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Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

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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.

Keywords:

2-O-sulfotransferase 6-Osulfotransferase Glucuronosyl C5-epimerase Heparan sulfate

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Published on: November 25, 2017

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.