reflecting the elevated presence of Fs disaccharide on these cells.ConclusionsGBGT1 expression is epigenetically silenced through promoter hypermethylation in ovarian cancer. Our findings not only suggest an involvement of DNA methylation in the synthesis of Fs antigen but may also explain earlier studies showing differential GBGT1 expression in various human tissue samples and disease stages.

Transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase

Although 6-gala series glycosphingolipids possessing R-Gal (alpha/beta) 1-6Gal beta 1-1'Cer have been found in some mollusks, pathogenic parasites, and fungi, their physiological functions and metabolic pathway are not fully understood. We described a novel method of detecting 6-gala series glyco- sphingolipids utilizing the specificity of endogalactosylceramidase (EGALC), which is capable of hydrolyzing 6-gala series glycosphingolipids to produce intact oligosaccharides and ceramides. EGALC catalyzes not only hydrolysis but also a transglycosylation reaction. In the latter reaction, EGALC transfers oligosaccharides from the glycosphingolipids to acceptors such as fluorescent 1-alkanols. Based on the transglycosylation reaction of EGALC, a specific, easy, fast, sensitive, and reproducible method of detecting 6-gala series glycosphingolipids was developed using NBD-pentanol as an acceptor. The fluorescent products, NBD-pentanol-conjugated 6-gala oligosaccharides, were separated and detected by TLC or HPLC with a fluorescent detector. Moreover, it was revealed that as well as glycosphingolipids, a glycoglycerolipid, digalactosyldiacylglycerol, was utilized by EGALC as a donor substrate. This method was successfully applied to detect 6-gala series glycosphingolipids in a fungus, Rhizopus oryzae, and a parasite, Taenia crassiceps. The method would be useful for studying glycosphingolipids and galactosyl glycerolipids which share the Gal (alpha/beta) 1-6Gal structure.

Identification of Further Elongation and Branching of Dimeric Type 1 Chain on Lactosylceramides from Colonic Adenocarcinoma by Tandem Mass Spectrometry Sequencing Analyses

Mammalian glycan chain elongation is mostly based on extending the type 2 chain, Galbeta1-4GlcNAc, whereas the corresponding type 1 chain, Galbeta1-3GlcNAc, is not normally extended. In a broader context of developing high sensitivity mass spectrometry methodologies for glycomic identification of Le(a) versus Le(x) and linear versus branched poly-N-acetyllactosamine (polyLacNAc), we have now shown that the dimeric type 1 glycan chain, as carried on the lactosylceramides of a human colonic adenocarcinoma cell line, Colo205, not only can be further extended linearly but can likewise be branched at C6 of 3-linked Gal in a manner similar to polyLacNAc. A combination of chemical and enzymatic derivatization coupled with advanced mass spectrometry analyses afforded unambiguous identification of a complex mixture of type 1 and 2 hybrids as well as those fucosylated variants founded exclusively on linear and branched trimeric type 1 chain. We further showed by in vitro enzymatic synthesis that extended type 1 and the hybrid chains can be branched by all three forms of the human I branching enzymes (IGnT) currently identified but with lower efficiency and stringency with respect to branching site preference. Importantly, it was found that a better substrate is one that carries a Gal site for branching that is extended at the non-reducing end by a type 2 and not a type 1 unit, whereas the IGnTs are less discriminative with respect to whether the targeted Gal site is itself beta3- or beta4-linked to GlcNAc at the reducing end.

Synthesis of fluorescent glycosphingolipids and neoglycoconjugates which contain 6-gala oligosaccharides using the transglycosylation reaction of a novel endoglycoceramidase (EGALC)

Endoglycoceramidase is a glycohydrolase capable of hydrolysing the O-glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. However, no endoglycoceramidase reported so far can hydrolyse 6-gala series glycosphingolipids which possess the common structure R-Gal beta1-6Gal beta1-1'Cer. Recently, we found a novel endoglycoceramidase (endogalactosylceramidase, EGALC) which specifically hydrolyses 6-gala series glycosphingolipids. Here, we report that EGALC catalyses the hydrolysis as well as transglycosylation. An intact sugar chain of neogalatriaosylceramide (Gal beta1-6Gal beta1-6Gal beta1-1'Cer) was found to be transferred by EGALC to a primary hydroxyl group of various alkanols and non-ionic detergents such as Triton X-100 generating corresponding alkyl- and Triton-trigalactooligosaccharides. Furthermore, fluorescent 6-gala series glycosphingolipids were synthesized by transglycosylation in a reaction with EGALC using fluorescent ceramides as acceptors. Because of high efficiency and broad acceptor specificity, EGALC would facilitate the synthesis of fluorescent glycosphingolipids and neoglycoconjugates which contain 6-gala oligosaccharides.

