Hydrolysis of glycosphingolipids by a sphingolipid ceramide N-deacylase (SCDase)

Authors：

Category

Glycosidases & related proteins

Protocol Name

Authors

Okino, Nozomu *
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

Lyso-glycosphingolipid Sphingolipid ceramide N-deacylase (SCDase) Glycosphingolipid

Reagents

●	Sphingolipid ceramide N-deacylase (SCDase) from Pseudomonas sp. TK4 (Takara Bio Inc., Otsu, Japan)
●	TLC plate (Silicagel 60, 20 × 20 cm, Merck Millipore, Billerica, MA)
●	chloroform (CHCl₃)
●	methanol (MeOH)
●	CHCl₃-MeOH mixture (C/M, volume/volume)
●	Orcinol reagent: Dissolve 250 mg of orcinol in 50 mL of H₂SO₄, and make up to 100 mL with DW. Store the reagent in a refrigerator.
●	Ninhydrin reagent: Dissolve 250 mg of ninhydrin in 100 mL of water-saturated n-butanol. Store the reagent in a refrigerator.

Instruments

●	TLC developing chamber (inside, to 24 cm × to 11 cm × to 21 cm)
●	Hot plate
●	TLC Chromatoscanner (CS-9300, Shimadzu Corp., Kyoto, Japan)

Methods

Hydrolysis of glycosphingolipids by a sphingolipid ceramide N-deacylase (SCDase)

1)	Dissolve the glycosphingolipids (GSLs, to 10 nmol) in 10 μL of 2x reaction buffer (50 mM acetate buffer, pH 6.0 containing 1.6% Triton X-100).	Comment 0

2)	Add 10 μL of SCDase (to 1 mU) and incubate at 37°C for an appropriate period (to 1 h). Stop the reaction by heating in a boiling water bath for 5 min. This step can be omitted depending on the purpose of the experiment.	Comment 0

3)	Dry the sample with a Speed Vac concentrator.	Comment 0

4)	Dissolve the dried sample in a small amount of CHCl₃/MeOH (1/2 or 2/1, v/v).	Comment 0

5)	Apply the sample to a TLC plate using a micro-syringe.	Comment 0

6)	Develop the TLC plate with an appropriate developing solvent in a TLC chamber.	Comment 0

7)	GSLs and lyso-GSLs can be visualized by orcinol reagent while only lyso-GSLs can be visualized by ninhydrin reagent. Dry the plate, spray on the orcinol reagent, and heat on a hot plate at 110°C until GSL and lyso-GSL bands can be visualized (to 5 min). Alternatively, heat the TLC plate on a hot plate at 110°C for 5 min, and then spray on the ninhydrin reagent.	Comment 0

To calculate the rate of hydrolysis, scan the TLC plate with a Shimadzu CS-9300 chromatoscanner with the reflectance mode set at 540 nm. (The extent of hydrolysis was calculated as follows: hydrolysis (%) = (peak area for lyso-GSLs generated) × 100/(peak area for remaining GSLs + peak area for lyso-GSLs generated). One enzyme unit is defined as the amount capable of catalyzing the release of 1 μmol of lyso-GM1/min from GM1 under the conditions indicated above.)

Comment 0

Notes

R_f values are smaller for lyso-GSLs than the parental GSLs. Since lyso-GSLs have one free amino group per molecule, they are also detectable with a ninhydrin reagent. In the case of the SCDase from Shewanella alga G8 ²⁾, a 50 mM acetate buffer, pH 6.0 containing 0.4% Triton X-100 and 10 mM CaCl₂ should be used for the 2x reaction buffer. The reverse hydrolysis reaction of the SCDase prevents the hydrolysis reaction, but use of an aqueous–organic biphasic system improves the efficiency of hydrolysis ³⁾. We have developed a new method of quantifying GlcCer and GalCer by normal-phase HPLC using O-phtalaldehyde derivatives prepared with SCDase ⁴⁾. (See “Simultaneous quantification of glucosylceramide and galactosylceramide by HPLC” of GlycoPOD)

Figure & Legends

Fig. 1. Action mode of SCDase.

Copyrights

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Date of registration：2014-07-31 09:59:24