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.

  • Compound

    (1R,2S,3R,4S,5S,6R)-7-(8-(4-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)butyl)-1H-1,2,3-triazol-1-yl)octyl)-3,4,5-trihydroxy-7-azabicyclo[4.1.0]heptane-2-carboxylic acid

  • Compound

    (1R,2S,3R,4S,5S,6R)-7-(8-(4-(4-(5,5-difluoro-3,7-bis(4-methoxyphenyl)-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)butyl)-1H-1,2,3-triazol-1-yl)octyl)-3,4,5-trihydroxy-7-azabicyclo[4.1.0]heptane-2-carboxylic acid

  • Compound

    1-(5-(((1-(8-((1R,2S,3R,4S,5S,6R)-2-carboxy-3,4,5-trihydroxy-7-azabicyclo[4.1.0]heptan-7-yl)octyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-5-oxopentyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium

  • Compound

    (1R,2S,3R,4S,5S,6R)-3,4,5-trihydroxy-7-(8-(4-((6-(4-((3aR,4S,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)butanamido)hexanamido)methyl)-1H-1,2,3-triazol-1-yl)octyl)-7-azabicyclo[4.1.0]heptane-2-carboxylic acid

  • Compound

    (1R,2S,3R,4S,5S,6R)-7-(8-azidooctyl)-3,4,5-trihydroxy-7-azabicyclo[4.1.0]heptane-2-carboxylic acid

  • Compound

    (1R,2S,3R,4S,5S,6R)-3,4,5-trihydroxy-7-azabicyclo[4.1.0]heptane-2-carboxylic acid

  • Compound

    (1R,2R,3R,4S,5R,6S)-3,4,5-trihydroxy-7-oxabicyclo[4.1.0]heptane-2-carboxylic acid

  • Compound

    (2S,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid

  • Compound

    (1S,2R,3S,4R,5R,6R)-5-((4-(4-(5,5-difluoro-3,7-bis(4-methoxyphenyl)-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)butyl)-1H-1,2,3-triazol-1-yl)methyl)-7-oxabicyclo[4.1.0]heptane-2,3,4-triol

  • Compound

    (2S,3S)-N2-((S)-1-(benzylamino)-1-oxo-3-phenylpropan-2-yl)-N3-(4-hydroxyphenethyl)oxirane-2,3-dicarboxamide

  • Compound

    benzyl ((S)-1-(((S)-1-(4-(tert-butoxy)phenyl)-4-diazo-3-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate

  • Compound

    (1R,2S,3S,4R,5R,6R)-7-(8-azidooctyl)-5-(hydroxymethyl)-7-azabicyclo[4.1.0]heptane-2,3,4-triol

  • Compound

    5,5-difluoro-10-(hex-5-yn-1-yl)-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine

  • Compound

    5,5-difluoro-10-(hex-5-yn-1-yl)-3,7-bis(4-methoxyphenyl)-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine

  • Compound

    3,3-dimethyl-1-(6-oxo-6-(prop-2-yn-1-ylamino)hexyl)-2-((1E,3E)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium

  • Compound

    6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)-N-(prop-2-yn-1-yl)hexanamide

  • Compound

    (1R,2R,5S,6S)-5,6-bis(benzyloxy)-2-(hydroxymethyl)cyclohex-3-en-1-ol

  • Compound

    (1R,2R,3R,4S,5R,6R)-4,5-bis(benzyloxy)-2-(hydroxymethyl)-7-oxabicyclo[4.1.0]heptan-3-ol

  • Compound

    (1R,2R,3R,4S,5R,6R)-4,5-bis(benzyloxy)-3-hydroxy-7-oxabicyclo[4.1.0]heptane-2-carboxylic acid

  • Compound

    (1R,2R,3R,4S,5S,6R)-4,5-bis(benzyloxy)-2-(hydroxymethyl)-7-azabicyclo[4.1.0]heptan-3-ol

  • Compound

    benzyl (1R,2S,3S,4R,5R,6R)-2,3-bis(benzyloxy)-4-hydroxy-5-(hydroxymethyl)-7-azabicyclo[4.1.0]heptane-7-carboxylate

  • Compound

    (1R,2S,3R,4S,5S,6R)-4,5-bis(benzyloxy)-7-((benzyloxy)carbonyl)-3-hydroxy-7-azabicyclo[4.1.0]heptane-2-carboxylic acid

  • Compound

    (3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2-ol

  • Compound

    (2S,3R,4R,5R)-2,3,4-tris(benzyloxy)-5,6-dihydroxyhexyl acetate

  • Compound

    (2S,3R,4R,5R)-2,3,4-tris(benzyloxy)-5-hydroxy-6-(trityloxy)hexyl acetate

  • Compound

    (2S,3R,4R,5R)-2,3,4-tris(benzyloxy)-6-(trityloxy)hexane-1,5-diol

  • Compound

    (2R,3R,4R,5S)-3,4,5-tris(benzyloxy)-2-((trityloxy)methyl)piperidine

  • Compound

    benzyl (2R,3R,4R,5S)-3,4,5-tris(benzyloxy)-2-((trityloxy)methyl)piperidine-1-carboxylate

  • Compound

    benzyl (2R,3R,4R,5S)-3,4,5-tris(benzyloxy)-2-(hydroxymethyl)piperidine-1-carboxylate

  • Compound

    (2S,3R,4R,5S)-3,4,5-tris(benzyloxy)-1-((benzyloxy)carbonyl)piperidine-2-carboxylic acid

  • Compound

    dibenzyl (2S,3R,4R,5S)-3,4,5-tris(benzyloxy)piperidine-1,2-dicarboxylate

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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.

Author information

Author notes

    • Liang Wu
    • , Jianbing Jiang
    •  & Yi Jin

    These authors contributed equally to this work.

Affiliations

  1. York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York, UK.

    • Liang Wu
    • , Yi Jin
    •  & Gideon J Davies
  2. Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands.

    • Jianbing Jiang
    • , Marta Artola
    • , Wei Dai
    • , Cas van Elk
    • , Gijsbert A van der Marel
    • , Jeroen D C Codée
    • , Bogdan I Florea
    •  & Herman S Overkleeft
  3. Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands.

    • Wouter W Kallemeijn
    • , Chi-Lin Kuo
    • , Marco van Eijk
    •  & Johannes M F G Aerts

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Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Herman S Overkleeft or Gideon J Davies.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 1–2, Supplementary Figures 1–12 and Supplementary Note 1

  2. 2.

    Supplementary Note 2

    Synthesis and compound characterization details

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    Supplementary Dataset 1

    Full mass spectrometry datasets for proteomics experiments.

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DOI

https://doi.org/10.1038/nchembio.2395

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