Ceramide Glycanase Research Articles

Approach for Profiling of Glycosphingolipid Glycosylation by Multiplexed Capillary Gel Electrophoresis Coupled to Laser-Induced Fluorescence Detection To Identify Cell-Surface Markers of Human Pluripotent Stem Cells and Derived Cardiomyocytes.

Application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as tissue transplants in regenerative medicine depends on cell-surface marker-based characterization and/or purification. Glycosphingolipids (GSLs) are a family of highly diverse surface-exposed biomolecules that have been neglected as potential surface markers for hiPSC-CMs due to significant analytical challenges. Here, we describe the development of a novel and high-throughput-compatible workflow for the analysis of GSL-derived glycans based on ceramide glycanase digestion, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling, and multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF). GSL glycans were detected with highly reproducible migration times after repeated analysis by xCGE-LIF. We built up a migration time database comprising 38 different glycan species, and we showed exemplarily that as few as 10 pg of fucosyl lactotetra was detectable. GSL glycan profiling could be performed with 105 human induced pluripotent stem cells, and we quantitatively dissected global alterations of GSL glycosylation of human induced pluripotent stem cells (hiPSCs) and hiPSC-CMs by employing xCGE-LIF. In our study, we observed a general switch from complex GSLs with lacto- and globo-series core structures comprising the well-known human pluripotent stem cell marker stage-specific embryonic antigen 3 (SSEA3) and SSEA4 in hiPSCs toward the simple gangliosides GM3 and GD3 in hiPSC-CMs. This is the first description of GM3 and GD3 being highly abundant GSLs on the cell surface of stem cell-derived cardiomyocytes.

Counter effects of Asiaticosids-D through putative neurotransmission on rotenone induced cerebral ganglionic injury in Lumbricus terrestris

Asiaticoside-D (AD) was shown to efficacy of ganglionic degenerated Lumbricus terrestris as a pioneering observation in our earlier research. Though, extract molecular mechanisms of AD for degenerative diseases (DDs) remains largely unknown. We investigated the neuroprotective effects of AD against ROT in cerebral ganglions (CGs) of degenerative L. terrestris. Worms were exposed to 0.4 ppm ROT for 7 days were subjected to co- treatment with 15 ppm of AD. After, CGs was removed. The levels oxidant, non-antioxidant, antioxidant status, ganglioside, ceramide and ceramide glycanase (CGase) were estimated. The m-RNA levels of dopamine transporter (DAT), octopamine transporter (OAT), innexins-9 (inx-9), ionotropic glutamate receptor 3 (iGlu3), heat shock proteins (hsp70), XPRLamide neuropeptide precursor, tyramine beta-hydroxylase (tbh-1) and β- adrenergic receptor kinase-2 (β-ARK2–3) by semi-qRT- PCR. The expression pattern of tyramine beta hydroxylase (TBH), glutamate receptor (iGluR), serotonin transporter (SERT), dopamine transporters (DAT), nerve growth factors (NGF), cytochrome C oxidase (COC), NADH dehydogenase subunit-1 (ND-1), neurotrophin receptor p75 (p75NTR), neuronal nitric oxiside synthase (nNOs) interleukin 1- beta (IL1-β) and tumor necrosis factor alpha (TNF-α) by western blotting. Glutaminergic, serotogenic and dopaminergic toxicity variations were also performed. The levels of oxidant, non-antioxidant, antioxidant status, lipids, proteins and m-RNAs were significantly altered (p < 0.001) on ROT-induced (group II) and their levels were significantly changes (p < 0.05) by ROT+AD in CGs. The sensitive study plan concluded the neuroprotective effects of AD against ROT induced degeneration in worms and suggest that the AD deserves future studies for its use as an effective alternative medicine that could minimize the morbidity of ganglionic degenerative diseases patients.

Degradation of glycosphingolipids in oyster: ceramide glycanase and ceramidase in the hepatopancreas of oyster, Crassostrea virginica.

