Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 285, Issue 15, Pages 11106-11113Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M109.081059
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Funding
- National Institutes of Health [AI50050, HL094463]
- American Heart Association
- MidAtlantic Affiliate [0855424E]
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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 H-3-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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