Characterization of the binding of cytosolic phospholipase A2 alpha and NOX2 NADPH oxidase in mouse macrophages.

Yulia Solomonov,Rachel Levy,Nurit Hadad

doi:10.1007/s11033-022-07191-w

Abstract

Previous studies have demonstrated that cytosolic phospholipase A2α (cPLA2α) is required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA2α translocates to the plasma membranes by binding to the assembled oxidase, forming a complex between its C2 domain and the PX domain of the cytosolic oxidase factor, p47phox in human phagocytes. Intravenous administration of antisense against cPLA2α that significantly inhibited its expression in mouse peritoneal neutrophils and macrophages also inhibited superoxide production, in contrast to cPLA2α knockout mice that showed normal superoxide production. The present study aimed to determine whether there is a binding between cPLA2α-C2 domain and p47phox-PX in mouse macrophages, to further support the role of cPLA2α in oxidase regulation also in mouse phagocytes. A significant binding of mouse GST-p47phox-PX domain fusion protein and cPLA2α in stimulated mouse phagocyte membranes was demonstrated by pull-down experiments, although lower than that detected by the human p47phox-PX domain. Substituting the amino acids Phe98, Asn99, and Gly100 to Cys98, Ser99, and Thr100 in the mouse p47phox-PX domain (present in the human p47phox-PX domain) caused strong binding that was similar to that detected by the human p47phox-PX domain CONCLUSIONS: The binding between cPLA2α-C2 and p47phox-PX domains exists in mouse macrophages and is not unique to human phagocytes. The binding between the two proteins is lower in the mice, probably due to the absence of amino acids Cys98, Ser 99, and Thr100in the p47phox-PX domain that facilitate the binding to cPLA2α.

Highlights

The multi-component electron carrier, NOX-2 NADPH oxidase, t transfers electrons from NADPH to molecular oxygen to form superoxides, a precursor of microbicidal oxidants
An affinity binding assay with human GST-cPLA2α-C2 domain which is identical to the mouse cPLA2α-C2 domain performed in membranes of stimulated peritoneal macrophages resulted with efficient binding to p47phox in stimulated human neutrophil membranes, but with much lower binding to p47phox in stimulated mouse macrophage membranes (Fig. 1A)
The results of the present study show that there is a binding between cPLA2α and p47phox mediated by cPLA2α-C2 domain and p47phox-PX in mouse macrophages, similar to that of human phagocytes with a lower affinity

Summary

Introduction

The multi-component electron carrier, NOX-2 NADPH oxidase, t transfers electrons from NADPH to molecular oxygen to form superoxides, a precursor of microbicidal oxidants. The cytosolic components translocate to the plasma membrane upon stimulation and associate with the flavocytochrome b558 to form the assembled active oxidase. Previous studies have demonstrated that Cytosolic phospholipase A2a (cPLA2a) is absolutely required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Upon stimulation, cPLA2a translocates to the plasma membranes of by binding to the assembled oxidase, forming a complex between its C2 domain and the PX domain of the oxidase cytosolic factor, p47phox in human phagocytes. The aim of the present study was to determine whether there is a binding between cPLA2a-C2 domain and p47phox-PX in mouse macrophages, to further support the role of cPLA2a in oxidase regulation in mouse phagocytes

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Conclusion