Abstract
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.
| Original language | English |
|---|---|
| Article number | 113488 |
| Journal | Analytical Biochemistry |
| Volume | 588 |
| DOIs | |
| Publication status | Published - Jan 1 2020 |
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Keywords
- Activity-based probe
- Cathepsin G
- CD39 Tregs
- Proteases
- T helper cells
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology
Cite this
Application of a novel FAM-conjugated activity-based probe to determine cathepsin G activity intracellularly. / Schroeder, Roman; Grzywa, Renata; Wirtz, Christian Rainer; Sienczyk, Marcin; Burster, Timo.
In: Analytical Biochemistry, Vol. 588, 113488, 01.01.2020.Research output: Contribution to journal › Article
}
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
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
UR - http://www.scopus.com/inward/record.url?scp=85073737019&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85073737019&partnerID=8YFLogxK
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 -