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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.
Carbohydrate Polymers
|March 6, 2023
Summary
Heparan sulfate biosynthesis involves enzyme interactions. Glucuronyl 5-epimerase (Hsepi) functionally interacts with sulfotransferases, revealing insights into heparan sulfate production.
Area of Science:
- Biochemistry
- Glycoscience
- Enzymology
Background:
- Heparan sulfate (HS) biosynthesis is a complex process involving multiple enzymes.
- Glucuronyl 5-epimerase (Hsepi) catalyzes the conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA), a key step in HS structure.
- Sulfotransferases, such as Hs2st and Hs6st, perform crucial polymer modification steps in HS synthesis.
Purpose of the Study:
- To investigate the functional interactions between Hsepi and key sulfotransferases (Hs2st and Hs6st) during heparan sulfate biosynthesis.
- To elucidate the kinetic mechanisms and efficiency of coupled epimerase and sulfotransferase reactions.
- To provide novel insights into the roles of enzyme complexes in the cellular context of HS production.
Main Methods:
- Utilized an isotope exchange approach with a D2O/H2O medium and a specific precursor substrate.
- Employed recombinant enzymes, including Hsepi, Hs2st, and Hs6st.
- Integrated computational modeling and homogeneous time-resolved fluorescence (HTRF) to support enzyme complex findings.
- Analyzed GlcA and IdoA deuterium-to-hydrogen (D/H) ratios to determine kinetic isotope effects.
Main Results:
- Demonstrated functional interaction between Hsepi and Hs6st, evidenced by selective deuterium incorporation into GlcA units adjacent to 6-O-sulfated glucosamine.
- Identified kinetic isotope effects reflecting the efficiency of coupled epimerase and sulfotransferase reactions.
- Showed that simultaneous 2-O- and 6-O-sulfation could not be achieved in vitro, suggesting topologically separated reactions within the cell.
Conclusions:
- Enzyme interactions play a critical role in the regulation and efficiency of heparan sulfate biosynthesis.
- A functional complex between Hsepi and Hs6st facilitates specific modifications during HS production.
- The findings suggest spatial compartmentalization of sulfotransferase activities in the cellular environment for HS synthesis.
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