Copyright Notice
Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Unported license. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Bookshelf ID: NBK593955PMID: 37590690
NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.
Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.
Introduction
High-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and capillary electrophoresis have been used for the separation and detection of oligosaccharides released from glycosphingolipids (GSLs). Since intact oligosaccharides exhibit no effective absorption in the UV-Vis region, chemical derivatization of oligosaccharides with 2-aminopyridine (AP), 2-aminobenzamide (AB), 2-aminobenzoic acid (AA), or 4-aminobenzoic ethyl ester (4-ABEE) is frequently used to confer fluorescence or UV-absorption to oligosaccharides for detection (1). However, chemical derivatization is somewhat time-consuming and requires purification that may lead to a loss of samples. In this section, the enzymatic derivatization of 6-gala (neogala) series glycolipid-derived oligosaccharides using a specific enzyme, endogalactosylceramidase (EGALC, EGCase III), is described.
6-Gala series GSLs possessing R-Gal (α/β)1-6Galβ1-1’Cer have been found in some mollusks, pathogenic parasites, and fungi, but their physiological functions and metabolic pathways are not fully understood. In this section, we describe a new method to detect 6-gala series GSLs utilizing the specificity of a novel enzyme, EGALC, which is capable of hydrolyzing 6-gala series GSLs to produce intact oligosaccharides and ceramides (2). EGALC catalyzes not only hydrolysis but also the transglycosylation reaction (3). In the latter reaction, EGALC transfers oligosaccharides from the GSLs to acceptors, such as fluorescent 1-alkanols. Utilizing the transglycosylation reaction of EGALC, a specific, easy, fast, sensitive, and reproducible method for detecting 6-gala series GSLs was developed using NBD-pentanol as an acceptor (Figure 1) (4). The fluorescent products (NBD-pentanol-conjugated 6-gala oligosaccharides) were separated and detected using TLC or HPLC with a fluorescent detector. Moreover, not only GSLs but also glycoglycerolipids having the R-Gal(α/β)1-6Galβ1-1’diacylglycerol structure, such as digalactosyldiacylglycerol (DGDG), can be detected using this method. This method was successfully applied to the detection of 6-gala series GSLs in a fungus (Rhizopus oryzae) and parasite (Taenia crassiceps) (4). The method would be useful for the study of glycolipids, which share the R-Gal (α/β)1-6Gal structure. Novel glycolipid having ether-linked phytol and Galα1-6Gal moiety was discovered in green algae using this method (5).
Protocol
This chapter describes the direct fluorescent labeling of 6-gala series GSL-oligosaccharides using transglycosylation reaction of EGALC, which hydrolyzes 6-gala series GSLs into intact oligosaccharides and ceramides. First, prepare fluorescent alkanols (NBD-pentanol), then incubate 6-gala series GSLs with EGALC in the presence of fluorescent alkanols, and finally analyze the fluorescent GSL-oligosaccharides using TLC or HPLC.
Materials
- 1.
Total lipid fraction prepared using the method described in “Extraction of glycolipids”
- 2.
Pre-coated Silica gel 60 TLC plates (Merck Millipore, Billerica, MA, USA)
- 3.
5-Amino-1-pentanol (TCI, Tokyo, Japan)
- 4.
4-Fluoro-7nitro-2,1,3-benzoxadiazole (NBD-F) (Sigma-Aldrich, MO, USA)
- 5.
Sodium acetate buffer, pH 5.5
- 6.
Triton X-100 (Sigma-Aldrich)
- 7.
Chloroform (Nacalai Tesque Inc., Kyoto, Japan)
- 8.
Methanol (Nacalai Tesque, Inc.)
- 9.
Acetonitrile HPLC grade (Kanto Chemical Co. Inc., Tokyo, Japan)
- 10.
EGALC (Note 1)
- 11.
Ninhydrin reagent (Sigma-Aldrich)
Instruments
- 1.
Sep-Pak silica cartridge (Waters, MA, USA)
- 2.
Sep-Pak C18 cartridge (Waters, if necessary)
- 3.
HPLC with fluorescence detector
- 4.
Asahipak NH2P 50 4E, 4.6 × 250 mm (Shodex, Tokyo, Japan)
- 5.
AE-6935B Visirays (ATTO, Tokyo, Japan)
- 6.
Shimadzu CS-9300 TLC chromatoscanner with the fluorescence detector (SHIMADZU, Kyoto, Japan)
- 7.
Block heater
- 8.
Speed vac concentrator
- 9.
TLC developing chamber
Methods
- 1.
Preparation of NBD-pentanol
- a.
Mix 25 μmol of NBD-F and 25 μmol of 5-amino-1-pentanol and incubate at 60°C for 1 min.
- b.
Dry the mixture with a speed vac concentrator, dissolve the residue in 2 mL of chloroform, and apply the solution to a Sep-Pak silica cartridge equilibrated with chloroform.
- c.
Elute NBD-pentanol from the cartridge with 5 mL of chloroform/methanol (9/1, v/v). Dry the eluent with speed vac concentrator and dissolve it in 2 mL of ethanol.
- d.
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 (Note 2).
- 2.
Transglycosylation reaction
- a.
Evaporate an appropriate amount of total lipids (>400 ng) and 20 nmol of NBD-pentanol in a 1.5-mL tube with a speed vac concentrator.
- b.
Prepare 10 μL of a reaction mixture containing 10 μU of EGALC and 0.1% Triton X-100 in 50 mM of sodium acetate buffer, pH 5.5, and incubate the mixture at 37°C for 2 h.
- c.
Add 80 μL of chloroform/methanol (2/1, v/v) and 10 μL of water. After vortexing for a few seconds, centrifuge the mixture at max speed for 3 min.
- d.
Collect the upper phase containing NBD-pentanol-conjugated oligosaccharides, add 100 μL of chloroform/methanol/water (86/14/1, v/v/v), and centrifuge the mixture at max speed for 3 min (Note 3).
- e.
Collect the second upper phase, combine the first and second upper phases, and dry it with speed vac concentrator. You can choose TLC or HPLC to detect the transglycosylation products (Note 4).
- 3.
TLC analysis of transglycosylation products
- a.
Dissolve the dried sample of Step 2e in 10 μL of methanol and apply it to a TLC plate.
- b.
Develop the TLC plate with chloroform/methanol/0.02% CaCl2 (5/4/1, v/v/v) for 30 min.
- c.
Visualize transglycosylation products by AE-6935B Visirays or UV transilluminator.
- d.
Quantify products with a Shimadzu CS-9300 TLC chromatoscanner with the fluorescence detector (excitation 475 nm).
- 4.
HPLC analysis of transglycosylation products
- a.
Set excitation and emission wavelengths of fluorescence detector to 470 and 530 nm, respectively.
- b.
Dissolve the dried sample of Step 2e in 120 μL of acetonitrile/water (9/1, v/v) and centrifuge it at max speed for 5 min.
- c.
Apply 100 μL of the supernatant to HPLC with Asahipak NH2P 50 4E column equilibrated with solvent A (acetonitrile/water [9/1, v/v]). HPLC gradient conditions are 0% solvent B (acetonitrile/water [1/1, v/v]) to 100% B in 25 min at a flow rate of 1.0 mL/min.
- d.
Before performing the next run, equilibrate the column with 100% solvent A for 10 min.
Notes
- 1.
EGALC is not commercially available presently. For requesting EGALC, please contact the author.
- 2.
If the purity of NBD-pentanol is not enough, further purification step using a C18 Sep-Pak cartridge is required.
- a.
Dry the mixture with a speed vac concentrator, dissolve the residue in 2 mL of water/methanol (9/1, v/v), and apply the solution on a Sep-Pak C18 cartridge conditioned by methanol and equilibrated with water/methanol (9/1, v/v).
- b.
Wash the cartridge with 5 mL of water/methanol (9/1, v/v) and water/methanol (5/1, v/v) then elute NBD-pentanol from the cartridge with 5 mL of methanol. Dry the eluent with a speed vac concentrator and dissolve it in 2 mL of ethanol.
- 3.
Removal of unreacted excess reagent is necessary for the appropriate separation and quantification of fluorescent derivatives. For this purpose, Folch’s partition can be used wherein NBD-pentanol is moved to the organic phase, while almost all fluorescent oligosaccharides are moved to the water phase without a drop in yield.
- 4.
The limit of detection (LOD) of fluorescent-GLSs by TLC is ~1.5 pmol (S/N = 3) and that of HPLC is 50 fmol (S/N = 5). The detection of the labeled oligosaccharide is linear from 4 pmol to 60 pmol in TLC and from 250 fmol to 60 pmol in HPLC.he limit of detection (LOD) of fluorescent-GLSs by TLC is ~1.5 pmol (S/N = 3) and that of HPLC is 50 fmol (S/N = 5). The detection of the labeled oligosaccharide is linear from 4 pmol to 60 pmol in TLC and from 250 fmol to 60 pmol in HPLC.
References
- 1.
- Pabst M, Kolarich D, Pöltl G, Dalik T, Lubec G, Hofinger A, Altmann F. Comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method. Anal Biochem. 2009 Jan 15;384(2):263–73. [PubMed: 18940176] [CrossRef]
- 2.
- Ishibashi Y, Nakasone T, Kiyohara M, Horibata Y, Sakaguchi K, Hijikata A, Ichinose S, Omori A, Yasui Y, Imamura A, Ishida H, Kiso M, Okino N, Ito M. A novel endoglycoceramidase hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides. J Biol Chem. 2007 Apr 13;282(15):11386–96. [PubMed: 17244618] [CrossRef]
- 3.
- Ishibashi Y, Kiyohara M, Okino N, Ito M. Synthesis of fluorescent glycosphingolipids and neoglycoconjugates which contain 6-gala oligosaccharides using the transglycosylation reaction of a novel endoglycoceramidase (EGALC). J Biochem. 2007 Aug;142(2):239–46. [PubMed: 17567653] [CrossRef]
- 4.
- Ishibashi Y, Nagamatsu Y, Meyer S, Imamura A, Ishida H, Kiso M, Okino N, Geyer R, Ito M. Transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase. Glycobiology. 2009 Jul;19(7):797–807. [PubMed: 19389917] [CrossRef]
- 5.
- Ishibashi Y, Nagamatsu Y, Miyamoto T, Matsunaga N, Okino N, Yamaguchi K, Ito M. A novel ether-linked phytol-containing digalactosylglycerolipid in the marine green alga, Ulva pertusa. Biochem Biophys Res Commun. 2014 Oct 3;452(4):873–80. [PubMed: 25157808] [CrossRef]
Footnotes
The authors declare no competing or financial interests.
Figures
Figure 1:
The scheme of transglycosylation-based fluorescent labeling of 6-gala series glycosphingolipids using endogalactosylceramidase.