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Monitoring Stub1-Mediated Pexophagy
Published on: May 12, 2023
Visualization of phagosomal hydrogen peroxide production by a novel fluorescent probe that is localized via SNAP-tag
Masahiro Abo1, Reiko Minakami, Kei Miyano
1Departments of Biochemistry and ‡Health Sciences, Kyushu University Graduate School of Medical Sciences , Fukuoka 812-8582, Japan.
Abstract:
Hydrogen peroxide (H2O2), a member of reactive oxygen species (ROS), plays diverse physiological roles including host defense and cellular signal transduction. During ingestion of invading microorganisms, professional phagocytes such as macrophages release H2O2 specifically into the phagosome to direct toxic ROS toward engulfed microbes. Although H2O2 is considered to exert discrete effects in living systems depending on location of its production, accumulation, and consumption, there have been limitations of techniques for probing this oxygen metabolite with high molecular specificity at the subcellular resolution. Here we describe the development of an O(6)-benzylguanine derivative of 5-(4-nitrobenzoyl)carbonylfluorescein (NBzF-BG), a novel H2O2-specific fluorescent probe; NBzF-BG is covalently and selectively conjugated with the SNAP-tag protein, leading to formation of the fluorophore-protein conjugate (SNAP-NBzF). SNAP-NBzF rapidly reacts with H2O2 and thereby shows a 9-fold enhancement in fluorescence. When SNAP-tag is expressed in HEK293T cells and RAW264.7 macrophages as a protein C-terminally fused to the transmembrane domain of platelet-derived growth factor receptor (PDGFR), the tag is presented on the outside of the plasma membrane; conjugation of NBzF-BG with the cell surface SNAP-tag enables detection of H2O2 added exogenously. We also demonstrate molecular imaging of H2O2 that is endogenously produced in phagosomes of macrophages ingesting IgG-coated latex beads. Thus, NBzF-BG, combined with the SNAP-tag technology, should be useful as a tool to measure local production of H2O2 in living cells.
Insights
Researchers developed a novel fluorescent probe, NBzF-BG, for detecting hydrogen peroxide (H2O2) with high specificity. This tool, combined with SNAP-tag technology, enables precise measurement of H2O2 at the subcellular level in living cells.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Imaging
Background:
- Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are crucial for cellular signaling and host defense.
- Macrophages utilize H2O2 within phagosomes to combat ingested microbes.
- Existing methods for detecting H2O2 at the subcellular level lack specificity and resolution.
Purpose of the Study:
- To develop a novel, highly specific fluorescent probe for detecting hydrogen peroxide (H2O2).
- To enable subcellular resolution imaging of H2O2 production in living cells.
Main Methods:
- Development of NBzF-BG, an O(6)-benzylguanine derivative of 5-(4-nitrobenzoyl)carbonylfluorescein, as an H2O2-specific probe.
- Covalent conjugation of NBzF-BG with SNAP-tag protein to create SNAP-NBzF fluorophore-protein conjugate.
- Utilizing SNAP-tag expressed on the cell surface of HEK293T cells and RAW264.7 macrophages for H2O2 detection.
Main Results:
- SNAP-NBzF exhibits a 9-fold fluorescence enhancement upon reaction with H2O2.
- Cell surface-expressed SNAP-tag allowed detection of exogenously added H2O2.
- Successful molecular imaging of endogenously produced H2O2 in macrophage phagosomes.
Conclusions:
- NBzF-BG, in conjunction with SNAP-tag technology, provides a valuable tool for measuring localized H2O2 production.
- This approach offers high molecular specificity and subcellular resolution for H2O2 detection in biological systems.
