Transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase.
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.
Ishibashi
Yohei
Y
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Nagamatsu
Yusuke
Y
Meyer
Sandra
S
Imamura
Akihiro
A
Ishida
Hideharu
H
Kiso
Makoto
M
Okino
Nozomu
N
Geyer
Rudolf
R
Ito
Makoto
M
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
D005456:Fluorescent Dyes; D006028:Glycosphingolipids; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase