Enzyme overexpression – an exercise toward understanding regulation of heparan sulfate biosynthesis

Jianping Fang,Ulf Lindahl,Jin-Ping Li,Tianyi Song

doi:10.1038/srep31242

Jianping Fang, Ulf Lindahl + Show 2 more

Open Access

https://doi.org/10.1038/srep31242

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Abstract

Biosynthesis of heparan sulfate (HS) involves conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) units catalyzed by glucuronyl C5-epimerase (Hsepi). IdoA units are the favored substrate for 2-O-sulfotransferase (2OST). We used HEK293 cells as a model to investigate the effects of overexpression of these enzymes on HS structure. Overexpression of Hsepi alone resulted in an unexpected increase in HS chain length. A Hsepi point-mutant (Y168A), devoid of catalytic activity, failed to affect chain length. Moreover, the effect of Hsepi overexpression on HS chain length was abolished by simultaneous overexpression of 2OST. These findings raise novel aspects on regulation of HS biosynthesis. We propose a hypothetical enzyme-binding protein (EBP) with distinct, specific and partly overlapping binding sites, the interactions of which will determine levels of enzymes available to the biosynthetic process.

Highlights

Heparan sulfate (HS) chain length, which was reverted by co-overexpression of 2OST
HS chains isolated from Clone 1, with the highest levels of mRNA expression as well as Hsepi catalytic activity, showed an elution profile on Superose-6 gel chromatography that was markedly shifted compared to elution pattern of HS from mock-transfected cells
Overexpression of Hsepi in HEK293 cells led to formation of longer HS chains compared to mock-transfected cells

Summary

Introduction

HS chain length, which was reverted by co-overexpression of 2OST. Possible mechanisms behind these findings are discussed, based on a notion of enzyme regulation through protein/protein interactions. Metabolic 35S-labeling of HS revealed an unexpected increase in chain length that correlated with Hsepi expression levels (Fig. 2C). HS chains isolated from Clone 1, with the highest levels of mRNA expression as well as Hsepi catalytic activity, showed an elution profile on Superose-6 gel chromatography that was markedly shifted compared to elution pattern of HS from mock-transfected cells.

Results

Conclusion