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

Background Previous studies have demonstrated that cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA(2)alpha 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, p47(Phox) in human phagocytes. Intravenous administration of antisense against cPLA(2)alpha that significantly inhibited its expression in mouse peritoneal neutrophils and macrophages also inhibited superoxide production, in contrast to cPLA(2)alpha knockout mice that showed normal superoxide production. The present study aimed to determine whether there is a binding between cPLA(2)alpha-C2 domain and p47(Phox)-PX in mouse macrophages, to further support the role of cPLA(2)alpha in oxidase regulation also in mouse phagocytes. Methods and results A significant binding of mouse GST-p47(Phox)-PX domain fusion protein and cPLA(2)alpha in stimulated mouse phagocyte membranes was demonstrated by pull-down experiments, although lower than that detected by the human p47(Phox)-PX domain. Substituting the amino acids Phe98, Asn99, and Gly100 to Cys98, Ser99, and Thr100 in the mouse p47(Phox)-PX domain (present in the human p47(Phox)-PX domain) caused strong binding that was similar to that detected by the human p47(Phox)-PX domain Conclusions The binding between cPLA(2)alpha-C2 and p47(Phox)-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 p47(Phox)-PX domain that facilitate the binding to cPLA(2)alpha.

Automated summary

The present study aimed to determine whether there is a binding between cPLA(2)alpha-C2 domain and p47(Phox)-PX in mouse macrophages, to further support the role of cPLA(2)alpha in oxidase regulation also in mouse phagocytes.