A Novel Endoglycoceramidase Hydrolyzes Oligogalactosylceramides to Produce Galactooligosaccharides and Ceramides

Enzymes capable of hydrolyzing the beta-glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids has been found in microorganisms and invertebrates and designated endoglycoceramidase (EC 3.2.1.123) or ceramide glycanase. Here we report the molecular cloning, characterization, and homology modeling of a novel endoglycoceramidase that hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides. The novel enzyme was purified from a culture supernatant of Rhodococcus equi, and the gene encoding 488 deduced amino acids was cloned using peptide sequences of the purified enzyme. Eight residues essential for the catalytic reaction in microbial and animal endoglycoceramidases were all conserved in the deduced amino acid sequence of the novel enzyme. Homology modeling of the enzyme using endocellulase E1 as a template revealed that the enzyme displays a (beta/alpha)8 barrel structure in which Glu234 at the end of beta-strand 4 and Glu341 at the end of beta-strand 7 could function as an acid/base catalyst and a nucleophile, respectively. Site-directed mutagenesis of these glutamates resulted in a complete loss of the activity without a change in their CD spectra. The recombinant enzyme hydrolyzed the beta-galactosidic linkage between oligosaccharides and ceramides of 6-gala series glycosphingolipids that were completely resistant to hydrolysis by the enzymes reported so far. In contrast, the novel enzyme did not hydrolyze ganglio-, globo-, or lactoseries glycosphingolipids. The enzyme is therefore systematically named "oligogalactosyl-N-acylsphingosine 1,1'-beta-galactohydrolase" or tentatively designated "endogalactosylceramidase."

Molecular Cloning of Globotriaosylceramide/CD77 Synthase, a Glycosyltransferase That Initiates the Synthesis of Globo Series Glycosphingolipids

The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (alpha1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. The isolated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 308 amino acids. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed alpha1, 4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. In Northern blotting, a 2.3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and placenta and weakly in colon, small intestine, and brain. Transfection of the cDNA into L cells resulted in the constitution of sensitivity to the apoptosis with Shiga-like toxins (verotoxins). Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids.

Novel Neogala Series Glycosphingolipids with Glucose at the Non-reducing Termini in the Earthworm, Pheretima sp.

Three novel neogala series glycosphingolipids each containing one or two glucose residues were isolated and purified from the earthworm, Pheretima sp. Their chemical structures were determined to be Glc α 1-4 Gal β 1-6 Gal β 1-Cer (glucosylneogalabiaosylceramide), Glc α 1-4 Gal β 1-6 Gal β 1-6 Gal β 1-Cer (glucosylneogalatriaosylceramide) and Glc α 1-4 Gal β 1-6 (Glc α 1-4) Gal β 1-6 Gal β 1-Cer (diglucosylneogalatriaosylceramide) based on the results of compositional analysis, methylation analysis, acid hydrolysis, fast atom bombardment mass spectrometry and proton nuclear magnetic resonance analysis. The ceramide components were behenic and lignoceric acids as the major fatty acids, and octadeca-4-sphingenine and its branched homologue as the sphingoid bases.

Occurrence of Novel Neogala Series Glycosphingolipids with a Mannose at the Non-reducing Terminal in Annelida (Earthworm, Pheretima hilgendorfi)

Two novel neogala series glycosphingolipids containing mannose has been shown present in the earthworm, Pheretima hilgendorfi. Their structures were determined as Man α 1-4Gal β 1-6 Gal β 1-Cer (mannosylneogalabiaosylceramide) and Gal α 1-6 (Man α 1-4) Gal β 1-6 Gal β 1-Cer (galactosylmannosyl-neogalabiaosylceramide) by GC analysis of component sugars, fatty acids and sphingoids, and by methylation analysis, enzymatic hydrolysis, 1H-nuclear magnetic resonance spectroscopy, and negative ion-mode fast atom bombardment mass spectrometer.The major ceramide fatty acids were predominantly saturated C22-, C23-and C24-acids. The sphingoids were octadeca-4-sphingenine and its branched homologue and branched nonadeca-4-sphingenine.

Specific staining on thin-layer chromatograms of glycosphingolipids of neolacto series and gangliosides with a terminal N-acetylneuraminyl residue by different procedures with wheat germ agglutinin

Sensitive staining methods with wheat germ agglutinin were developed for the detection of glycosphingolipids of neolacto series (A) and gangliosides with a terminal N-acetylneuraminyl residue (B) on thin-layer chromatograms. (A) Neolacto series glycosphingolipids were treated by β-galactosidase on the chromatograms in the presence of taurodeoxycholate. Then the chromatograms were incubated with biotinated wheat germ agglutinin followed by incubation with a complex of avidin and biotinated horseradish peroxidase, and the reaction was detected by 4-chloro-1-naphthol. In the case of gangliosides, sialidase treatment on the chromatograms was performed before the β-galactosidase treatment. The sensitivity of the method for Lc 3Cer, nLc 4Cer, sialyl-nLc 4Cer, and sialyl-nLc 6Cer was 4 pmol, 7.6 pmol, 2.9 pmol and 1.4 pmol, respectively. (B) The gangliosides on the chromatograms were oxidized by periodic acid and reduced by NaBH 4. Then the chromatograms were stained with wheat germ agglutinin as mentioned above. As little as 0.5 pmol of G M3, NeuAc-nLc 4Cer, and NeuAc-nLc 6Cer was detected by this method, whereas the detected limits for these gangliosides were 10 pmol, 10 pmol and 2 pmol, respectively, when periodate oxidation was omitted. G M4, G D3 and G Dla were an order less reactive than G M3. G M2, G M1 or G Dlb were not stained under the same condition. In contrast to NeuAc-containing gangliosides, any gangliosides with N-glycolylneuraminic acid were not stained by the method in (B).