Abstract
Humans express at least two distinct β-glucuronidase enzymes that are involved in disease: exo-acting β-glucuronidase (GUSB), whose deficiency gives rise to mucopolysaccharidosis type VII, and endo-acting heparanase (HPSE), whose overexpression is implicated in inflammation and cancers. The medical importance of these enzymes necessitates reliable methods to assay their activities in tissues. Herein, we present a set of β-glucuronidase-specific activity-based probes (ABPs) that allow rapid and quantitative visualization of GUSB and HPSE in biological samples, providing a powerful tool for dissecting their activities in normal and disease states. Unexpectedly, we find that the supposedly inactive HPSE proenzyme proHPSE is also labeled by our ABPs, leading to surprising insights regarding structural relationships between proHPSE, mature HPSE, and their bacterial homologs. Our results demonstrate the application of β-glucuronidase ABPs in tracking pathologically relevant enzymes and provide a case study of how ABP-driven approaches can lead to discovery of unanticipated structural and biochemical functionality.
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Acknowledgements
We thank the Diamond Light Source for access to beamlines i02, i03 and i04 (proposals mx-9948 and mx-13587), which contributed to the results presented here. We acknowledge the Netherlands Organization for Scientific Research (NWO, ChemThem Grant to J.M.F.G.A. and H.S.O.), the China Scholarship Council (CSC, PhD Grant to J.J.), the European Research Council (ErC-2011-AdG-290836 to H.S.O.; ErC-2012-AdG-322942 to G.J.D.), and the Royal Society (Ken Murray Research Professorship to G.J.D.) for financial support.
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L.W., J.M.F.G.A., H.S.O. and G.J.D. conceived and designed the experiments. J.J., M.A., W.D. and C.v.E. carried out synthesis of probes, with guidance from G.A.v.d.M. and J.D.C.C. L.W. and Y.J. carried out protein expression and structural studies on enzyme-probe complexes. J.J., L.W., W.W.K. and C.-L.K. carried out gel labeling experiments. J.J. and B.I.F. carried out proteomics experiments. C.-L.K. and W.W.K. determined IC50 and kinetic parameters for ABP inhibition. M.v.E. obtained tissue samples. L.W., J.J., H.S.O., and G.J.D. wrote the manuscript with input from all authors.
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Supplementary Results, Supplementary Tables 1–2, Supplementary Figures 1–12 and Supplementary Note 1 (PDF 4578 kb)
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Synthesis and compound characterization details (PDF 6444 kb)
Supplementary Dataset 1
Full mass spectrometry datasets for proteomics experiments. (XLSX 2614 kb)
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Wu, L., Jiang, J., Jin, Y. et al. Activity-based probes for functional interrogation of retaining β-glucuronidases. Nat Chem Biol 13, 867–873 (2017). https://doi.org/10.1038/nchembio.2395
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DOI: https://doi.org/10.1038/nchembio.2395
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