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Activity-Based Profiling of Retaining β-Glucosidases: A Comparative Study

Authors

  • Dr. Martin D. Witte,

    1. Leiden Institute of Chemistry and the Netherlands Proteomics Centre, Leiden University, P.O. Box 9502, 2300 RA Leiden (The Netherlands), Fax: (+31) 71-5274307
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    • These authors contributed equally to this work.

  • Marthe T. C. Walvoort,

    1. Leiden Institute of Chemistry and the Netherlands Proteomics Centre, Leiden University, P.O. Box 9502, 2300 RA Leiden (The Netherlands), Fax: (+31) 71-5274307
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    • These authors contributed equally to this work.

  • Kah-Yee Li,

    1. Leiden Institute of Chemistry and the Netherlands Proteomics Centre, Leiden University, P.O. Box 9502, 2300 RA Leiden (The Netherlands), Fax: (+31) 71-5274307
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  • Wouter W. Kallemeijn,

    1. Department of Medical Biochemistry, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam (The Netherlands)
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  • Wilma E. Donker-Koopman,

    1. Department of Medical Biochemistry, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam (The Netherlands)
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  • Dr. Rolf G. Boot,

    1. Department of Medical Biochemistry, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam (The Netherlands)
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  • Prof. Dr. Johannes M. F. G. Aerts,

    1. Department of Medical Biochemistry, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam (The Netherlands)
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  • Dr. Jeroen D. C. Codée,

    1. Leiden Institute of Chemistry and the Netherlands Proteomics Centre, Leiden University, P.O. Box 9502, 2300 RA Leiden (The Netherlands), Fax: (+31) 71-5274307
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  • Prof. Dr. Gijsbert A. van der Marel,

    Corresponding author
    1. Leiden Institute of Chemistry and the Netherlands Proteomics Centre, Leiden University, P.O. Box 9502, 2300 RA Leiden (The Netherlands), Fax: (+31) 71-5274307
    • Leiden Institute of Chemistry and the Netherlands Proteomics Centre, Leiden University, P.O. Box 9502, 2300 RA Leiden (The Netherlands), Fax: (+31) 71-5274307
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  • Prof. Dr. Herman S. Overkleeft

    Corresponding author
    1. Leiden Institute of Chemistry and the Netherlands Proteomics Centre, Leiden University, P.O. Box 9502, 2300 RA Leiden (The Netherlands), Fax: (+31) 71-5274307
    • Leiden Institute of Chemistry and the Netherlands Proteomics Centre, Leiden University, P.O. Box 9502, 2300 RA Leiden (The Netherlands), Fax: (+31) 71-5274307
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Abstract

Activity-based protein profiling (ABPP) is a versatile strategy to report on enzyme activity in vitro, in situ, and in vivo. The development and use of ABPP tools and techniques has met with considerable success in monitoring physiological processes involving esterases and proteases. Activity-based profiling of glycosidases, on the other hand, has proven more difficult, and to date no broad-spectrum glycosidase activity-based probes (ABPs) have been reported. In a comparative study, we investigated both 2-deoxy-2-fluoroglycosides and cyclitol epoxides for their utility as a starting point towards retaining β-glucosidase ABP. We also investigated the merits of direct labeling and two-step bio-orthogonal labeling in reporting on glucosidase activity under various conditions. Our results demonstrate that 1) in general cyclitol epoxides are the superior glucosidase ABPs, 2) that direct labeling is the more efficient approach but it hinges on the ability of the glucosidase to be accommodated in the active site of the reporter (BODIPY) entity, and 3) that two-step bio-orthogonal labeling can be achieved on isolated enzymes but translating this protocol to cell extracts requires more investigation.

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