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Degradation of Glycolipids by Endoglycoceramidase
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Degradation of Glycolipids by Endoglycoceramidase

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

Degradation of Glycolipids by Endoglycoceramidase

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
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Triton X-100 (Sigma-Aldrich, St. Louis, MO)

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

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Orcinol-H2SO4 reagent (see “Thin-layer chromatography of glycolipids”)

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Sodium acetate buffer, pH 5.5

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Chloroform (Nacalai Tesque Inc., Kyoto, Japan)

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Methanol (Nacalai Tesque, Inc.)

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EGCase II (Recombinant enzyme expressed in E. coli is commercially available from Takara Bio Inc., Otsu, Japan)

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EGCase I* (see Notes)

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EGALC* (see Notes)

Instruments
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Shimadzu CS-9300 TLC chromatoscanner (Shimadzu Corp., Kyoto, Japan)
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Speed vac concentrator (Thermo Fisher Scientific Inc., Waltham, MA)
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TLC developing chamber
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Hot plate
Methods
1.

Degradation of GSLs by EGCase

1) 

 Evaporate 2 nmol of each GSL in an Eppendorf tube with a speed bac concentrator when the sample is dissolved in an organic solvent.

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2) 

 Dissolve the sample in 50 mM sodium acetate buffer, pH 5.5 containing 0.1% (w/v) Triton X-100, and sonicate for 10 sec.

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3) 

 Add 0.5 mU of EGCase I or EGCase II for lacto- and ganglio-series GSLs, 10 mU of EGCase I for globo-series GSLs, and 1 mU of EGALC for 6-gala series GSLs. The volume of the reaction mixture should be 10–50 μL. One milliunit of enzyme is defined as that capable of catalyzing the hydrolysis of 1 nmol of GM1a per min.

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4) 

 Incubate the reaction mixture at 37°C for 1 h for the hydrolysis of ganglio- and 6-gala series GSLs and for 10 h for the hydrolysis of lacto- and globo-series GSLs. Stop the reaction by heating in a boiling water bath for 5 min.

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5) 

 Evaporate the reaction mixture with a speed bac concentrator. Dissolve the sample in 10 μL of methanol and then apply to a TLC plate (see “Thin-layer chromatography of glycolipids”).

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6) 

 Develop the TLC plate with chloroform/methanol/0.02% CaCl2 (5/4/1, v/v/v) for globo-, ganglio-, and lacto-series GSLs and chloroform/methanol/0.02% CaCl2 (2/3/1, v/v/v) for 6-gala-series GSLs.

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2.

Detection of products by TLC chromatoscanner.

1) 

 Visualize the GSLs remaining and oligosaccharides generated by spraying the TLC plate with orcinol-H2SO4 reagent.

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2) 

 Scan the band intensities of TLC using a Shimadzu CS-9300 chromatoscanner with the reflection mode set at 540 nm.

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3) 

 Calculate the extent of hydrolysis as follows: hydrolysis (%) = (peak area for oligosaccharide) × 100 / (peak area for oligosaccharide + peak area for remaining substrate).

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Notes

The specificity of EGCase I is somewhat different from that of EGCase II as shown in Table I. Kinetic study revealed that the Michaelis constant (Km) toward GM1a (ganglio series) and Gb3Cer (globo series) of EGCase I is almost the same of that of EGCase II (0.4 mM). On the other hand, rate of the turnover number (kcat) of EGCase I for GM1a and Gb3Cer is 22- and 1,209-fold that of EGCase II, respectively. The nonionic detergent Triton X-100 enhances the activity of EGCase I, II and EGALC at a concentration of 0.1%. No metal ions are required for the reactions of these enzymes. Not only 6-gala series GSLs but also DGDG can be hydrolyzed by EGALC 4).

 

[Reagents]

*If you need EGCase I and EGALC, please contact Dr. Makoto Ito at Kyushu University.

Figure & Legends

Figure & Legends

Fig. 1. Action points of EGCase I, II and III (EGALC) on GSLs.

 

 

Table 1. Substrate specificities of EGCase I, II and III (EGALC).

Various GSLs (2 nmol) were incubated at 37°C for 12 h with 1 mU (10 mU) of enzyme in 20 mL of 50 mM sodium acetate buffer, pH 5.5, containing 0.1% Triton X-100. Values for EGALC were from ref. 3.

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