Purification and characterization of glycosphingolipid-specific endoglycosidases (endoglycoceramidases) from a mutant strain of Rhodococcus sp. Evidence for three molecular species of endoglycoceramidase with different specificities.
Two molecular species of endoglycoceramidase (designated as endoglycoceramidases I and II) were purified 32,700 and 43,000 times with overall recoveries of 4.8 and 2.9%, respectively, from a culture fluid of the mutant strain M-750 of Rhodococcus sp., cultivated in the absence of inducers (ganglioside). After being stained with Coomassie Brilliant Blue or a silver-staining solution, each purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The apparent molecular weights, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 55,900 for endoglycoceramidase I and 58,900 for endoglycoceramidase II, and their pIs were 5.3 and 4.5, respectively. both were capable of hydrolyzing the glucosylceramide linkage of ganglio-type, lacto-type, and globo-type glycosphingolipids to afford intact oligosaccharides and ceramides. Globo-type glycosphingolipids were strongly resistant to hydrolysis by endoglycoceramidase II in comparison with endoglycoceramidase I. Neither could hydrolyze gala-type glycosphingolipids, cerebrosides, sulfatides, glycoglycerolipids, or sphingomyelins. In addition to these two enzymes, the strain M-750 produced a third minor molecular species of endoglycoceramidase designated as endoglycoceramidase III. It was found capable of specifically hydrolyzing the galactosylceramide linkage of gala-type glycosphingolipids that were not hydrolyzable at all by endoglycoceramidases I or II. The molecular weights of the oligosaccharide and ceramide released from asialo GM1, incubated either in normal H2O or H2(18)O with the enzyme, were compared by fast atom bombardment-mass spectrometry. The result clearly indicated that both endoglycoceramidases I and II hydrolyze the glycosidic linkage between the oligosaccharide and ceramide. Thus, a systematic name of the endoglycoceramidase should be glycosyl-N-acyl-sphingosine 1,1-beta-D-glucanohydrolase.
Ito
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Laboratory of Glycoconjugate Research, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Yamagata
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T
D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D006028:Glycosphingolipids / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D017830:Octoxynol; D011092:Polyethylene Glycols; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D006028:Glycosphingolipids; D011092:Polyethylene Glycols; D017830:Octoxynol; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase