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

Wing,; Garner, Brett; Hunnam, Victoria; Reinkensmeier, Gabriele; Andersson, Ulrika; Harvey, David J.; Dwek, Raymond A.; Platt, Frances M.; Butters, Terry D.

doi:10.1006/abio.2001.5393

Cited by 57 publications

(57 citation statements)

References 32 publications

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“…The oligosaccharides released by EGCases could be labeled by aromatic amine-based fl uorescent reagents (18)(19)(20)(21)(22) and analyzed sensitively by HPLC and MS or LC-MS ( 23 ). Recently, Fujitani et al ( 24 ) reported the cellular glycomics of GSLs based on the preparation of oligosaccharides with rhodococcal EGCase followed by glycoblotting and MALDI-TOF/TOF-MS. Jellyfi sh EGCase and rhodococcal EGALC were also utilized for the synthesis of fl uorescent GSLs and neoglycoconjugates by utilizing their transglycosylation reactivity ( 25,26 ).…”

Section: Expression and Purifi Cation Of The Recombinant Egcase Imentioning

confidence: 99%

Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Ishibashi

Kobayashi

Hijikata

et al. 2012

Journal of Lipid Research

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This article is available online at http://www.jlr.org Glycosphingolipids (GSLs), amphipathic compounds consisting of oligosaccharides and ceramide (Cer) moieties, are ubiquitous in the outer leafl et of the plasma membrane. More than 500 species of GSLs differing in oligosaccharides and Cer moieties have been characterized, some of which represent potential biomarkers for diseases and cell development. For instance, Forssman antigen is specifi cally detected in some gastric, colonic, and lung cancers ( 1 ), and stage-specifi c embryonic antigen (SSEA)-3 and SSEA-4, highly expressed at a stage of embryonic development, are utilized as a marker of embryonic stem cells ( 2 ) and induced pluripotent stem cells ( 3 ).The classifi cation of GSLs, into ganglio-series, globoseries and lacto-series GSLs, is entirely based on the structure of the saccharide moiety. However, the Cer moiety, composed of a sphingoid base and fatty acyl chain linked by an N -acyl linkage, shows heterogeneity in carbon chain length, and saturation and hydroxylation status, etc. The complexity of the Cer moiety prevents the comprehensive analysis of cellular GSLs by MS and/or LC-MS. Removal of the Cer moiety makes the analysis of GSLs much simpler and easier. For this purpose, chemical methods such as ozonolysis ( 4 ) and osmium-catalyzed periodate oxidation ( 5 ) have been developed. These chemical methods, however, require complicated and time-consuming procedures with relatively low yield ( 6 ). Alternatively, enzymes capable of detaching intact oligosaccharides from various GSLs Abstract Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the ␤ -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specifi city were found in the culture fl uid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (k cat ) ( k cat ) of the recombinant EGCase I to be 22-and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the K m of both enzymes was almost the same for these substrates.

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Section: Expression and Purifi Cation Of The Recombinant Egcase Imentioning

confidence: 99%

Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Ishibashi

Kobayashi

Hijikata

et al. 2012

Journal of Lipid Research

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“…Glycan separation was achieved using a gradient of 50 mM ammonium formate, pH 4.4, in acetonitrile as previously described (3, 13). The major murine plasma GSL detected using this method is N-glycolyl GM2, which accounts for ?90% of total plasma gangliosides (18).…”

Section: Analysis Of Plasma Lipidsmentioning

confidence: 99%

“…Because free glucose interferes with the HPLC assay for glucosylceramide (GlcCer) (18), plasma samples were pooled (from 10 mice, to give a sample volume of 0.3 ml), and the isolated GSLs were analyzed by TLC to assess changes in GlcCer concentration. The total cholesterol content of the two pooled plasma samples was also measured and found to vary by ,3%.…”

Section: Analysis Of Plasma Lipidsmentioning

confidence: 99%

Reduction of plasma glycosphingolipid levels has no impact on atherosclerosis in apolipoprotein E-null mice