The hepatopancreas of oyster, Crassostrea virginica, was found to contain two unique glycosphingolipid (GSL) cleaving enzymes, ceramide glycanase (CGase) and ceramidase. These two enzymes were found to be tightly associated together through the consecutive purification steps including gel filtration, hydrophobic interaction and cation-exchange chromatographies. They were separated only by preparatory SDS-PAGE. The purified CGase was found to have a molecular mass of 52kDa and pH optimum of 3.2-3.3. This enzyme prefers to hydrolyze the acidic GSLs, II3SO3LacCer and gangliosides over the neutral GSLs. Oyster ceramidase was found to have a molecular mass of 88kDa and pH optimum of 4-4.5. Since oyster ceramidase greatly prefers ceramides with C6 to C8 fatty acids, C6-ceramide (N-hexanoyl-D-sphingosine) was used as the substrate for its purification and characterization. The oyster acid ceramidase also catalyzed the synthesis of ceramide from a sphingosine and a fatty acid. For the synthesis, C16 and C18 fatty acids were the best precursors. The amino acid sequences of the two cyanogenbromide peptides derived from the purified ceramidase were found to have similarities to those of several neutral and alkaline ceramidases reported. The tight association of CGase and ceramidase may indicate that CGase in oyster hepatopancreas acts as a vehicle to release ceramide from GSLs for subsequent generation of sphingosines and fatty acids by ceramidase to serve as signaling factors and energy source.

Glycan profiling of urine, amniotic fluid and ascitic fluid from galactosialidosis patients reveals novel oligosaccharides with reducing end hexose and aldohexonic acid residues

Urine, amniotic fluid and ascitic fluid samples of galactosialidosis patients were analyzed and structurally characterized for free oligosaccharides using capillary high-performance anion-exchange chromatography with pulsed amperometric detection and online mass spectrometry. In addition to the expected endo-beta-N-acetylglucosaminidase-cleaved products of complex-type sialylated N-glycans, O-sulfated oligosaccharide moieties were detected. Moreover, novel carbohydrate moieties with reducing-end hexose residues were detected. On the basis of structural features such as a hexose-N-acetylhexosamine-hexose-hexose consensus sequence and di-sialic acid units, these oligosaccharides are thought to represent, at least in part, glycan moieties of glycosphingolipids. In addition, C(1)-oxidized, aldohexonic acid-containing versions of most of these oligosaccharides were observed. These observations suggest an alternative catabolism of glycosphingolipids in galactosialidosis patients: oligosaccharide moieties from glycosphingolipids would be released by a hitherto unknown ceramide glycanase activity. The results show the potential and versatility of the analytical approach for structural characterization of oligosaccharides in various body fluids.

New method for quantitative analysis of GD2 ganglioside in plasma of neuroblastoma patients.

Neuroblastoma, the most common extracranial solid tumour of childhood, is a malignancy of unknown origin and non-specific symptoms. One of the markers of the disease is GD2 ganglioside (disialoganglioside), which is abundantly expressed on the surface of neuroblastoma cells. Gangliosides are known to be shed by tumour cells and this phenomenon can be significant in cancer progression as they inhibit a number of immune responses both in vitro and in vivo. In search for novel markers useful in monitoring and prognosis of neuroblastoma, we developed and validated a new quantitative method of GD2 ganglioside analysis in human blood plasma. We evaluated the level of gangliosides in blood serum of 34 neuroblastoma patients using high-performance liquid chromatography. The technique was used to detect fluorescently labelled oligosaccharides derived from serum glycosphingolipids by enzymatic digestion with ceramide glycanase. The developed method allowed determination of GD2 concentrations at the picomole level and required only 40 microl of plasma, which should be particularly useful when the quantity of clinical material is limiting. Moreover, this method can be applied to study concentration of other gangliosides, as shown for GD3 ganglioside. Analysis of plasma samples from the 34 neuroblastoma patients did not reveal any correlations between the concentration of GD2 ganglioside and clinical parameters, including the results of therapy; it showed, however, that the concentration of GD2 ganglioside in the plasma of neuroblastoma patients decreased substantially in the course of treatment.

