title: Application of a novel highly sensitive activity-based probe for detection of cathepsin G.
abstract: Cathepsins are crucial in antigen processing in the major histocompatibility complex class II (MHC II) pathway. Within the proteolytic machinery, three classes of proteases (i.e., cysteine, aspartic, and serine proteases) are present in the endocytic compartments. The combined action of these proteases generates antigenic peptides from antigens, which are loaded to MHC II molecules for CD4+ T cell presentation. Detection of active serine proteases in primary human antigen-presenting cells (APCs) is restricted because of the small numbers of cells isolated from the peripheral blood. For this purpose, we developed a novel highly sensitive α-aminoalkylphosphonate diphenyl ester (DAP) activity-based probe to detect the serine protease cathepsin G (CatG) in primary APCs and after Epstein-Barr virus (EBV) exposure. Although CatG activity was not altered after short-term exposure of EBV in primary myeloid dendritic cells 1 (mDC1s), the aspartic protease cathepsin D (CatD) was reduced, suggesting that EBV is responsible for mitigating the presentation of a model antigen tetanus toxoid C-fragment (TTCF) by reduction of CatD. In addition, CatG activity was reduced to background levels in B cells during cell culture; however, these findings were independent of EBV transformation. In conclusion, our activity-based probe can be used for both Western blot and 96-well-based high-throughput CatG detection when cell numbers are limited.
authors.affiliation: Division of Endocrinology and Diabetes, Center for Internal Medicine, University Medical Center Ulm, 89081 Ulm, Germany.
authors.forename: Fang
authors.initials: F
authors.lastname: Zou
authors.forename: Michael
authors.initials: M
authors.lastname: Schmon
authors.forename: Marcin
authors.initials: M
authors.lastname: Sienczyk
authors.forename: Renata
authors.initials: R
authors.lastname: Grzywa
authors.forename: David
authors.initials: D
authors.lastname: Palesch
authors.forename: Bernhard O
authors.initials: BO
authors.lastname: Boehm
authors.forename: Zi Lin
authors.initials: ZL
authors.lastname: Sun
authors.forename: Colin
authors.initials: C
authors.lastname: Watts
authors.forename: Reinhold
authors.initials: R
authors.lastname: Schirmbeck
authors.forename: Timo
authors.initials: T
authors.lastname: Burster
mesh_terms: D015153:Blotting, Western; D056649:Cathepsin G / Q000032:analysis*; D003124:Colorimetry; D004705:Endocytosis; D005434:Flow Cytometry; D006801:Humans; D057230:Limit of Detection; D015335:Molecular Probes
chemical_list: D015335:Molecular Probes; D056649:Cathepsin G