Anion binding to a cationic europium(III) probe enables the first real-time assay of heparan sulfotransferase activity.

David G Fernig,Colum Breen,Edwin A Yates,Igor L Barsukov,Simon Wheeler,Sarah H Hewitt,Yong Li,Stephen J Butler,Erin Robertson

doi:10.1039/d1ob02071d

David G Fernig, Colum Breen + Show 7 more

Open Access

https://doi.org/10.1039/d1ob02071d

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Abstract

Sulfotransferases constitute a ubiquitous class of enzymes which are poorly understood due to the lack of a convenient tool for screening their activity. These enzymes use the anion PAPS (adenosine-3′-phosphate-5′-phosphosulfate) as a donor for a broad range of acceptor substrates, including carbohydrates, producing sulfated compounds and PAP (adenosine-3′,5′-diphosphate) as a side product. We present a europium(iii)-based probe that binds reversibly to both PAPS and PAP, producing a larger luminescence enhancement with the latter anion. We exploit this greater emission enhancement with PAP to demonstrate the first direct real-time assay of a heparan sulfate sulfotransferase using a multi-well plate format. The selective response of our probe towards PAP over structurally similar nucleoside phosphate anions, and over other anions, is investigated and discussed. This work opens the possibility of investigating more fully the roles played by this enzyme class in health and disease, including operationally simple inhibitor screening.

Highlights

Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]
We postulated that substituents on the distal benzene ring might affect the emission enhancements we observed in the presence of PAP and PAPS and complex EuBn, lacking the boronate ester group of EupBOH2, serves as an appropriate control complex to test our hypothesis
We have demonstrated proof-of-concept for the use of a new europium(III)-based anion receptor to monitor in real-time the activity of a heparan sulfotransferase

Summary

Introduction

Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. They exist in numerous isoforms with varying substrate preferences.[19] The sulfated products of this reaction play important roles in cell communication[20] and feature in various pathologies including those of cancer,[21] Alzheimer’s22 and the mucopolysaccharidoses.[23] Heparan sulfotransferases belong to the wider group of sulfotransferases found across nature that use the universal sulfate donor compound PAPS. Supramolecular approaches utilising cucurbituril and calixarene host molecules have been developed for monitoring enzyme reactions, such as hydrolases and methyltransferases[31,32,33] while we have previously used supramolecular anion recognition of phosphate-based species to monitor kinases.[14,15] Here we report the initial results of our investigations into heparan sulfotransferases and disclose a europium(III)-based probe capable of monitoring one such enzyme in real-time, in principle independent of both substrate and product and so applicable to any sulfotransferase catalysed reaction

Results and Discussion

Conclusion

Conflicts of interest