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.

Preparation of homogenous oligosaccharide chains from glycosphingolipids

After the discovery of glycosphingolipid (GSL) glycan detaching enzymes, Rhodococcal endoglycoceramidase (EGCase) and leech ceramide glycanase (CGase), the method for enzymatically releasing glycans from GSLs has become the method of choice for preparing intact ceramide-free oligosaccharide chains from GSLs. This paper describes (1) the preparation of the intact oligosaccharides from GM1 (II(3)NeuAcGgOse(4)Cer) and GbOse(4)Cer as examples to show the use of CGase to prepare intact glycan chains from GSLs, and (2) the specificity and detergent requirements of Rhodococcal EGCases for the release of glycan chains from different GSLs.

N-Azidoacetylmannosamine-mediated chemical tagging of gangliosides

Peracetylated N-alpha-azidoacetylmannosamine (Ac(4)ManNAz) is metabolized by cells to CMP-azidosialic acid. It has been demonstrated previously that in this way azidosialic acid-containing glycoproteins are formed that can be labeled on the cell surface by a modified Staudinger ligation. Here, we first demonstrate that the same procedure also results in the formation of azidosialic acid-containing gangliosides. Deoxymannojirimycin, an inhibitor of N-glycan processing in proteins, decreases the total cell surface labeling in Jurkat cells by approximately 25%. Inhibition of ganglioside biosynthesis with N-[5-(adamantan-1-yl-methoxy)-pentyl]1-deoxynojirimycin reduces cell surface labeling by approximately 75%. In conclusion, exposure of cells to Ac(4)ManNAz allows in vivo chemical tagging of gangliosides.

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."

Integrated mass spectrometric strategy for characterizing the glycans from glycosphingolipids and glycoproteins: direct identification of sialyl Lex in mice

The current interest in applying systems biology approaches to studying an organism's form or function promises to reveal further insights into the role of glycosylation in cells and whole organisms. This has prompted the development of a rapid, sensitive method of profiling the glycan component of both glycosphingolipids and glycoproteins from a single sample. Here we report a new mass spectrometric screening strategy for characterizing glycosphingolipid-derived oligosaccharides, which can be integrated into an existing highly sensitive glycoprotein glycomics strategy. Using ceramide glycanase to release the glycans from glycosphingolipids, this method provides a reliable profile of the glycosphingolipid-derived glycans present in a sample and has revealed new glycan structures. Glycoproteins are also efficiently recovered using this method, allowing the subsequent analysis of glycoprotein-derived glycans by mass spectrometry. The high sensitivity of this glycomic screening method allowed us to directly characterize the sialyl Le(x) epitope from mouse brain for the first time, where it was observed on an O-mannose structure. Thus, we present a mass spectrometric method that allows glycomic screening of N- and O-glycans as well as glycosphingolipid-derived glycans from a single tissue.

Cellular effects of deoxynojirimycin analogues: uptake, retention and inhibition of glycosphingolipid biosynthesis.

Deoxynojirimycin (DNJ) analogues are inhibitors of ceramide glucosyltransferase (CGT), which catalyses the first step in the glucosphingolipid (GSL) biosynthetic pathway. We have synthesized a series of DNJ analogues to study the contribution of N-alk(en)yl side chains (C4, C9 or C18) to the behaviour of these analogues in cultured HL60 cells. When cells were treated for 16 h at non-cytotoxic concentrations of inhibitor, a 40-50% decrease in GSL levels was measured by HPLC analysis of GSL-derived oligosaccharides following ceramide glycanase digestion of GSL and 2-aminobenzamide labelling of the released oligosaccharides. Using a novel technique for short-term [14C]galactose labelling of cellular GSL, we used compound inhibition of GSL biosynthesis as a marker for compound uptake into cells. Surprisingly, the uptake of all three of the DNJ analogues was extremely rapid and was not dependent upon the length of the N-alk(en)yl moiety. Compound uptake occurred in less than 1 min, as shown by the complete inhibition of GSL labelling in cells treated with all the DNJ analogues. Greatly increased cellular retention of N-cis-13-octadecenyl-DNJ was observed relative to the shorter-chain compounds, N-butyl-DNJ and N-nonyl-DNJ, as indicated by complete inhibition of CGT 24 h after removal of inhibitor from the culture medium. The present study further characterizes the properties of N-alk(en)ylated DNJs, and demonstrates that increasing the length of the side chain is a simple way of improving imino sugar retention and therefore inhibitory efficacy for CGT in cultured cells.

Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling

Interest in cellular glycosphingolipid (GSL) function has necessitated the development of a rapid and sensitive method to both analyze and characterize the full complement of structures present in various cells and tissues. An optimized method to characterize oligosaccharides released from glycosphingolipids following ceramide glycanase digestion has been developed. The procedure uses the fluorescent compound anthranilic acid (2-aminobenzoic acid; 2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform and is based on the published method of Anumula and Dhume [Glycobiology 8 (1998) 685], originally used to analyze N-linked oligosaccharides. It is less time consuming than a previously published 2-aminobenzamide labeling method [Anal. Biochem. 298 (2001) 207] for analyzing GSL-derived oligosaccharides, as the fluorescent labeling is performed on the enzyme reaction mixture. The purification of 2-AA-labeled products has been improved to ensure recovery of oligosaccharides containing one to four monosaccharide units, which was not previously possible using the Anumula and Dhume post-derivatization purification procedure. This new approach may also be used to analyze both N- and O-linked oligosaccharides.

Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling

Interest in cellular glycosphingolipid (GSL) function has necessitated the development of a rapid and sensitive method to both analyze and characterize the full complement of structures present in various cells and tissues. An optimized method to characterize oligosaccharides released from glycosphingolipids following ceramide glycanase digestion has been developed. The procedure uses the fluorescent compound anthranilic acid (2-aminobenzoic acid; 2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform and is based on the published method of Anumula and Dhume [Glycobiology 8 (1998) 685], originally used to analyze N-linked oligosaccharides. It is less time consuming than a previously published 2-aminobenzamide labeling method [Anal. Biochem. 298 (2001) 207] for analyzing GSL-derived oligosaccharides, as the fluorescent labeling is performed on the enzyme reaction mixture. The purification of 2-AA-labeled products has been improved to ensure recovery of oligosaccharides containing one to four monosaccharide units, which was not previously possible using the Anumula and Dhume post-derivatization purification procedure. This new approach may also be used to analyze both N- and O-linked oligosaccharides.

Lactotetraosylceramide, a Novel Glycosphingolipid Receptor for Helicobacter pylori, Present in Human Gastric Epithelium

The binding of Helicobacter pylori to glycosphingolipids was examined by binding of (35)S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. In addition to previously reported binding specificities, a selective binding to a non-acid tetraglycosylceramide of human meconium was found. This H. pylori binding glycosphingolipid was isolated and, on the basis of mass spectrometry, proton NMR spectroscopy, and degradation studies, were identified as Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer (lactotetraosylceramide). When using non-acid glycosphingolipid preparations from human gastric epithelial cells, an identical binding of H. pylori to the tetraglycosylceramide interval was obtained in one of seven samples. Evidence for the presence of lactotetraosylceramide in the binding-active interval was obtained by proton NMR spectroscopy of intact glycosphingolipids and by gas chromatography-electron ionization mass spectrometry of permethylated tetrasaccharides obtained by ceramide glycanase hydrolysis. The lactotetraosylceramide binding property was detected in 65 of 74 H. pylori isolates (88%). Binding of H. pylori to lactotetraosylceramide on thin-layer chromatograms was inhibited by preincubation with lactotetraose but not with lactose. Removal of the terminal galactose of lactotetraosylceramide by galactosidase hydrolysis abolished the binding as did hydrazinolysis of the acetamido group of the N-acetylglucosamine. Therefore, Galbeta3GlcNAc is an essential part of the binding epitope.

