Glycobiology Advance Access originally published online on January 22, 2007
Glycobiology 2007 17(4):367-373; doi:10.1093/glycob/cwm006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Purification and structural characterization of de-N-acetylated form of GD3 ganglioside present in human melanoma tumors
2 Laboratory of Dermatological Research, University of Lyon-1 and Edouard Herriot Hospital, 69437 Lyon Cx 03, France
3 Laboratory of Biological Chemistry, CNRS UMR 8576, USTL, 59655 Villeneuve d'Ascq Cx, France
4 Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113, Japan
5 Department of Dermatology, Hotel-Dieu Hospital, Lyon, France
6 Institute of Macromolecular Chemistry Petru Poni, Aleea Gr. Ghica Voda 41A, Iassy 6600, Romania
1 To whom correspondence should be addressed; Tel: +33-4 72 11 03 07; Fax: +33-4 72 11 02 90; e-mail portoukalian{at}lyon.inserm.fr
Received on May 6, 2006; revised on December 22, 2006; accepted on January 11, 2007
The presence of gangliosides containing de-N-acetylated sialic acids in human tissues has been so far shown by using mouse monoclonal antibodies specific for the de-N-acetylated forms, but the isolation and chemical characterization of such compounds have not yet been performed. Since indirect evidence suggested that de-N-acetylGD3 ganglioside could be present in human melanoma tumors, we analyzed the gangliosides purified from a 500-g pool of those tumors. The de-N-acetylGD3 that was found to migrate just below GD2 in thin-layer chromatography was isolated from the disialogangliosides by high-pressure liquid chromatography using the specific antibody SGR37 to monitor the elution. The amount of antigen was found to be 320 ng per gram of fresh tumor or 0.1% of total gangliosides. Gas chromatography–mass spectrometry analysis of the antibody-positive ganglioside showed that sialic acids were formed of one molecule of N-acetylneuraminic acid and one molecule of neuraminic acid. Radioactive re-N-acetylation of the antigen yielded a GD3-like ganglioside with the radioactive label on the external sialic acid. The constitutive fatty acids were found to differ markedly from those of GD3 and 9-O-acetylGD3 isolated from the same pool of tumors. The major fatty acids were C16:0 and C18:0 in de-N-acetylGD3, whereas GD3 and its 9-O-acetylated derivative contained a large amount of C24:1. These data show that de-N-acetylGD3 ganglioside is indeed present in human melanoma tumors, and the fatty acid content suggests the existence of a de-N-acetylase mostly active on the molecular species of gangliosides with short-chain fatty acids.
Key words: melanoma / gangliosides / GD3 / de-N-acetylation / mass spectrometry