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

Abstract

Background: Previous studies have demonstrated that Cytosolic phospholipase A 2 a (cPLA 2 a) is absolutely required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA 2 a 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, p47 phox in human phagocytes. Intravenous administration of an antisense against cPLA 2 a that signi cantly inhibited its expression in mouse peritoneal neutrophil and macrophages also inhibited superoxide production, in contrast to cPLA 2 a knockout mice that showed normal superoxide production.The aim of the present study was to determine whether there is a binding between cPLA 2 a-C2 domain and p47 phox -PX in mouse macrophages, to further support the role of cPLA 2 a in oxidase regulation also in mouse phagocytes.Methods and Results: A signi cant binding of mouse GST-p47 phox -PX domain fusion protein and cPLA 2 a in stimulated mouse phagocyte membranes was demonstrated by pull down experiments, although lower than that detected by human p47 phox -PX domain. Substituting the amino acids Phe98, Asn99 and Gly100 to Cys98 Ser99 and Thr100 in mouse p47 phox -PX domain (that are present in 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 a-C2 and p47 phox -PX domains exist 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 Ser99 and Thr100 in p47 phox -PX domain that facilitate the binding to cPLA 2 a.