Molecular cloning and expression of a novel glycolipid sulfotransferase in Mycobacterium tuberculosis The GenBank/EMBL/DDBJ accession number for the sequence (gene Rv1373) reported in this paper is Z81011.

Sulfated trehalose glycolipids are among the most characteristic cell wall molecules of virulent strains of Mycobacterium tuberculosis. They comprise a family of trehalose-2-sulfate esters with an array of acyl fatty acids at various positions of the trehalose moiety. Although their structure has been well characterized, most of the enzymes involved in their biosynthesis, such as sulfotransferases, are unknown. It is demonstrated here by metabolic labelling with 35S abundant incorporation into sulfolipids of M. tuberculosis strains, in comparison to Mycobacterium avium, Mycobacterium bovis BCG and Mycobacterium smegmatis. The most abundant sulfolipid, sulfolipid I, is present in virulent strains H37Rv and Erdman, but absent in attenuated H37Ra. Sulfotransferase assays with the donor substrate 3'-phosphoadenosine-5'-[35S]phosphosulfonate and whole cell lysates of H37Ra resulted in the synthesis of four major sulfolipids (I, II, IV and VI). A search for sulfotransferase gene sequences in M. tuberculosis yielded gene Rv1373, a 981 bp gene slightly homologous (24% identity) to eukaryotic aryl-sulfotransferases. Rv1373 was cloned by PCR and expressed as a 39 kDa recombinant his-tagged protein. The recombinant M. tuberculosis aryl-sulfotransferase exhibited activity towards the cerebroside glycolipids glucosyl- and galactosylceramide. No activity was detected with sulfatide (3'-sulfated galactosylceramide), suggesting that sulfation of galactosylceramide may occur at C-3 of the galactose. Treatment of sulfated products with ceramide glycanase resulted in the release of 35S-labelled material showing that sulfation was at the saccharide moiety (galactose or glucose) of the ceramide. Assays with the M. tuberculosis aryl-sulfotransferase and total H37Ra glycolipids showed one major product corresponding to sulfolipid IV. These results demonstrate that Rv1373 encodes a novel glycolipid sulfotransferase with activity towards typical ceramide glycolipids and mycobacterial trehalose glycolipids.

An Engineered Biocatalyst for the Synthesis of Glycoconjugates: Utilization of β1,3-N-Acetyl-D-glucosaminyltransferase from Streptococcus agalactiae Type Ia Expressed in Escherichia coli as a Fusion with Maltose-Binding Protein

A fusion protein composed of β1,3-N-acetyl-D-glucosaminyltransferase (β1,3-GlcNAcT) from Streptococcus agalactiae type Ia and maltose-binding protein (MBP) was produced in Escherichia coli as a soluble and highly active form. Although this fusion protein (MBP-β1,3-GlcNAcT) did not show any sugar-elongation activity to some simple low-molecular weight acceptor substrates such as galactose, Galβ(14)Glc (lactose), Galβ(14)GlcNAc (N-acetyllactosamine), Galβ(14)GlcNAcβ(13)Galβ(14)Glc (lacto-N-tetraose), and Galβ(14)GlcβCer (lactosylceramide, LacCer), the multivalent glycopolymer having LacCer-mimic branches (LacCer mimic polymer, LacCer primer) was found to be an excellent acceptor substrate for the introduction of a β-GlcNAc residue at the O-3 position of the non-reducing galactose moiety by this engineered enzyme. Subsequently, the polymer having GlcNAcβ(13)Galβ(14)Glc was subjected to further enzymatic modifications by using recombinant β1,4-D-galactosyltransferase (β1,4-GalT), α2,3-sialyltransferase (α2,3-SiaT), α1,3-L-fucosyltransferase (α1,3-FucT), and ceramide glycanase (CGase) to afford a biologically important ganglioside; Neu5Aα(23)Galβ(14)[Fucα(13)]GlcNAcβ(13)Galβ(14)GlcCerα(IV3Neu5Acα,III3Fucα-nLc4Cer) in 40% yield (4 steps). Interestingly, it was suggested that MBP-β1,3-GlcNAcT could also catalyze a glycosylation reaction of the LacCer mimic polymer with N-acetyl-D-galactosamine served from UDP-GalNAc to afford a polymer carrying trisaccharide branches, GalNAcβ(13)Galβ(14)Glc. The versatility of the MBP-β1,3-GlcNAcT in the practical synthesis was preliminarily demonstrated by applying this fusion protein as an immobilized biocatalyst displayed on the amylose resin which is known as a solid support showing potent binding-affinity with MBP.

