TY - JOUR
T1 - Application of a novel FAM-conjugated activity-based probe to determine cathepsin G activity intracellularly
AU - Schroeder, Roman
AU - Grzywa, Renata
AU - Wirtz, Christian Rainer
AU - Sienczyk, Marcin
AU - Burster, Timo
N1 - Funding Information:
We appreciate Nadine Turiello for active-site label and Professor Dr. Markus Huber-Lang, Ulm University Medical Center, Ulm, Germany for the flow cytometer. All materials included in the manuscript will be made freely available to any researchers who wish to use them for non-commercial purposes. RG and MS are thankful to the Wroc?aw University of Science and Technology (Wroclaw, Poland) for support (Statute Funds 0401/0145/18). 5(6)-Carboxyfluorescein used for synthesis of MARS116-FAM and MARS116*-FAM was kindly donated by Dr. ?ukasz Winiarski.
Funding Information:
We appreciate Nadine Turiello for active-site label and Professor Dr. Markus Huber-Lang, Ulm University Medical Center, Ulm, Germany for the flow cytometer. All materials included in the manuscript will be made freely available to any researchers who wish to use them for non-commercial purposes. RG and MS are thankful to the Wrocław University of Science and Technology (Wroclaw, Poland) for support (Statute Funds 0401/0145/18 ). 5(6)-Carboxyfluorescein used for synthesis of MARS116-FAM and MARS116*-FAM was kindly donated by Dr. Łukasz Winiarski.
PY - 2020/1/1
Y1 - 2020/1/1
N2 - Cathepsin G (CatG) is responsible for several distinct immune processes of adaptive and innate immunity depending on extra- or intracellular occurrence of CatG. Recently, we established a method to detect CatG activity at the cell surface of natural killer cells by using the activity-based probe MARS116-Bt in flow cytometry. MARS116-Bt consists of biotin, spacer, amino acid sequence, and a phosphonate warhead which binds covalently to the serine amino acid residue within the active center of CatG. Herein, MARS116 was conjugated to 5(6)-carboxyfluorescein (FAM) in order to limit non-specific signal-to-noise ratio generally resulting from binding of fluorescein-labelled avidin (avidin-FAM) to biotinylated, intracellular proteins; since MARS116-Bt is incubated with avidin-FAM in a second labelling step. MARS116-FAM was capable to detect intracellular CatG activity, in contrast to the control compound MARS116*-FAM which lacks the functional phosphonate warhead crucial for binding to the active-site of CatG and contains a carboxyl group instead. Furthermore, intracellular CatG activity was determined in CD4+ T cells, CD8+ T cells as well as in T regulatory cell (Treg) subsets. Thus, MARS116-FAM is a convenient activity-based probe to detect intracellular CatG activity in a flow cytometry approach.
AB - Cathepsin G (CatG) is responsible for several distinct immune processes of adaptive and innate immunity depending on extra- or intracellular occurrence of CatG. Recently, we established a method to detect CatG activity at the cell surface of natural killer cells by using the activity-based probe MARS116-Bt in flow cytometry. MARS116-Bt consists of biotin, spacer, amino acid sequence, and a phosphonate warhead which binds covalently to the serine amino acid residue within the active center of CatG. Herein, MARS116 was conjugated to 5(6)-carboxyfluorescein (FAM) in order to limit non-specific signal-to-noise ratio generally resulting from binding of fluorescein-labelled avidin (avidin-FAM) to biotinylated, intracellular proteins; since MARS116-Bt is incubated with avidin-FAM in a second labelling step. MARS116-FAM was capable to detect intracellular CatG activity, in contrast to the control compound MARS116*-FAM which lacks the functional phosphonate warhead crucial for binding to the active-site of CatG and contains a carboxyl group instead. Furthermore, intracellular CatG activity was determined in CD4+ T cells, CD8+ T cells as well as in T regulatory cell (Treg) subsets. Thus, MARS116-FAM is a convenient activity-based probe to detect intracellular CatG activity in a flow cytometry approach.
KW - Activity-based probe
KW - Cathepsin G
KW - CD39 Tregs
KW - Proteases
KW - T helper cells
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U2 - 10.1016/j.ab.2019.113488
DO - 10.1016/j.ab.2019.113488
M3 - Article
C2 - 31654611
AN - SCOPUS:85073737019
VL - 588
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
M1 - 113488
ER -