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Published online before print January 29, 2007

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(Journal of Leukocyte Biology. 2007;81:990-1001.)
© 2007 by Society for Leukocyte Biology

Endocytosis targets exogenous material selectively to cathepsin S in live human dendritic cells, while cell-penetrating peptides mediate nonselective transport to cysteine cathepsins

Michael Reich^*, Paul F. van Swieten, Vinod Sommandas^*, Marianne Kraus^*, Rainer Fischer, Ekkehard Weber, Hubert Kalbacher^||, Herman S. Overkleeft and Christoph Driessen^*,1

^* Department of Medicine II,
Institute for Cell Biology, and
^|| Medical and Natural Sciences Research Center, University of Tübingen, Tübingen, Germany;
Institute of Physiological Chemistry, University of Halle, Halle, Germany;
Leiden Institute of Chemistry, University of Leiden, Leiden, The Netherlands

¹ Correspondence: Kantonspital St. Gallen, Dept. of Oncology and Hematology, Rorschachstr. 95, CH-9007 St. Gallen, Switzerland. E-mail: christoph.driessen{at}kssg.ch

The way the MHC II-associated proteolytic system of APC handles exogenous antigen is key to the stimulation of the T cell in infections and immunotherapy settings. Using a cell-impermeable, activity-based probe (ABP) for papain cathepsins, the most abundant type of endocytic proteases, we have simulated the encounter between exogenous antigen and endocytic proteases in live human monocyte-derived dendritic cells (MO-DC). Although cathepsin S (CatS), -B, -H, and -X were active in DC-derived endocytic fractions in vitro, the peptide-size tracer was routed selectively to active CatS after internalization by macropinocytosis. Blocking of the vacuolar adenosine triphosphatase abolished this CatS-selective targeting, and LPS-induced maturation of DC resulted in degradation of active CatS. Conjugation of the ABP to a protein facilitated the delivery to endocytic proteases and resulted in labeling of sizable amounts of CatB and CatX, although CatS still remained the major protease reached by this construct. Conjugation of the probe to a cell-penetrating peptide (CPP) routed the tracer to the entire panel of intracellular cathepsins, independently from endocytosis or LPS stimulation. Thus, different means of internalization result in differential targeting of active cathepsins in live MO-DC. CPP may serve as vehicles to target antigen more efficiently to protease-containing endocytic compartments.

Key Words: antigen processing • lysosomal cysteine proteases • antigen-presenting cell • endocytic compartment • activity-base probe

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