A facile method for controlling the reaction equilibrium of sphingolipid ceramide N-deacylase for lyso-glycosphingolipid production

Huang, Feng-Tao; Han, Yunbin; Feng, Yue; Yang, Guangyu

doi:10.1194/jlr.d061176

Cited by 10 publications

(8 citation statements)

References 46 publications

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“…The SCDase de-N-acylated derivative of GM1a (Lyso-GM1a) and SCDase de-N-acylated derivative of cellular GSLs (lyso-GSLs) were prepared by treatment with SCDase under the manufacturer's recommendation with minor modifications. 23 Briefly, GSLs were resuspended in 35 mM sodium acetate buffer (pH 5.8) containing 100 mM calcium chloride and 0.28% TDC, followed by the use of 20 mU of SCDase at 37 °C for 16 h. Lyso-GSLs were purified with Sep-Pak C 18 cartridges and then labeled with Amersham Cy3Mono-Reactive Dye under the manufacturer's recommendation. The redundant Cy3 was removed with Sephadex G-25 cartridges.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

“…In this study, we developed a novel method that can directly detect and reveal glycopatterns of GSL extracts by a lectin microarray without the need for glycan release from complex biological or clinical samples (Table S1). There were six steps to perform analysis of GSL–glycans: (i) extraction of GSLs from cell pellets; , (ii) quantification of GSL–glycans using orcinol–sulfuric acid reaction; (iii) preparation of lyso-GSLs by using the sphingolipid ceramide N - deacylase (SCDase); , (iv) fluorescence labeling of lyso-GSLs; (v) detection by a lectin microarray (Figure A); (vi) data acquisition and analysis. Simultaneously, a supplemental verification analysis for GSL–glycans was performed by MALDI-TOF MS.…”

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confidence: 99%

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Analysis of Glycosphingolipid Glycans by Lectin Microarrays

et al. 2019

Anal. Chem.

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Glycosphingolipids (GSLs) are ubiquitous glycoconjugates of cell membranes. Identification of unknown GSL− glycan structures is still a major challenge. To address this challenge, we developed a novel strategy for analysis of GSL−glycans from cultured cells based on a lectin microarray that can directly detect and reveal glycopatterns of GSL extracts without the need for glycan release. There were six steps to perform the analysis of GSL−glycans: (i) extraction of GSLs from cell pellets, (ii) quantification of GSL−glycans using orcinol−sulfuric acid reaction, (iii) preparation of lyso-GSLs by using sphingolipid ceramide N-deacylase, (iv) fluorescence labeling of lyso-GSLs, (v) detection by a lectin microarray, (vi) data acquisition and analysis. Simultaneously, a supplementary verification analysis for GSL−glycans was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Optimized experimental conditions, which consisted of the blocking buffer, incubation buffer, and appropriate GSL concentration, were investigated by analyzing the glycopatterns of a standard ganglioside (GM1a) via lectin microarray. The analysis of GSL−glycans from human hepatocarcinoma cell lines (MHCC97L, MHCC97H, and HCCLM3) showed that there were 27 lectins (e.g., WFA, MAL-II, and LTL) to give significantly different signals compared with a normal human liver cell line (HL-7702), indicating up-and/or down-regulations of corresponding glycopatterns such as α1−2 fucosylation and α2−3 sialylation, and changes of certain glycostructures such as Galβ1− 3GalNAcβ1−4(NeuAcα2−3)Galβ1−4Glc:Cer and GalNAcα1−3(Fucα1−2)Galβ1−3GlcNAcβ1−3Galβ1−4Glc:Cer. The lectin microarray analysis of lyso-GSLs labeled by fluorescence has proven to be credible, which can provide the glycopatterns and detailed linkage information on GSL−glycans.

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Section: ■ Experimental Sectionmentioning

confidence: 99%

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confidence: 99%

Analysis of Glycosphingolipid Glycans by Lectin Microarrays

et al. 2019

Anal. Chem.

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“…Because N-acylated fatty acids contribute to Gb3 heterogeneity, we removed the fatty acids from Gb3 with sphingolipid ceramide N-deacylase (SCDase (25); Fig. 1), which has previously been used for the deacylation of gangliosides (26). Then, we identified deacylated products (lyso-Gb3 and its analogs) using ultra-performance (UP)LC-MS/MS (27), enabling the determination of all types of Gb3 isoforms and analogs.…”

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confidence: 99%

Determination of globotriaosylceramide analogs in the organs of a mouse model of Fabry disease

Ishii

Taguchi

Okino

et al. 2020

Journal of Biological Chemistry

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Fabry disease is a heritable lipid disorder caused by the low activity of α-galactosidase A and characterized by the systemic accumulation of globotriaosylceramide (Gb3). Recent studies have reported a structural heterogeneity of Gb3 in Fabry disease, including Gb3 isoforms with different fatty acids and Gb3 analogs with modifications on the sphingosine moiety. However, Gb3 assays are often performed only on the selected Gb3 isoforms. To precisely determine the total Gb3 concentration, here we established two methods for determining both Gb3 isoforms and analogs. One was the deacylation method, involving Gb3 treatment with sphingolipid ceramide N-deacylase, followed by an assay of the deacylated products, globotriaosylsphingosine (lyso-Gb3) and its analogs, by ultra-performance LC coupled to tandem MS (UPLC-MS/MS). The other method was a direct assay established in the present study for 37 Gb3 isoforms and analogs/isoforms by UPLC-MS/MS. Gb3s from the organs of symptomatic animals of a Fabry disease mouse model were mainly Gb3 isoforms and two Gb3 analogs, such as Gb3(+18) containing the lyso-Gb3(+18) moiety and Gb3(−2) containing the lyso-Gb3(−2) moiety. The total concentrations and Gb3 analog distributions determined by the two methods were comparable. Gb3(+18) levels were high in the kidneys (24% of total Gb3) and the liver (13%), and we observed Gb3(−2) in the heart (10%) and the kidneys (5%). These results indicate organ-specific expression of Gb3 analogs, insights that may lead to a deeper understanding of the pathophysiology of Fabry disease.

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“…GM1 was hydrolyzed by hydrolase SCDase to form lysoGM1. 47 PLGA-lysoGM1 was synthesized by amide condensation reaction between PLGA and lysoGM1. 48 Typically, PLGA (10 KD, 1 g) and lysoGM1 were dissolved in 10 mL of dry N, N-D i m e t h y l f o r m a m i d e ( D M F ) .…”

Section: Synthesis and Characterization Of Plga-lysogm1mentioning

confidence: 99%