How Much H₂O₂ Is Produced by Recombinant D-Amino Acid Oxidase in Mammalian Cells?

Introduction

Cells produce reactive oxygen species (ROS) during metabolism and ROS emerge as crucial signaling molecules. Hydrogen peroxide (H₂O₂) plays an important regulatory role in cell signaling cascades by oxidizing critical allosteric and catalytic Cys residues of a range of proteins (9). At high concentrations it also becomes involved in pathological processes, damaging cellular structures through Fenton chemistry. H₂O₂ is rapidly degraded by antioxidant enzymes that localize ROS signaling in time and space and minimize their harmful effects (9).

In many studies of ROS-mediated signaling, H₂O₂ and other pro-oxidative agents are applied externally, without estimating their actual intracellular concentration. This may result in multiple protein oxidation and cause oxidative stress; conversely, apparent lack of effect may be due to numerous reasons, ranging from the oxidant degradation in the media to poor cell membrane permeability. Together, this complicates interpretation of experimental results. Besides, this approach does not allow targeted oxidation of specific proteins, which would be very useful for evaluating the role of ROS in a specific pathway. Therefore, developing a targeted ROS production system would provide a novel approach for redox signaling studies. It would be further advantageous if this approach would allow tracing of intracellular H₂O₂ dynamics.

D-amino acid oxidase (DAO) is an enzyme that oxidizes the NH₂- group of D-amino acid, producing corresponding α-keto acid, ammonia, and hydrogen peroxide (7). The yeast DAO gene from Rhodotorula gracilis (red yeast) has been previously cloned and the enzyme characterized in vitro (5). It is highly specific to D-amino acids, has relatively low affinity to D-alanine (D-Ala) (about 1 mM) and high enzymatic activity in comparison with known DAO from other organisms (8). DAO can serve as a convenient genetically encoded source for controlled D-Ala-dependent H₂O₂ production in mammalian cells (3). However, the estimation of the intracellular H₂O₂ production requires extracellular measurements of the released oxidant (3). The rate of H₂O₂ release as well as the rate of the initial D-amino acid uptake may vary between cells. Therefore, the requirement of measuring the outcome of DAO reaction would be better suited by an intracellular reporter of H₂O_2..

To trace intracellular DAO-driven H₂O₂ production, we used the genetically encoded fluorescent indicator HyPer (1). HyPer is based on the circularly permutated yellow fluorescent protein inserted into a regulatory domain of Escherichia coli H₂O₂-sensitive transcription factor OxyR. The indicator is sensitive to physiological concentrations of H₂O₂ and has a ratiometric fluorescent readout, making it a useful tool for studying redox signaling and oxidative stress in cultured cells (3) and live animals (2). Therefore, we examined whether H₂O₂ produced by DAO in mammalian cells can be visualized by combining the generator (DAO) and the indicator (HyPer) to produce a single fusion protein.

Results, Discussion, and Future Directions

To produce a genetically encoded system for visually controlled H₂O₂ production, we engineered a fusion protein from DAO and HyPer polypeptides. Initially, we produced both the HyPer-DAO and DAO-HyPer variants and expressed them in cultured HeLa-Kyoto cells. DAO-HyPer was not responsive to externally added D-Ala, while having unperturbed response to H₂O₂, thus indicating that DAO, but not the HyPer, function was impaired in the fusion protein. In contrast, HyPer-DAO (Fig. 1A) responded to D-Ala addition (Fig. 1B) indicating that N-terminal fusion of DAO preserved the function of the enzyme intact. Therefore, we used HyPer-DAO in further experiments and compared it to DAO and HyPer coexpressed.

We first examined whether the HyPer response to DAO was pH driven. HyPer is pH sensitive and since the DAO reaction produces ammonia ions, changes in the HyPer ratio might reflect alkalinization of the cytoplasm rather than changes in H₂O₂ production. To determine whether ammonia released by DAO affects intracellular pH, we used a version of HyPer with C199S substitution, which makes the protein nonsensitive to H₂O₂, but preserves its sensitivity to pH. We observed no change in signal intensity in HyPerC199S-DAO-transfected cells in response to 8 mM D-Ala, whereas HyPer-DAO responded to the amino acid. These results indicate that production of H₂O₂, but not the change in local pH, was the cause of the signal change (Fig. 1B). Moreover, they indicate that the amount of ammonia released from the DAO is not sufficient to produce detectable changes of pH.

Because of the close proximity of HyPer to the source of the oxidant in the HyPer-DAO fusion, its response kinetics may differ from that of the DAO and HyPer coexpression system. To examine this, we cotransfected HeLa-Kyoto (Fig. 2) and NIH-3T3 (Fig. 3) cells with DAO and HyPer plasmids and monitored H₂O₂ production using the fluorescent ratiometric readout of the indicator. In both cell lines, DAO produced detectable amounts of H₂O₂ in response to 4–8 mM D-Ala added to the imaging medium (Figs. 2A, B and 3A, B), with no response to the addition of 8 mM L-alanine (not shown). Since the signals were within the previously determined range of HyPer sensitivity (1), we conclude that in our experiments, the amount of H₂O₂ produced in cells did not exceed 50–100 nM, a value that is within the physiological range for H₂O₂. As we did not observe any signs of cellular stress like cell shrinkage or plasma membrane blebbing during 45 min after addition of D-Ala, products of the DAO activity are likely not toxic to the cells. Notably, in contrast to previous observation (3), D-Ala alone was sufficient to induce sustained H₂O₂ production by the cells and did not required external addition of DAO cofactor flavin adenine dinucleotide to the cells.

