title: Transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase.
abstract: Although 6-gala series glycosphingolipids possessing R-Gal (alpha/beta) 1-6Gal beta 1-1'Cer have been found in some mollusks, pathogenic parasites, and fungi, their physiological functions and metabolic pathway are not fully understood. We described a novel method of detecting 6-gala series glyco- sphingolipids utilizing the specificity of endogalactosylceramidase (EGALC), which is capable of hydrolyzing 6-gala series glycosphingolipids to produce intact oligosaccharides and ceramides. EGALC catalyzes not only hydrolysis but also a transglycosylation reaction. In the latter reaction, EGALC transfers oligosaccharides from the glycosphingolipids to acceptors such as fluorescent 1-alkanols. Based on the transglycosylation reaction of EGALC, a specific, easy, fast, sensitive, and reproducible method of detecting 6-gala series glycosphingolipids was developed using NBD-pentanol as an acceptor. The fluorescent products, NBD-pentanol-conjugated 6-gala oligosaccharides, were separated and detected by TLC or HPLC with a fluorescent detector. Moreover, it was revealed that as well as glycosphingolipids, a glycoglycerolipid, digalactosyldiacylglycerol, was utilized by EGALC as a donor substrate. This method was successfully applied to detect 6-gala series glycosphingolipids in a fungus, Rhizopus oryzae, and a parasite, Taenia crassiceps. The method would be useful for studying glycosphingolipids and galactosyl glycerolipids which share the Gal (alpha/beta) 1-6Gal structure.
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Ishibashi
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nagamatsu
authors.forename: Sandra
authors.initials: S
authors.lastname: Meyer
authors.forename: Akihiro
authors.initials: A
authors.lastname: Imamura
authors.forename: Hideharu
authors.initials: H
authors.lastname: Ishida
authors.forename: Makoto
authors.initials: M
authors.lastname: Kiso
authors.forename: Nozomu
authors.initials: N
authors.lastname: Okino
authors.forename: Rudolf
authors.initials: R
authors.lastname: Geyer
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006028:Glycosphingolipids / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry*; D006031:Glycosylation; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D006028:Glycosphingolipids; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase