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Characterization of the acid stability of glycosidically linked neuraminic acid: use in detecting de-N-acetyl-gangliosides in human melanoma.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2002 May 17; Vol. 277 (20), pp. 17502-10. Date of Electronic Publication: 2002 Mar 07. - Publication Year :
- 2002
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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.
- Subjects :
- Acetylation
Animals
Antibodies, Monoclonal
CHO Cells
Chromatography, High Pressure Liquid
Cricetinae
Cross Reactions
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay
G(M3) Ganglioside metabolism
Humans
Hydrogen-Ion Concentration
Magnetic Resonance Spectroscopy
Neuraminic Acids chemistry
Tumor Cells, Cultured
Gangliosides metabolism
Melanoma metabolism
Neuraminic Acids metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 277
- Issue :
- 20
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 11884388
- Full Text :
- https://doi.org/10.1074/jbc.M110867200