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Fluorescent labeling of 6-gala (neogala) series glycosphingolipids by EGALC
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Fluorescent labeling of 6-gala (neogala) series glycosphingolipids by EGALC

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Introduction Protocol References Credit lines
Category
Glycolipids and related compounds
Protocol Name

Fluorescent labeling of 6-gala (neogala) series glycosphingolipids by EGALC

Authors
Ishibashi, Yohei *
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University

Ito, Makoto
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
*To whom correspondence should be addressed.
KeyWords
Reagents

Total lipids are prepared by the method described in the section "Extraction of glycolipids"

5-Amino-1-pentanol (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan)

4-Fluoro-7nitoro-2,1,3-benzoxadiazole (NBD-F) (Sigma-Aldrich, St. Louis, MO)

Pre-coated Silica gel 60 TLC plates (Merck Millipore, Billerica, MA)

0.5 M Sodium acetate buffer, pH 5.5

HPLC grade acetonitrile (Kanto Chemical Co., Ltd.)

Chloroform (Nacalai Tesque, Inc., Kyoto, Japan)

Methanol (Nacalai Tesque, Inc.)

Endogalactosylceramidase (EGALC) (To obtain the enzyme, contact Prof. Makoto Ito, Kyushu University, makotoi@agr.kyushu-u.ac.jp)

Instruments

Sep-Pak silica cartridge (Waters Corporation, Milford, MA)

HPLC with fluorescent detector

Asahipak NH2P 50-4E, 4.6 × 250 mm (Showa Denko K. K., Tokyo, Japan)

AE-6935B Visirays (Atto Corp., Tokyo, Japan)

Shimadzu CS-9300 TLC chromatoscanner with the fluorescent detector (Shimadzu Corp., Kyoto, Japan)

Block heater

Speed vac concentrator

Methods
1.

Preparation of NBD-pentanol

1) 

 Mix 25 μmol of NBD-F and 25 μmol of 5-amino-1-pentanol. Incubate at 60°C for 1 min.

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2) 

 Dry the mixture with a speed vac concentrator, dissolve in 2 mL of chloroform, and apply to a Sep-Pak silica cartridge equilibrated with chloroform.

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3) 

 Elute NBD-pentanol from the cartridge with chloroform/methanol (9/1, v/v). Dry the eluent with a speed vac concentrator, and dissolve it in ethanol.

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4) 

 Check the quality and quantity of purified NBD-pentanol by TLC with AE-6935B Visirays and with ninhydrin reagent to detect unreacted 5-amino-1-pentanol.

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2.

Transglycosylation reaction

1) 

 Prepare 10 μL of a reaction mixture containing an appropriate amount of total lipid (>400 ng), 20 nmol of NBD-pentanol, and 10 μU of EGALC in 50 mM sodium acetate buffer, pH 5.5, and incubate at 37°C for 2 h.

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2) 

 Add 80 μL of chloroform/methanol (2/1, v/v) and 10 μL of water. After vortexing for a few seconds, centrifuge at max speed for 3 min.

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3) 

 Collect the upper phase containing NBD-pentanol-conjugated oligosaccharides, add chloroform/methanol/water (86/14/1, v/v/v), and centrifuge at max speed for 3 min.

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4) 

 Collect the upper phase, and dry it with a speed vac concentrator. TLC or HPLC is available to detect the transglycosylation products.

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3.

TLC analysis of transglycosylation products

1) 

 Dissolve a dried sample in 10 μL of methanol, and apply it to a TLC plate.

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2) 

 Develop the TLC plate with chloroform/methanol/0.02% CaCl2 (5/4/1, v/v/v) for 30 min.

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3) 

 Visualize transglycosylation products by AE-6935B Visirays or UV transiluminator.

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4) 

 Quantify products with a Shimadzu CS-9300 TLC chromatoscanner with the fluorescent detector (excitation 475 nm).

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4.

HPLC analysis of transglycosylation products

1) 

 Set excitation and emission wavelengths of the fluorescent detector to 470 and 530 nm, respectively.

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2) 

 Dissolve a dried sample in 120 μL of acetonitrile/water (9/1, v/v), and centrifuge at max speed for 5 min.

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3) 

 Apply 100 μL of the supernatant to Asahipak NH2P 50-4E equilibrated with solvent A (acetonitrile/water (9/1, v/v)). HPLC gradient conditions; 0% solvent B (acetonitrile/water (1/1, v/v)) to 100% B in 25 min at a flow rate of 1.0 mL/min.

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4) 

 Before performing the next run, equilibrate the column with 100% solvent A for 10 min.

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Notes

  This method using EGALC can be applied to glycolipids having the R-Gal(α/β)1-6Galβ1-1ceramide/diacylglycerol structure but not other GSLs such as ganglio-series, globo-series, and lacto-series GSLs.

  An excess amount of NBD-pentanol in the reaction could be removed by Folch's partition, by which NBD-pentanol and fluorescent oligosaccharides were moved to organic and aqueous phases, respectively. Then, the aqueous phase was withdrawn and used for further analysis.

  The limit of detection (LOD) of TLC was approximately 1.5 pmol (S/N=3) while that of HPLC was 50 fmol (S/N=5). The detection of the labeled oligosaccharides was linear from 4 pmol to 60 pmol for TLC, and from 250 fmol to 60 pmol for HPLC.

Figure & Legends

Figure & Legends

Fig. 1. The scheme of transglycosylation-based fluorescent labeling of 6-gala series GSLs by EGALC.

This figure was originally published in Glycobiology. 19(7):797–807. 2009 "Transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase" Ishibashi Y. Ito M. et al. Oxford University Press. 

 

 

Fig. 2. Separation of NBD-pentanol-conjugated oligosaccharides generated from CDS (Ceramide disaccharide), TGC (Trigalactosylceramide) and TeGC (Tetragalactosylceramide) on an amino column (NH2P 50-4E).

This figure was originally published in Glycobiology. 19(7):797–807. 2009 "Transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase" Ishibashi Y. Ito M. et al. Oxford University Press.

 

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