An Engineered Biocatalyst for the Synthesis of Glycoconjugates: Utilization of β1,3-N-Acetyl-D-glucosaminyltransferase from Streptococcus agalactiae Type Ia Expressed in Escherichia coli as a Fusion with Maltose-Binding Protein

A fusion protein composed of β1,3-N-acetyl-D-glucosaminyltransferase (β1,3-GlcNAcT) from Streptococcus agalactiae type Ia and maltose-binding protein (MBP) was produced in Escherichia coli as a soluble and highly active form. Although this fusion protein (MBP-β1,3-GlcNAcT) did not show any sugar-elongation activity to some simple low-molecular weight acceptor substrates such as galactose, Galβ(1→4)Glc (lactose), Galβ(1→4)GlcNAc (N-acetyllactosamine), Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)Glc (lacto-N-tetraose), and Galβ(1→4)GlcβCer (lactosylceramide, LacCer), the multivalent glycopolymer having LacCer-mimic branches (LacCer mimic polymer, LacCer primer) was found to be an excellent acceptor substrate for the introduction of a β-GlcNAc residue at the O-3 position of the non-reducing galactose moiety by this engineered enzyme. Subsequently, the polymer having GlcNAcβ(1→3)Galβ(1→4)Glc was subjected to further enzymatic modifications by using recombinant β1,4-D-galactosyltransferase (β1,4-GalT), α2,3-sialyltransferase (α2,3-SiaT), α1,3-L-fucosyltransferase (α1,3-FucT), and ceramide glycanase (CGase) to afford a biologically important ganglioside; Neu5Aα(2→3)Galβ(1→4)[Fucα(1→3)]GlcNAcβ(1→3)Galβ(1→4)GlcCerα(IV3Neu5Acα,III3Fucα-nLc4Cer) in 40% yield (4 steps). Interestingly, it was suggested that MBP-β1,3-GlcNAcT could also catalyze a glycosylation reaction of the LacCer mimic polymer with N-acetyl-D-galactosamine served from UDP-GalNAc to afford a polymer carrying trisaccharide branches, GalNAcβ(1→3)Galβ(1→4)Glc. The versatility of the MBP-β1,3-GlcNAcT in the practical synthesis was preliminarily demonstrated by applying this fusion protein as an immobilized biocatalyst displayed on the amylose resin which is known as a solid support showing potent binding-affinity with MBP.

High-Performance Liquid Chromatography Analysis of Ganglioside Carbohydrates at the Picomole Level after Ceramide Glycanase Digestion and Fluorescent Labeling with 2-Aminobenzamide

