Quantifying Cathepsin S Activity in Antigen Presenting Cells Using a Novel Specific Substrate

Abstract

Cathepsin S (CatS) is a lysosomal cysteine protease belonging to the papain superfamily. Because of the relatively broad substrate specificity of this family, a specific substrate for CatS is not yet known. Based on a detailed study of the CatS endopeptidase specificity, using six series of internally quenched fluorescent peptides, we were able to design a specific substrate for CatS. The peptide series was based on the sequence GRWHTVGL-RWE-Lys(Dnp)-DArg-NH 2 , which shows only one single cleavage site between Gly and Leu and where every substrate position between P-3 and P-3 was substituted with up to 15 different amino acids. The endopeptidase specificity of CatS was mainly determined by the P-2, P-1, and the P-3 substrate positions. Based on this result, systematically modified substrates were synthesized. Two of these modified substrates, Mca-GRWPP-MGLPWE-Lys(Dnp)-DArg-NH 2 and Mca-GRWHPMGAPWELys(Dnp)-DArg-NH 2 , did not react with the purified cysteine proteases cathepsin B (CatB) and cathepsin L (CatL). Using a specific CatS inhibitor, we could further show that these two peptides were not cleaved by endosomal fractions of antigen presenting cells (APCs), when CatS was inhibited and related cysteine proteases cathepsin B, H, L and X were still active. Although aspartic proteases like cathepsin E and cathepsin D were also present, our substrates were suitable to quantify cathepsin S activity specifically in APCs, including B cells, macrophages, and dendritic cells without the use of any protease inhibitor. We find that CatS activity differs significantly not only between the three types of professional APCs but also between endosomal and lysosomal compartments.Lysosomal cysteine proteases of the papain family were long believed to be exclusively involved in nonspecific proteolysis within the lysosome. However, the use of specific protease inhibitors and the study of gene knock-out mice suggested that some of them are involved in many other processes, such as cellular homeostasis, autophagy, apoptosis, and antigen presentation (1-3). Antigen presentation by MHC 2 class II molecules requires the entry of antigens into the endosomal-lysosomal compartment. These antigens are then processed by proteolytic enzymes, of which the lysosomal cysteine proteases of the papain family constitute an important subset. The generated peptides bind to MHC class II molecules, which are then displayed at the surface of professional APCs including macrophages, dendritic cells (DCs), and B cells. The variety of MHC class II peptide products that can activate CD4 ϩ T cells is on the one hand determined by allelic variation in MHC molecule binding specificity and on the other by the identity and level of activity of processing proteases present in APCs. Although CatS is one of the major proteases involved in antigen processing (4 -6), specific determination of its activity in antigen presenting cells is currently complicated because a specific substrate is not yet known. One reason for this is that CatB, CatL, and CatS show relati...

Targeting proteins to distinct subcellular compartments reveals unique requirements for MHC class I and II presentation

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Involvement of a cysteine protease in the secretion process of human xylosyltransferase I

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The Small GTPase Rac2 Controls Phagosomal Alkalinization and Antigen Crosspresentation Selectively in CD8+ Dendritic Cells

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