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Updated: Aug 8, 2025

Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity
Published on: February 17, 2017
Interacting polymer-modification enzymes in heparan sulfate biosynthesis
Tianji Zhang1, Mingjia Yu2, Honglian Li3
1Division of Chemistry and Analytical Science, National Institute of Metrology, Beijing, China; College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China; Key Laboratory of Chemical Metrology and Applications on Nutrition and Health for State Market Regulation, China.
Abstract:
Glucuronyl 5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) into L-iduronic acid (IdoA) units, through a mechanism involving reversible abstraction of a proton at C5 of hexuronic acid residues. Incubations of a [4GlcAβ1-4GlcNSO3α1-]n precursor substrate with recombinant enzymes in a D2O/H2O medium enabled an isotope exchange approach to the assessment of functional interactions of Hsepi with hexuronyl 2-O-sulfotransferase (Hs2st) and glucosaminyl 6-O-sulfotransferase (Hs6st), both involved in the final polymer-modification steps. Enzyme complexes were supported by computational modeling and homogeneous time resolved fluorescence. GlcA and IdoA D/H ratios related to product composition revealed kinetic isotope effects that were interpreted in terms of efficiency of the coupled epimerase and sulfotransferase reactions. Evidence for a functional Hsepi/Hs6st complex was provided by selective incorporation of D atoms into GlcA units adjacent to 6-O-sulfated glucosamine residues. The inability to achieve simultaneous 2-O- and 6-O-sulfation in vitro supported topologically separated reactions in the cell. These findings provide novel insight into the roles of enzyme interactions in heparan sulfate biosynthesis.
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