Glycobiology Advance Access originally published online on July 13, 2005
Glycobiology 2005 15(12):1286-1301; doi:10.1093/glycob/cwj011
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Structure elucidation of neutral, di-, tri-, and tetraglycosylceramides from High Five cells: identification of a novel (non-arthro-series) glycosphingolipid pathway
2 Department of Biology, Georgia Institute of Technology, 309 Cherry Emerson Building, Atlanta, GA 30332-0230; 3 Faculty of Health Sciences, Department of Medical Biochemistry and Genetics, University of Copenhagen, Blegdamsvej 3, DK-2200, Copenhagen N, Denmark; and 4 Department of Chemistry, University of New Hampshire, Durham, NH 03824
1 To whom correspondence should be addressed; e-mail: slevery{at}cisunix.unh.edu
Received on March 28, 2005; revised on July 6, 2005; accepted on July 8, 2005
The major neutral glycosphingolipids (GSLs) of High Five insect cells have been extracted, purified, and characterized. It was anticipated that GSLs from High Five cells would follow the arthro-series pathway, known to be expressed by both insects and nematodes at least through the common tetraglycosylceramide (Glcß1Cer 
Manß4Glcß1Cer [MacCer] GlcNAcß3Manß4Glcß1Cer [At3Cer] GalNAcß4- GlcNAcß3Manß4Glcß1Cer [At4Cer]). Surprisingly, the structures of the major neutral High Five GSLs already diverge from the arthro-series pathway at the level of the triglycosylceramide. Studies by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS) showed the structure of the predominant High Five triglycosylceramide to be Galß3Manß4Glcß1Cer, whereas the predominant tetraglycosylceramide was characterized as GalNAc
4Galß3Manß4- Glcß1Cer. Both of these structures are novel products for any cell or organism so far studied. The GalNAc4 and Galß3 units are found in insect GSLs, but always as the fifth and sixth residues linked to GalNAcß4 in the arthro-series penta- and hexaglycosylceramide structures (At5Cer and At6Cer, respectively). The structure of the High Five tetraglycosylceramide thus requires a reversal of the usual order of action of the glycosyltransferases adding the GalNAc4 and Galß3 residues in dipteran GSL biosynthesis and implies the existence of an insect Galß3-T capable of using Manß4Glcß1Cer as a substrate with high efficiency. The results demonstrate the potential appearance of unexpected glycoconjugate biosynthetic products even in widely used but unexamined systems, as well as a potential for core switching based on MacCer, as observed in mammalian cells based on the common LacCer intermediate.
Key words: electrospray ionization / glycolipid / insect / mass spectrometry / NMR spectroscopy