Using engineered 2-O-sulfotransferase to determine the activity of heparan sulfate C5-epimerase and its mutants.
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.
Li
Kai
K
Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Bethea
Heather N
HN
Liu
Jian
J
D002238:Carbohydrate Epimerases / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D004187:Disaccharides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D020723:Glucuronic Acid / Q000737:chemistry; D006801:Humans; D007067:Iduronic Acid / Q000737:chemistry; D008956:Models, Chemical; D016296:Mutagenesis; D009154:Mutation; D011134:Polysaccharides / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D015238:Sulfotransferases / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry
D004187:Disaccharides; D011134:Polysaccharides; D007067:Iduronic Acid; D014443:Tyrosine; D020723:Glucuronic Acid; D015238:Sulfotransferases; D002238:Carbohydrate Epimerases; C430881:heparin-heparan sulfate-glucuronic acid C5-epimerase