| Using engineered 2-O-sulfotransferase to determine the activity of heparan sulfate C5-epimerase and its mutants. | |
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MedLine Citation:
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PMID: 20118238 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Heparan sulfate (HS) is involved in essential physiological and pathophysiological functions. HS is a highly sulfated polysaccharide consisting of glucuronic acid (or iduronic acid) linked to glucosamine carrying various sulfo groups. Biosynthesis of HS involves sulfotransferases and an epimerase. The HS C(5)-epimerase converts glucuronic acid to iduronic acid. The method for determining the activity has been cumbersome due to the use of a site-specifically (3)H-labeled polysaccharide substrate. Here, we report a two-enzyme coupling assay to determine the activity of C(5)-epimerase. HS 2-O-sulfotransferase (2OST) transfers the sulfo group to the 2-OH-position of glucuronic or iduronic acid. Unlike the wild type protein, 2-O-sulfotransferase mutant (2OST Y94I) transfers sulfate to the iduronic acid but not to the glucuronic acid. Thus, 2OST Y94I cannot sulfate N-sulfated heparosan, a polysaccharide containing glucuronic acid. Incubating N-sulfated heparosan with C(5)-epimerase converts some of the glucuronic acid to iduronic acid, thus becoming a substrate for 2OST Y94I. The susceptibility of the C(5)-epimerase-treated N-sulfated heparosan to 2OST Y94I modification directly correlates to the amount of the activity of C(5)-epimerase, proving that this two-enzyme coupling system can be used to assay for C(5)-epimerase. The method was further used to determine the activities of various C(5)-epimerase mutants. Our approach will significantly reduce the complexity for assaying the activity of C(5)-epimerase and facilitate the structural and functional analysis of C(5)-epimerase. |
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Authors:
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Kai Li; Heather N Bethea; Jian Liu |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't Date: 2010-01-29 |
Journal Detail:
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Title: The Journal of biological chemistry Volume: 285 ISSN: 1083-351X ISO Abbreviation: J. Biol. Chem. Publication Date: 2010 Apr |
Date Detail:
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Created Date: 2010-04-05 Completed Date: 2010-05-04 Revised Date: 2011-07-28 |
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: 11106-13 Citation Subset: IM |
Affiliation:
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Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Carbohydrate Epimerases
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chemistry* Catalysis Catalytic Domain Disaccharides / chemistry Dose-Response Relationship, Drug Glucuronic Acid / chemistry Humans Iduronic Acid / chemistry Models, Chemical Mutagenesis Mutation Polysaccharides / chemistry Protein Engineering / methods* Sulfotransferases / chemistry* Tyrosine / chemistry |
| Grant Support | |
ID/Acronym/Agency:
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AI50050/AI/NIAID NIH HHS; HL094463/HL/NHLBI NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Disaccharides; 0/Polysaccharides; 3402-98-0/Iduronic Acid; 55520-40-6/Tyrosine; 576-37-4/Glucuronic Acid; EC 2.8.2.-/Sulfotransferases; EC 5.1.3.-/Carbohydrate Epimerases; EC 5.1.3.-/heparin-heparan sulfate-glucuronic acid C5-epimerase |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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