tailieunhanh - Báo cáo khoa học: A new approach for distinguishing cathepsin E and D activity in antigen-processing organelles

Cathepsin E (CatE) and D (CatD) are the major aspartic proteinases in the endolysosomal pathway. They have similar specificity and therefore it is difficult to distinguish between them, as known substrates are not exclu-sively specific for one or the other. | ễFEBS Journal A new approach for distinguishing cathepsin E and D activity in antigen-processing organelles Nousheen Zaidi1 Timo Herrmann1 5 Daniel Baechle2 Sabine Schleicher3 Jeannette Gogel4 Christoph Driessen4 Wolfgang Voelter5 and Hubert Kalbacher1 5 1 Medicaland NaturalSciences Research Centre University of Tubingen Germany 2 PANATecs GmbH Tubingen Germany 3 Children s HospitalDepartment I University of Tubingen Germany 4 Department of Medicine II University of Tubingen Germany 5 Interfacultary Institute of Biochemistry University of Tubingen Germany Keywords antigen-presenting cells cathepsin D cathepsin E enzyme activity assay fluorescent substrate Correspondence H. Kalbacher Ob dem Himmelreich 7 72074 Tubingen Germany Fax 49 7071 294507 Tel 49 7071 2985212 E-mail kalbacher@ Website http Received 27 March 2007 revised 24 April 2007 accepted 25 April2007 doi Cathepsin E CatE and D CatD are the major aspartic proteinases in the endolysosomal pathway. They have similar specificity and therefore it is difficult to distinguish between them as known substrates are not exclusively specific for one or the other. In this paper we present a substratebased assay which is highly relevant for immunological investigations because it detects both CatE and CatD in antigen-processing organelles. Therefore it could be used to study the involvement of these proteinases in protein degradation and the processing of invariant chain. An assay combining a new monospecific CatE antibody and the substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys Dnp -D-Arg-NH2 where MOCAc is 7-methoxycoumarin-4-yl acetyl and Dnp is dinitrophenyl is presented. This substrate is digested by both proteinases and therefore can be used to detect total aspartic proteinase activity in biological samples. After depletion of CatE by immunoprecipitation the remaining activity is due to CatD and the decrease in activity can be