Activity-Based Proteomics DOI: 10.1002/ange.200805529 Receptor-Mediated Targeting of Cathepsins in Professional Antigen Presenting Cells** Ulrik Hillaert, Martijn Verdoes, Bogdan I. Florea, Anastasios Saragliadis, Kim L. L. Habets, Johan Kuiper, Serge Van Calenbergh, Ferry Ossendorp, Gijsbert A. van der Marel, Christoph Driessen, and Hermen S. Overkleeft* Activity-based profiling probes (ABPs) have received considerable attention in recent years as tools for the detection of enzyme activities both in cell extracts and in living cells. [1] An eminent example of an ABP is the biotinylated peptide epoxysuc- cinate DCG-04 (1; Figure 1). [2] Peptide epoxysuccinates covalently and irreversibly inhibit cysteine proteases of the papain family, and attachment of an affinity or identification handle enables visualization and/or enrichment of the thus modified proteases for ensuing studies in a proteo- mics setting. [3] In this way, and depending on the research setting, the action of known cysteine proteases may be monitored in the appropriate physiological conditions, and new proteolytic activities may be unearthed. The cathepsins, of which the majority are cysteine proteases, reside mostly in the endo- somal and lysosomal compartments. [4] Here they partake in the processing and degradation of proteins brought to these compartments by endocytosis and phagocytosis events, but also by specific targeting of newly synthesized proteins through the secretory pathway. Cathepsin activity is however not restricted to the endo/lysosomal pathway. For instance, cathepsin L is excreted to the extracellular space by some tissues and cathepsin activities are found in other subcellular compartments as well. Measuring cathepsin activity in cell lysates therefore may not accurately reflect the relevant activity being studied in a given subcellular compartment, even more so since the optimum pH value for a given cathepsin (encountered upon gradual acidification in the course of endosome maturation) may differ from that of the next cathepsin. Information on lysosomal and endosomal cysteine pro- tease activity is of particular interest in the context of processing of antigenic peptides and their presentation on both MHC (major histocompatibility complex) class II mol- ecules and MHC class I molecules [5] (the latter in a process referred to as cross-presentation [6] ). For this purpose, cathe- psin-directed ABPs that specifically target the endosomal/ lysosomal compartments would be of particular interest. Some years ago Ploegh and co-workers reported a strategy in which streptavidin-coated latex beads were loaded with the biotinylated ABP, DCG-04. [7] The resulting constructs appeared suitable for phagocytosis by macrophages after which the encountered cysteine proteases were trapped for post-lysis analysis and identification by strip domain structure polyacrylamide gel electrophoresis (SDS-PAGE). We report herein the design of fluorescent epoxysuccinate construct 2 (Figure 1) equipped with a mannose cluster, [8] with which a broad panel of cysteine proteases are selectively targeted in the endosomal pathway in both dendritic cells and macro- Figure 1. Structures of epoxysuccinate DCG-04 (1) and the envisioned mannosylated fluorescent epoxysuccinate construct 2. [*] Dr. U. Hillaert, M. Verdoes, Dr. B.I. Florea, A. Saragliadis, Prof. G. A. van der Marel, Prof. H. S. Overkleeft Leiden Institute of Chemistry, Leiden University P.O. Box 9052, 2300 RA Leiden (The Netherlands) Fax: (+ 31) 71-527-4307 E-mail: h.s.overkleeft@chem.leidenuniv.nl K. L. L. Habets, Dr. J. Kuiper Dept of Biopharmaceutics LACDR, Leiden University (The Netherlands) Prof. C. Driessen Department of Oncology and Hematology, St. Gallen, (Switzerland) Prof. S. Van Calenbergh University of Ghent (Belgium) Dr. F. Ossendorp Department IHB, Leiden University Medical Center (The Netherlands) [**] We thank the Netherlands Organisation for Scientific Research (NWO), the Netherlands Proteomics Center (NPC), and the Marie Curie Research Training Network (MRTN-CT-2004-512585) for their financial support. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.200805529. Angewandte Chemie 1657 Angew. Chem. 2009, 121, 1657 –1660  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim