Glycobiology Advance Access originally published online on April 23, 2009
Glycobiology 2009 19(7):797-807; doi:10.1093/glycob/cwp051
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Transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase
2 Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
3 Institute of Biochemistry, Medical Faculty, Justus-Liebig-University Giessen, Friedrichstrasse 24, Giessen D-35392, Germany
4 Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193
5 Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Sakyo-ku, Kyoto 606-8507
6 New Energy and Industrial Technology Development Organization (NEDO), MUZA Kawasaki Central Tower 1310, Saiwai-ku, Kanagawa 212-8554
7 Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
1 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 on March 13, 2009; accepted on March 31, 2009
Although 6-gala series glycosphingolipids possessing R-Gal (
/β) 1-6Galβ1-1'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 (/β) 1-6Gal structure.
Key words: endogalactosylceramidase / fluorescence derivatization / transglycosylation reaction / 6-gala series glycosphingolipids