Published TCIMAIL newest issue No.196
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Endoglycoceramidase (EGCase) is a glycolipid-specific hydrolase that cleaves the glycosidic linkage between oligosaccharide and ceramide of various glycosphingolipids (GSLs).
Endoglycoceramidases (EGCases) derived from the genus Rhodococcus are endo-type hydrolases reported by Ito et al.,1) and work under detergent-containing reactive conditions in vitro. For preparation of analytical oligosaccharide samples cleaved from glycolipids extracted from cells, recombinant EGCase (rEGCase II) [R0242] is the enzyme that has generally been used.2) Moreover, rEGCase I [R0240] is capable of releasing ceramides from organic glucosylceramide.3) To strip oligosaccharides from the surface of living cells without detergent reagents, the mixture reagents, rEGCase II assisted by Activator II [R0243] and rEGCase I assisted by Activator II [R0241] are helpful because Activator II replaces the surfactant as the enzyme-activating protein.4,5)
Products
- R0240
- rEGCase I
- R0242
- rEGCase II
- R0241
- rEGCase I assisted by Activator II
- R0243
- rEGCase II assisted by Activator II
The products were commercialized under a license from Kyushu University.
Activity of EGCase (Example: Ganglioside GM1)
Hydrolysis Reaction of Ganglioside GM1 using EGCase
Recombinant EGCase (rEGCase) is activated under a detergent-dependent condition.
On the other hand, Activator II is capable of inducing activity of EGCases without any detergent reagents.
By the use of Activator II, GSLs on cell surfaces of living cells could be hydrolyzed without cell disruption caused by detergent.
Substrate Specificities of rEGCase I and rEGCase II
Each substrate (4 nmol) was incubated with EGCase and 0.1% Triton™ X-100 at 37°C.
These data were provided by Prof. Makoto Ito and Dr. Yohei Ishibashi, Kyushu University.
Practical Use of EGCase I for Cleavage of Crude Glycolipids from Natural Resource
Sample: Glycolipid extract derived from plant
Extract of plant-type glycolipid includes natural ceramides composed of several glycosphingolipids like glucosylceramide as a main component.
In terms of sphingosine and fatty acid, structural diversity of ceramides is formed depend on the number of carbon and the existence of double bond.
As shown below, several types of glycosphingolipids were simultaneously hydrolyzed by EGCase I with detergent, and resulting data showed potential molecular weights of various ceramide parts accompanied with reduction of glucose part from glucosylceramide.
References
- 1) A Novel Glycosphingolipid-degrading Enzyme Cleaves of the Linkage between the Oligosaccharide and Ceramide of Neutral and Acidic Glycosphingolipids
- 2) Purification and Characterization of Glycosphingolipid-specific Endoglycosidases (Endoglycoceramidases) from a Mutant Strain of Rhodococcus sp.
- 3) Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system
- 4) Specific hydrolysis of intact erythrocyte cell‐surface glycosphingolipids by endoglycoceramidase. Lack of modulation of erythrocyte glucose transporter by endogenous glycosphingolipids
- 5) Kinetics of endoglycoceramidase action toward cell‐surface glycosphingolipids of erythrocytes
