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

Yulia Solomonov, Nurit Hadad, Rachel Levy

Research output: Contribution to journalArticlepeer-review

Abstract

Background: 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. Methods and results: 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.

Original languageEnglish
Pages (from-to)3511-3518
Number of pages8
JournalMolecular Biology Reports
Volume49
Issue number5
DOIs
StateAccepted/In press - 29 Jan 2022

Keywords

  • C2 domain
  • Cytosolic phospholipase Aα
  • NOX2 NADPH oxidase
  • PX-domain

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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