Impacts of vesicular environment on Nox2 activity measurements in vitro
Graphical abstract
Introduction
The phagocyte NADPH oxidase has an essential function in the innate immune system (reviews [1,2]), in the destruction of pathogens due to a massive production of superoxide anions (O2•-) in the phagosome [3]. This is called the oxidative burst. This enzyme is composed of two transmembrane proteins forming the flavocytochrome b558 (the catalytic core Nox2 and p22phox), and four regulatory cytosolic proteins (p40phox, p47phox, p67phox and Rac1/2), that must bind to the dehydrogenase domain of Nox2 and p22phox to form an active complex (for reviews see [[4], [5], [6]]). In the resting state, neutrophils do not produce superoxide, the cytosolic proteins being distributed in the cytosol, alone or pre-assembled with some partners and the flavocytochrome b558 (cyt b558) being in the plasma and granule membranes.
Superoxide anions production from dioxygen reduction using NADPH as electron donor is the sole catalytic function of NADPH oxidases. The substrate NADPH binds on the dehydrogenase domain of Nox2, delivers two electrons to the cofactor FAD that transfers them sequentially through two hemes to the final acceptors, two dioxygen molecules, on the other side of the membrane. The global reaction is:
The phagocyte NADPH oxidase complex activity is studied in cell-free assays by mixing recombinant cytosolic proteins with native membrane fractions (MF), which are vesicles from the cell membrane of disrupted neutrophils. The system is activated by anionic amphiphilic molecules, commonly the all cis-arachidonic acid. The action of arachidonic acid consists in modifying the structure of most of the proteins of the complex [7,8] by enabling them to interact with each other and with phospholipids, resulting in the formation of an active enzyme [[9], [10], [11]]. In such cell-free assays, the enzyme activity is usually determined indirectly by measuring the rate of cytochrome c reduction by the produced superoxide anion [12]. Alternatively, measurement of the rate of NADPH oxidation is another approach to quantify the enzyme activity.
In the cell-free assay system used in this study, the catalytic core of the phagocyte NADPH oxidase is embedded in the plasma membrane of neutrophils. After disruption of this membrane, the membrane fraction (MF) consists in closed vesicles [13,14]. This membrane fraction contains vesicles with the NADPH oxidase oriented across the membrane in one direction and vesicles with the NADPH oxidase in the other orientation, unlike in phagosomes in which only the “inside-out” orientation occurs. The cytosolic proteins and NADPH can only bind to the accessible dehydrogenase domain of Nox2, exposed to the external side of the MF vesicles. Thus, these vesicles are inside-out compared to plasma membrane. Consequently, the NADPH oxidation necessarily occurs on the outside of the MF vesicles and the superoxide anion production inside. This vesicular context can lead to difficulties to accurately measure the superoxide production. Indeed, the low permeability of membranes for negatively charged molecules such as O2•- leads to their sequestration, convoluted with side reactions such as superoxide disproportionation. According to reaction 1, in principle, the enzyme oxidizes one NADPH molecule to reduce two dioxygen molecules into two superoxide anions. Thus, the rate of superoxide production should be twice that of NADPH oxidation. However, experimentally, this is not the case and significant differences of this stoichiometry have been reported in literature [[15], [16], [17]]. Our aim with this paper is to elucidate these discrepancies. First, we identified what the cyt c approach really measures in order to evaluate the accuracy of the method for the determination of the initial rate of O2•- production by Nox-es. Second, we pointed out difficulties to get reliable enzymatic parameters from substrates or products quantifications.
Section snippets
Materials
SP-Sepharose Fast-Flow (FF), Q-Sepharose-FF, Glutathione Sepharose-FF, and Ni-Sepharose-FF resins were purchased from GE-Healthcare-Bioscience. All cis-arachidonic acid, Dextran, phosphate buffer saline (PBS) Dulbecco PBS (dPBS), and equine heart cytochrome c were purchased from Sigma-Aldrich, and NADPH from ACROS.
The plasmids coding for the human cytosolic proteins, pET15b-Hisp67phox, pET15b-Hisp47phox, pGEX2T-GST-Rac1Q61L were provided by Dr. M.C. Dagher, Grenoble, France. All the plasmids
Nox2 activity assessed by cyt c reduction versus NADPH oxidation in standard conditions
In vitro Nox2 activity is commonly assessed by measuring the cyt c reduction rate by produced superoxide ions. The dependence of human Nox2 activity measured by this assay as a function of arachidonic acid (AA) concentration has a typical bell shape. It shows first an activation effect, up to 50 μM AA, followed by an inhibition effect for higher concentrations (Fig. 1). The pattern of this bell shape may slightly vary with the origin of cells (i.e. the blood donor). In order to take into
Discussion
This study reveals a much more complex situation than it was first expected. Since the 1980s, the complicated regulation mechanism of Nox2 has been deciphered, the cofactors and cytosolic regulatory proteins have been identified, purifications of recombinant cytosolic proteins and the activation protocols have been established and improved. In general, in order to test the NADPH oxidase activity, the community felt safer to follow the SOD-inhibited production of superoxide indirectly by cyt c
Conclusion
These results altogether indicate that the cyt c reduction method underestimates the true Nox2 activity especially at short incubation times in a vesicle context. Nevertheless, this method is a good semi-quantitative tool to check the relative production of the end enzymatic product, the superoxide anion. Due to compartmentation, superoxide anions first have to diffuse through the membrane before being able to react with cyt c. In such a system, diffusion and disproportionation competes. Since
Funding
This work was supported by Electricité de France, Grant RB 2016–21.
CRediT authorship contribution statement
Xavier Serfaty: Investigation, Writing - original draft. Pauline Lefrançois: Investigation. Chantal Houée-Levin: Validation. Stéphane Arbault: Methodology. Laura Baciou: Conceptualization, Funding acquisition. Tania Bizouarn: Conceptualization, Writing - review & editing, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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