| Characterization of the acid stability of glycosidically linked neuraminic acid: use in detecting de-N-acetyl-gangliosides in human melanoma. | |
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MedLine Citation:
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PMID: 11884388 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Justin L Sonnenburg; Herman van Halbeek; Ajit Varki |
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Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, P.H.S. Date: 2002-03-07 |
Journal Detail:
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Title: The Journal of biological chemistry Volume: 277 ISSN: 0021-9258 ISO Abbreviation: J. Biol. Chem. Publication Date: 2002 May |
Date Detail:
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Created Date: 2002-05-13 Completed Date: 2002-07-16 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 2985121R Medline TA: J Biol Chem Country: United States |
Other Details:
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Languages: eng Pagination: 17502-10 Citation Subset: IM |
Affiliation:
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Glycobiology Research and Training Center, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0687, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Acetylation Animals Antibodies, Monoclonal CHO Cells Chromatography, High Pressure Liquid Cricetinae Cross Reactions Electrophoresis, Polyacrylamide Gel Enzyme-Linked Immunosorbent Assay G(M3) Ganglioside / metabolism Gangliosides / metabolism* Humans Hydrogen-Ion Concentration Magnetic Resonance Spectroscopy Melanoma / metabolism* Neuraminic Acids / chemistry, metabolism* Tumor Cells, Cultured |
| Grant Support | |
ID/Acronym/Agency:
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P01 CA58689/CA/NCI NIH HHS; R01-GM323373/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Antibodies, Monoclonal; 0/G(M3) Ganglioside; 0/Gangliosides; 0/Neuraminic Acids; 1113-83-3/N-glycolylneuraminic acid |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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