Next, we analyzed the dependence of H₂O₂ production in HeLa-Kyoto and NIH-3T3 cells on extracellular D-Ala concentrations. In NIH-3T3 fibroblasts, we observed a prominent HyPer signal at 1–2 mM external D-Ala and above, while no H₂O₂ production was detected in HeLa-Kyoto cells at these concentrations of D-Ala. H₂O₂ production in HeLa-Kyoto cells was observed when D-Ala concentrations in the media were elevated to 4 mM and above. This may indicate that different cell lines have different D-Ala uptake rates, which would explain the differences in the indicator response between the cell lines.

In both lines, further increasing the D-Ala concentration leads to an increased number of responding cells, but did not necessarily increase the rate of hydrogen peroxide production. In fact, in 3T3 cells, we observed some inhibition of H₂O₂ production at concentrations of D-Ala above 4 mM, probably due to the substrate-dependent inhibition of the DAO enzyme. Taken together, our results suggest that optimal D-Ala concentrations should be determined specifically for each cell type at given experimental conditions.

Since active oxygen forms degrade rapidly in the cytoplasm, we asked whether a fusion protein, containing both DAO and HyPer, would lead to increased HyPer oxidation and thus be a more efficient indicator of DAO activity. As expected, signals from cells expressing the HyPer-DAO fusion protein were about 1.5-2-fold higher on average compared to the cells coexpressing separate DAO and HyPer proteins (Figs. 2C, D and 3C, D). This improvement was not due to the altered dynamic range of HyPer in fusion with DAO as both HyPer and HyPer-DAO respond equally to external H₂O₂ (Fig. 4). Therefore, fusing DAO with a protein of interest makes the target oxidation selectively more efficient compared to other cytoplasmic proteins. This approach may be useful for targeted oxidation of a specific protein of interest, for separation of a specific cascade from other redox signaling pathways, or for limiting the oxidative damage to a specific cell structure.

There was a clear heterogeneity in hydrogen peroxide production dynamics among individual cells (Figs. 2 and 3). From our observation, H₂O₂ production not only depends on the HyPer-DAO fusion protein expression level, but also on some other cell properties, perhaps on the activity of amino acid transporters. Together, our results indicate that it is important to use either HyPer/DAO cotransfection or HyPer-DAO fusion in live cell imaging to ensure that H₂O₂ production actually takes place in studied cells.

How much H₂O₂ is produced by the DAO in mammalian cells? Despite millimolar concentrations of D-Ala added, H₂O₂ concentration in the cytoplasm does not exceed the 50–100 nM range as the ratio of HyPer coexpressed with DAO changes for up to 1.5-fold. These ratio changes correspond to ∼50 nM H₂O₂, as determined by in vitro calibration of purified HyPer protein [Fig. 1B in (1)]. This further suggests that even in the presence of millimolar D-Ala, oxygen is a limiting substrate, since the concentration of O₂ in cell culture media is ∼200 μM and even less in animal tissues (∼13–15 μM).

Finally, can the HyPer-DAO system be used in vivo? We suggest that it can be used without any restrictions in relatively small organisms suitable for whole body imaging, such as Danio rerio and Caenorhabditis elegans. HyPer demonstrated superior performance in both species (4, 6). D-amino acids can be delivered with food. Even higher levels of precision of H₂O₂ production/detection can be achieved by using cell type-specific promoters and subcellular localization tags. In bigger organisms, such as mice, HyPer-DAO in vivo imaging may be more problematic due to the low transparency of tissues and relatively poor brightness of HyPer. We believe that substitution of HyPer with its brighter versions in the future would enable a wider use of the HyPer-DAO system in vivo.

In summary, we successfully combined DAO and HyPer indicator to make a genetically encoded system for controlled H₂O₂ production/detection. This system can be useful in in situ and in vivo studies of redox signaling. The presence of the H₂O₂ indicator allows visualizing H₂O₂ production by DAO and makes it easier to adjust the optimal D-Ala concentration for specific experimental conditions. Additional benefits and more efficient oxidation of a protein of interest may be achieved by generating a fusion construct of this protein with DAO.

Notes

Acknowledgments

We thank Arcady Fradkov for his help with the codon-optimized DAO gene design. The work was supported by the Russian Foundation for Basic Research (EMBL-RFBR grant 12-04-92427 and 13-04-40333-H to V.V.B.), the National Institute of Aging (grant R01AG040209 to G.E.), Russian Ministry of Education and Science (grant 11.G34.31.0071 to G.E.; contract #8269), and Molecular and Cell Biology Program of Russian Academy of Sciences.