Glaros

Kim

Rye

et al. 2008

Journal of Lipid Research

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Glycosphingolipids (GSLs) have been implicated as potential atherogenic lipids. Studies in apolipoprotein E-null (apoE 2/2 ) mice indicate that exacerbated tissue GSL accumulation resulting from a-galactosidase deficiency promotes atherosclerosis, whereas the serine palmitoyl transferase inhibitor myriocin (which reduces plasma and tissue levels of several sphingolipids, including sphingomyelin, ceramide, sphingosine-1-phosphate, and GSLs) inhibits atherosclerosis. It is not clear whether GSL synthesis inhibition per se has an impact on atherosclerosis. To address this issue, apoE 2/2 mice maintained on a high-fat diet were treated with a potent glucosylceramide synthesis inhibitor, D-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4), 10 mg/kg/day for 94 days, and lesion development was compared in mice that were treated with vehicle only. EtDO-P4 reduced plasma GSL concentration by approximately 50% but did not affect cholesterol or triglyceride levels. Assessment of atherosclerotic lesions at four different sites indicated that EtDO-P4 had no significant impact on lesion area. Thus, despite the previously observed positive correlations between plasma and aortic GSL concentrations and the development of atherosclerosis, and the in vitro evidence implying that GSLs may be pro-atherogenic, our current data indicate that inhibition of GSL synthesis does not inhibit atherosclerosis in vivo.-Glaros, E. N., W. S. Kim, K-A. Rye, J. A. Shayman, and B. Garner . Reduction of plasma glycosphingolipid levels has no impact on atherosclerosis in apolipoprotein E-null mice.

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“…3.2.1.123; Calbiochem) in incubation buffer was added to cleave the glycans. Glucosylceramide is partially digested because of the specificity of the glycanase (Wing et al, 2001). After this period, 10 l of water was added to each enzyme digest followed by 80 l of anthranilic acid and sodium cyanoborohydride to each digest and incubated in the 80°C oven for 1 h. Derivatized oligosaccharides were then purified on DPA-6S (Supelco, Bellefonte, PA) columns preequilibrated with 2 ϫ 1 ml CH 3 CN.…”

Section: Quantitation Of Gsl Derivatives By High-performance Liquid Cmentioning

confidence: 99%

The Association of Shiga-like Toxin with Detergent-resistant Membranes Is Modulated by Glucosylceramide and Is an Essential Requirement in the Endoplasmic Reticulum for a Cytotoxic Effect

Smith

Sillence

Jarvis

et al. 2006

MBoC

103

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Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the productive intoxication of susceptible mammalian cells by Shiga-like toxin-1 (SLTx). Recently, it has been proposed that the observed association of certain ER-directed toxins and viruses with detergent-resistant membranes (DRM) may provide a general mechanism for their retrograde transport to endoplasmic reticulum (ER). Here, we show that DRM recruitment of SLTx bound to its globotriosylceramide (Gb 3 ) receptor is mediated by the availability of other glycosphingolipids. Reduction in glucosylceramide (GlcCer) levels led to complete protection against SLTx and a reduced cell surface association of bound toxin with DRM. This reduction still allowed efficient binding and transport of the toxin to the ER. However, toxin sequestration within DRM of the ER was abolished under reduced GlcCer conditions, suggesting that an association of toxin with lipid microdomains or rafts in the ER (where these are defined by detergent insolubility) is essential for a later step leading to or involving retro-translocation of SLTx across the ER membrane. In support of this, we show that a number of ER residents, proteins intimately involved in the process of ER dislocation of misfolded proteins, are present in DRM.

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

Cited by 57 publications

References 32 publications

Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Reduction of plasma glycosphingolipid levels has no impact on atherosclerosis in apolipoprotein E-null mice

The Association of Shiga-like Toxin with Detergent-resistant Membranes Is Modulated by Glucosylceramide and Is an Essential Requirement in the Endoplasmic Reticulum for a Cytotoxic Effect

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