Document Detail


Using engineered 2-O-sulfotransferase to determine the activity of heparan sulfate C5-epimerase and its mutants.
MedLine Citation:
PMID:  20118238     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Kai Li; Heather N Bethea; Jian Liu
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2010-01-29
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  285     ISSN:  1083-351X     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2010 Apr 
Date Detail:
Created Date:  2010-04-05     Completed Date:  2010-05-04     Revised Date:  2011-07-28    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  11106-13     Citation Subset:  IM    
Affiliation:
Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
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MeSH Terms
Descriptor/Qualifier:
Carbohydrate Epimerases / 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:
AI50050/AI/NIAID NIH HHS; HL094463/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
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

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