Structure elucidation of neutral, di-, tri-, and tetraglycosylceramides from High Five cells: identification of a novel (non-arthro-series) glycosphingolipid pathway.
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 (Glcbeta1Cer --> Manbeta4Glcbeta1Cer [MacCer] --> GlcNAcbeta3Manbeta4Glcbeta1Cer [At(3)Cer] --> GalNAcbeta4- GlcNAcbeta3Manbeta4Glcbeta1Cer [At(4)Cer]). 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 Galbeta3Manbeta4Glcbeta1Cer, whereas the predominant tetraglycosylceramide was characterized as GalNAcalpha4Galbeta3Manbeta4- Glcbeta1Cer. Both of these structures are novel products for any cell or organism so far studied. The GalNAcalpha4 and Galbeta3 units are found in insect GSLs, but always as the fifth and sixth residues linked to GalNAcbeta4 in the arthro-series penta- and hexaglycosylceramide structures (At(5)Cer and At(6)Cer, respectively). The structure of the High Five tetraglycosylceramide thus requires a reversal of the usual order of action of the glycosyltransferases adding the GalNAcalpha4 and Galbeta3 residues in dipteran GSL biosynthesis and implies the existence of an insect Galbeta3-T capable of using Manbeta4Glcbeta1Cer 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.
Fuller
Matthew D
MD
Department of Biology, Georgia Institute of Technology, 309 Cherry Emerson Building, Atlanta, GA 30332-0230, USA.
Schwientek
Tilo
T
Wandall
Hans H
HH
Pedersen
Johannes W
JW
Clausen
Henrik
H
Levery
Steven B
SB
D000818:Animals; D002460:Cell Line; D002518:Ceramides / Q000737:chemistry*; D002725:Chloroform / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006028:Glycosphingolipids / Q000737:chemistry*; D007313:Insecta; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D021241:Spectrometry, Mass, Electrospray Ionization
D002518:Ceramides; D006028:Glycosphingolipids; D002725:Chloroform; D004121:Dimethyl Sulfoxide