Journal of Biochemistry Advance Access originally published online on December 13, 2006
Journal of Biochemistry 2007 141(1):1-7; doi:10.1093/jb/mvm014
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© 2006 The Japanese Biochemical Society.
Fucosyl-GM1a, an Endoglycoceramidase-resistant Ganglioside of Porcine Brain
1Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan; 2Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; 3Faculty of Agriculture, Saga University, 1 Honjo, Saga 840-8502, Japan; 4Kinki University, 3-4-1 Kowakae, Higashiosaka, Osaka 577-8502, Japan; and 5Bio-architecture Center, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan
*To whom correspondence should be addressed. Tel: +81-92-642-2898, Fax: +81-92-642-2907, E-mail: makotoi{at}agr.kyushu-u.ac.jp
Received July 5, 2006; Accepted September 25, 2006
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Abstract |
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The use of bovine brain has been prohibited in many countries because of the world-wide prevalence of mad cow disease, and thus porcine brain is expected to be a new source for the preparation of gangliosides. Here, we report the presence of a ganglioside in porcine brain which is strongly resistant to hydrolysis by endoglycoceramidase, an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. Five major gangliosides (designated PBG-1, 2, 3, 4, 5) were extracted from porcine brain by Folch's partition, followed by mild alkaline hydrolysis and PBA column chromatography. We found that PBG-2, but not the others, was strongly resistant to hydrolysis by the enzyme. After the purification of PBG-2 with Q-Sepharose, Silica gel 60 and Prosep-PB chromatographies, the structure of PBG-2 was determined by GC, GC-MS, FAB-MS and NMR spectroscopy as Fuc
1-2Galß1-3GalNAcß1-4(NeuAc2-3)Galß1-4Glcß1-1’Cer (fucosyl-GM1a). The ceramide was mainly composed of C18:0 and C20:0 fatty acids and d18:1 and d20:1 sphingoid bases. The apparent kcat/Km for fucosyl-GM1a was found to be 30 times lower than that for GM1a, indicating that terminal fucosylation makes GM1a resistant to hydrolysis by the enzyme. This report indicates the usefulness of endoglycoceramidase to prepare fucosyl-GM1a from porcine brain.
Key Words: endoglycoceramidase, fucosyl-GM1a, ganglioside, glycosphingolipid, porcine brain
Abbreviations: BBG, bovine brain ganglioside; CBB, coomassie brilliant blue; EGCase, endoglycoceramidase; FAB-MS, fast-atom bombardment mass spectrometry; Fuc-GM1a, fucosyl-GM1a; GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry; GSL, glycosphingolipid; NMR, nuclear magnetic resonance; PBG, porcine brain ganglioside; rEGCase II, recombinant endoglycoceramidase II; TLC, thin-layer chromatography. Nomenclature system of gangliosides is followed by that of Svennerhorm (24)