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Glycobiology Advance Access published online on July 13, 2005
Glycobiology, doi:10.1093/glycob/cwj011
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org
Received March 28, 2005
Revised July 6, 2005
Accepted July 8, 2005

Article

Structure elucidation of neutral di-, tri-, and tetraglycosylceramides from High FiveTM cells. Identification of a novel (non-arthro-series) glycosphingolipid pathway

Matthew D. Fuller 1, Tilo Schwientek 2, Hans H. Wandall 2, Johannes W. Pedersen 2, Henrik Clausen 2, and Steven B. Levery 3*

1 Department of Biology, 309 Cherry Emerson Building, Georgia Institute of Technology, Atlanta GA 30332-0230, USA
2 Faculty of Health Sciences, Department of Medical Biochemistry and Genetics, University of Copenhagen, Blegdamsvej 3, DK-2200, Copenhagen N, Denmark
3 Department of Chemistry, University of New Hampshire, Durham, NH 03824, USA

* To whom correspondence should be addressed.
Steven B. Levery, E-mail: slevery{at}cisunix.unh.edu


  Abstract

The major neutral glycosphingolipids of High FiveTM insect cells have been extracted, purified, and characterized. It was anticipated that glycosphingolipids from High FiveTM cells would follow the arthro-series pathway, known to be expressed by both insects and nematodes at least through the common tetraglycosylceramide (Glc{beta}1Cer -> Man{beta}4Glc{beta}1Cer [MacCer] -> GlcNAc{beta}3Man{beta}4Glc{beta}1Cer [Ap3Cer] -> GalNAc{beta}4GlcNAc{beta}3Man{beta}4Glc{beta}1Cer [Ap4Cer]). Surprisingly, the structures of the major neutral High FiveTM glycosphingolipids already diverge from the arthro-series pathway at the level of the triglycosylceramide. Studies by 1- and 2-D nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry showed the structure of the predominant High FiveTM triglycosylceramide to be Gal{beta}3Man{beta}4Glc{beta}1Cer, while the predominant tetraglycosylceramide was characterized as GalNAc{alpha}4Gal{beta}3Man{beta}4Glc{beta}1Cer. Both of these structures are novel products for any cell or organism so far studied. The GalNAc{alpha}4 and Gal{beta}3 units are found in insect glycosphingolipids, but always as the fifth and sixth residues linked to GalNAc{beta}4 in the arthro-series penta- and hexaglycosylceramide structures (Ap5Cer and Ap6Cer, respectively). The structure of the High FiveTM tetraglycosylceramide thus requires a reversal of the usual order of action of the glycosyltransferases adding the GalNAc{alpha}4 and Gal{beta}3 residues in dipteran glycosphingolipid biosynthesis, and implies the existence of an insect Gal{beta}3-T capable of using Man{beta}4Glc{beta}1Cer 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.


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