Abstract
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.
Highlights
Agulant drug, is a special form of Heparan sulfate (HS) with higher iduronic acid content and sulfation levels
A recent study on the dermatan sulfate epimerase, an enzyme that catalyzes the conversion of the GlcUA to the IdoUA unit of chondroitin sulfate/dermatan sulfate, provides some evidence to support the idea [32]
His202, Tyr257, and His406, are found in the active site of the heparin lyase II from Pedobacter heparinus and may play catalytic roles based on the crystal structure [34]
Summary
Agulant drug, is a special form of HS with higher iduronic acid content and sulfation levels. Following the N-sulfation step, C5-epimerase (C5-epi) transforms GlcUA units into IdoUA (Fig. 1A). Because of the reversibility of the reaction catalyzed by C5-epi, the resultant polysaccharide contains both GlcUA and IdoUA units. We report a two-enzyme coupled method to measure the activity of C5-epi using a 2OST mutant (Fig. 1C). The mutant 2OST does not react with N-sulfoheparosan unless the GlcUA unit is converted to an IdoUA unit by C5-epi (Fig. 1C). This method is able to screen the activity of C5-epi mutants in relatively large numbers. Our method provides a unique application of engineered 2OST This approach should enhance the study of the mechanism of action of C5-epi
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