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. 2014 Mar 1;20(7):1039-44.
doi: 10.1089/ars.2013.5618. Epub 2013 Oct 23.

How Much H(2)O(2) Is Produced by Recombinant D-amino Acid Oxidase in Mammalian Cells?

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How Much H(2)O(2) Is Produced by Recombinant D-amino Acid Oxidase in Mammalian Cells?

Mikhail E Matlashov et al. Antioxid Redox Signal. .
Free PMC article

Abstract

Yeast D-amino acid oxidase (DAO) can serve as a genetically encoded producer of reactive oxygen species (ROS) in redox signaling studies. However, dynamics of hydrogen peroxide production and its sensitivity to externally added D-alanine (D-Ala) in cells have not been determined. Here we show that DAO, fused to a genetically encoded H2O2 indicator HyPer, can be used for controlled production of ROS in living eukaryotic cells. We found a clear heterogeneity in ROS production dynamics between individual cells. Moreover, different cell lines demonstrated distinct sensitivity to added D-Ala. Finally, by comparing signals generated by the HyPer-DAO fusion protein versus coexpressed HyPer and DAO proteins, we show that the fusion system is more sensitive to hydrogen peroxide production. Our results show the utility of the HyPer-DAO genetically encoded system for redox signaling studies and suggest that H2O2 produced by DAO in the cytoplasm acts locally in close proximity to the enzyme.

Figures

<b>FIG. 1.</b>
FIG. 1.
HyPer-D-amino acid oxidase (DAO) produces and detects H2O2 in response to D-alanine (D-Ala). (A) Scheme of HyPer-DAO fusion. (B) Time course of H2O2 production by HyPer-DAO in HeLa-Kyoto cells upon addition of 4 mM D-Ala. Mean values represent measurements of seven individual cells, with error bars showing standard error of the mean. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 2.</b>
FIG. 2.
HyPer-DAO fusion versus HyPer & DAO coexpression in HeLa-Kyoto cells. (A) Widefield ratiometric images of HeLa-Kyoto cells cotransfected with HyPer and DAO encoding vectors. Numbers indicate time points in minutes. (B) Time course of HyPer ratio changes in HeLa-Kyoto cells coexpressing HyPer and DAO upon addition of different concentrations of D-Ala. (C) Time course of HyPer-DAO fusion ratio changes in HeLa-Kyoto cells upon addition of different concentrations of D-Ala. (D) Widefield ratiometric images of HeLa-Kyoto cells transfected with HyPer-DAO fusion encoding vector. Numbers indicate time points in minutes. In (B) and (C), mean values represent at least five individual cells, with error bars showing standard error of the mean. Lookup table reflects the HyPer or HyPer-DAO ratio. Scale bars are 20 μm. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 3.</b>
FIG. 3.
HyPer-DAO fusion versus HyPer & DAO coexpression in NIH-3T3 cells. (A) Widefield ratiometric images of NIH-3T3 cells cotransfected with HyPer and DAO encoding vectors. Numbers indicate time points in minutes. (B) Time course of HyPer ratio changes in NIH-3T3 cells coexpressing HyPer and DAO upon addition of different concentrations of D-Ala. (C) Time course of HyPer-DAO fusion ratio changes in NIH-3T3 cells upon addition of different concentrations of D-Ala. (D) Widefield ratiometric images of NIH-3T3 cells transfected with HyPer-DAO fusion encoding vector. Numbers indicate time points in minutes. In (B) and (C), mean values represent at least five individual cells, with error bars showing standard error of the mean. Lookup table reflects HyPer or HyPer-DAO ratio. Scale bars are 20 μm. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 4.</b>
FIG. 4.
Dynamic range of HyPer is not altered in fusion with DAO. Addition of 100 μM H2O2 to HeLa-Kyoto cells either (A) coexpressing DAO and HyPer or (B) expressing HyPer-DAO leads to similar HyPer ratio changes. Black and red lines show the response of individual cells. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

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