Application of a novel FAM-conjugated activity-based probe to determine cathepsin G activity intracellularly

Roman Schroeder, Renata Grzywa, Christian Rainer Wirtz, Marcin Sienczyk, Timo Burster

Research output: Contribution to journalArticle

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 languageEnglish
Article number113488
JournalAnalytical Biochemistry
Volume588
DOIs
Publication statusPublished - Jan 1 2020

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Cathepsin G
Avidin
Organophosphonates
T-cells
Flow cytometry
Flow Cytometry
T-Lymphocytes
Amino Acids
Signal-To-Noise Ratio
T-Lymphocyte Subsets
Adaptive Immunity
Biotin
Fluorescein
Innate Immunity
Natural Killer Cells
Labeling
Serine
Amino Acid Sequence
Catalytic Domain
Signal to noise ratio

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 journalArticle

Schroeder, Roman ; Grzywa, Renata ; Wirtz, Christian Rainer ; Sienczyk, Marcin ; Burster, Timo. / Application of a novel FAM-conjugated activity-based probe to determine cathepsin G activity intracellularly. In: Analytical Biochemistry. 2020 ; Vol. 588.
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