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Fungal Metabolite Gliotoxin Targets Flavocytochrome b₅₅₈ in the Activation of the Human Neutrophil NADPH Oxidase

Department of Infectious Diseases, National Research Institute for Child Health and Development, Setagaya, Tokyo,¹ Department of Pediatrics, Miyazaki Medical College, University of Miyazaki, Kiyotake, Miyazaki,³ Department of Anatomy and Cell Biology, Kochi Medical School, Kochi University, Nankoku, Kochi,⁴ Japan Science and Technology Corp., Kawaguchi, Saitama, Japan²

Received 6 July 2004/ Returned for modification 13 August 2004/ Accepted 15 September 2004

Fungal gliotoxin (GT) is a potent inhibitor of the O₂^–-generating NADPH oxidase of neutrophils. We reported that GT-treated neutrophils fail to phosphorylate p47^phox, a step essential for the enzyme activation, because GT prevents the colocalization of protein kinase C ßII with p47^phox on the membrane. However, it remains unanswered whether GT directly affects any of NADPH oxidase components. Here, we examine the effect of GT on the NADPH oxidase components in the cell-free activation assay. The O₂^–-generating ability of membranes obtained from GT-treated neutrophils is 40.0 and 30.6% lower, respectively, than the untreated counterparts when assayed with two distinct electron acceptors, suggesting that flavocytochrome b₅₅₈ is affected in cells by GT. In contrast, the corresponding cytosol remains competent for activation. Next, GT addition in vitro to the assay consisting of flavocytochrome b₅₅₈ and cytosolic components (native cytosol or recombinant p67^phox, p47^phox, and Rac2) causes a striking inhibition (50% inhibitory concentration = 3.3 µM) when done prior to the stimulation with myristic acid. NADPH consumption is also prevented by GT, but the in vitro assembly of p67^phox, p47^phox, and Rac2 with flavocytochrome b₅₅₈ is normal. Posterior addition of GT to the activated enzyme is ineffective. The separate treatment of membranes with GT also causes a marked loss of flavocytochrome b₅₅₈'s ability to reconstitute O₂^– generation, supporting the conclusion at the cellular level. The flavocytochrome b₅₅₈ heme spectrum of the GT-treated membranes stays, however, unchanged, showing that hemes remain intact. These results suggest that GT directly harms site(s) crucial for electron transport in flavocytochrome b₅₅₈, which is accessible only before oxidase activation.

Editor: T. R. Kozel

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