Plant Physiology 135:1170-1178 (2004)
© 2004 American Society of Plant Biologists
BREAKTHROUGH TECHNOLOGIES
Activity Profiling of Papain-Like Cysteine Proteases in Plants1
Renier A. L. van der Hoorn*,
Michiel A. Leeuwenburgh,
Matthew Bogyo,
Matthieu H. A. J. Joosten and
Scott C. Peck
Laboratory of Phytopathology, Wageningen University, 6709 PD, Wageningen, The Netherlands (R.A.L.v.d.H., M.H.A.J.J.); Sainsbury Laboratory, John Innes Centre, NR4–7UH, Norwich, United Kingdom (R.A.L.v.d.H., S.C.P.); Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA, Leiden, The Netherlands (M.A.L.); and Department of Pathology, Stanford University Medical School, Stanford, California 94305–5324 (M.B.)
Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in plants. This technology is based on the use of biotinylated, irreversible protease inhibitors that react with active proteases in a mechanism-based manner. Using a biotinylated derivative of the Cys protease inhibitor E-64, we display simultaneous activities of many papain-like Cys proteases in extracts from various tissues and from different plant species. Labeling is pH dependent, stimulated with reducing agents, and inhibited specifically by Cys protease inhibitors but not by inhibitors of other protease classes. Using one-step affinity capture of biotinylated proteases followed by sequencing mass spectrometry, we identified proteases that include xylem-specific XCP2, desiccation-induced RD21, and cathepsin B- and aleurain-like proteases. Together, these results demonstrate that this technology can identify differentially activated proteases and/or characterize the activity of a particular protease within complex mixtures.
1 This work was supported by the Dutch Organisation for Scientific Research (N.W.O.; Veni grant to R.A.L.v.d.H., Vidi grant to M.H.A.J.J., and Talent grant to R.A.L.v.d.H.) and by the Gatsby Charitable Foundation.
www.plantphysiol.org/cgi/doi/10.1104/pp.104.041467.
* Corresponding author; e-mail renier.vanderhoorn{at}wur.nl; fax 31–317–483412.
Received February 24, 2004;
returned for revision April 13, 2004;
accepted May 12, 2004.
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