The functional importance of glycolipids has emphasized the need for more sensitive methods of detection, characterization, and quantification than has often been possible using traditional thin-layer chromatographic techniques. We describe the use of ceramide glycanase and HPLC to identify and quantify gangliosides in which the carbohydrate is in Glcβ1→ linkage with ceramide. Detection of released carbohydrate was by fluorescent labeling with 2-aminobenzamide at the reducing terminal prior to HPLC analysis. Under the conditions described, ceramide glycanase hydrolyzed all of the common gangliosides studied, offering a broad spectrum of specificity. Release and detection of carbohydrate were linear over a wide range (over two orders of magnitude) of micromolar glycolipid substrate concentrations. Use of an N-linked glycan as an internal standard allowed accurate quantification and a recovery of 93% was achieved. The method additionally maintained the sensitivity (chromatographic peaks containing 1 pmol were readily detected from tissue samples) and comparable resolution to related assays. This was shown by the separation, not only of isomeric carbohydrates from the “a” and “b” series, but also of ganglioside carbohydrate differing only by the presence of either N-acetyl- or N-glycolylneuraminic acid. Application of the method to neutral glycosphingolipids and to tissue samples, including 10-μl quantities of plasma, is illustrated. Glycan structures were confirmed by exoglycosidase digestion and/or matrix-assisted laser desorption/ionization mass spectrometry.

31] Properties of animal ceramide glycanases

Ceramide glycanase (CGase), the endoglycosidase that cleaves various glycosphingolipids in a one-step process liberating ceramide and the corresponding oligosaccharides, is characterized from different annelids, bacteria, and mollusk. The same CGase activity has also been detected and purified from mammalian sources. The discovery of the mammalian CGases raises the possibility of glycosphingolipids (GSLs) being involved in the signal transduction and/or apoptotic processes as a secondary messenger. The ceramide and its breakdown product sphingosine that is very much in the limelight of apoptosis and signal transduction cascades have so far been predicted to come from the breakdown of the major membrane lipid sphingomyelin by the action of sphingomyelinase. Substantial CGase activities have been observed in different mammalian organs, although the highest activity was found in the mammary tissues. A parallel between CGase activity and the gestation as well as lactation has also been observed. However, the significance of this observation has not been established as it was for bovine mammary glucosidase. CGase activities in the kidneys and the mammary tissues of newborn rats parallel that found in the lactating mammary tissue. This chapter discusses the study of CGase characterized from mammalian sources.

Ceramide glycanase activities in human cancer cells.

Ceramide glycanase (CGase) activities have been detected in different human tumor cells (colon, carcinoma Colo-205; neuroblastoma, IMR-32; breast cancer lines, SKBr3 and MCF7). However, the level of enzymatic activity is lower in these cells compared to that present in other mammalian tissues reported before (Basu, M., Kelly, P., Girzadas, M. A., Li, Z., and Basu, S. Methods Enzymol. (in press)). The majority of CGase activity was found in the 100,000 g soluble supernatant fraction isolated from all these cell lines and tissues. Using the soluble enzyme, the requirement for optimum CGase activity was found to be consistent with previous observations found for rat and rabbit tissues (Basu, M., Dastgheib, S., Girzadas, M. A., O'Donnell, P. H., Westervelt, C. W., Li, Z., Inokuchi, J. I., and Basu, S. (1998) Acta Pol. Biochim. 42:327). The CGase activities from both Colo-205 and IMR-32 cells are optimum at a protein to detergent ratio of one. All the mammalian CGases, including human cancer cells, show an optimum pH between 5.5 and 5.8 in sodium acetate buffer. The CGase activities from cancer cells are found to be cation-independent; however, mercury, zinc, and copper ions seem to inhibit the enzyme activity substantially in both tumor cells lines. The mercury ion inhibition of CGase activities from all different sources indicates a possible structural homology in the CGase proteins. Radiolabeled substrates, labeled at the sphingosine double bond or at the 3-position of sphingosine without modifying double bond of sphingosine were used in this investigation. Both were active substrates with all enzyme preparations isolated from different cancer cells (apparent Km, 500 microM for nLcOse5[3H-DT]Cer and 350 microM for GgOse4[sph-3-3H]Cer with Colo-205 enzyme). Structural analogues of ceramide and sphingosine (L-PPMP. L-PDMP, alkylamines, and Tamoxifen) inhibited cancer cell CGase activities in vitro.