title: Characterization of the acid stability of glycosidically linked neuraminic acid: use in detecting de-N-acetyl-gangliosides in human melanoma.
abstract: The glycosidic linkage of sialic acids is much more sensitive to acid hydrolysis than those of other monosaccharides in vertebrates. The commonest sialic acids in nature are neuraminic acid (Neu)-based and are typically N-acylated at the C5 position. Unsubstituted Neu is thought to occur on native gangliosides of certain tumors and cell lines, and synthetic de-N-acetyl-gangliosides have potent biological properties in vitro. However, claims for their natural existence are based upon monoclonal antibodies and pulse-chase experiments, and there have been no reports of their chemical detection. Here we report that one of these antibodies shows nonspecific cross-reactivity with a polypeptide epitope, further emphasizing the need for definitive chemical proof of unsubstituted Neu on naturally occurring gangliosides. While pursuing this, we found that alpha2-3-linked Neu on chemically de-N-acetylated G(M3) ganglioside resists acid hydrolysis under conditions where the N-acetylated form is completely labile. To ascertain the generality of this finding, we investigated the stability of glycosidically linked alpha- and beta-methyl glycosides of Neu. Using NMR spectroscopy to monitor glycosidic linkage hydrolysis, we find that only 47% of Neualpha2Me is hydrolyzed after 3 h in 10 mm HCl at 80 degrees C, whereas Neu5Acalpha2Me is 95% hydrolyzed after 20 min under the same conditions. Notably, Neubeta2Me is hydrolyzed even slower than Neualpha2Me, indicating that acid resistance is a general property of glycosidically linked Neu. Taking advantage of this, we modified classical purification techniques for de-N-acetyl-ganglioside isolation using acid to first eliminate conventional gangliosides. We also introduce a phospholipase-based approach to remove contaminating phospholipids that previously hindered efforts to study de-N-acetyl-gangliosides. The partially purified sample can then be N-propionylated, allowing acid release and mass spectrometric detection of any originally existing Neu as Neu5Pr. These advances allowed us to detect covalently bound Neu in lipid extracts of a human melanoma tumor, providing the first chemical proof for naturally occurring de-N-acetyl-gangliosides.
authors.affiliation: Glycobiology Research and Training Center, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0687, USA.
authors.forename: Justin L
authors.initials: JL
authors.lastname: Sonnenburg
authors.forename: Herman
authors.initials: H
authors.lastname: van Halbeek
authors.forename: Ajit
authors.initials: A
authors.lastname: Varki
mesh_terms: D000107:Acetylation; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005679:G(M3) Ganglioside / Q000378:metabolism; D005732:Gangliosides / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008545:Melanoma / Q000378:metabolism*; D009438:Neuraminic Acids / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D005679:G(M3) Ganglioside; D005732:Gangliosides; D009438:Neuraminic Acids; C032592:N-glycolylneuraminic acid