Aims: Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A(2) (PLA(2)) activities, has been demonstrated as playing a critical role in antioxidant defense of the lung. Our aim was to evaluate the relative role of each activity in Prdx6-mediated protection of mouse pulmonary microvascular endothelial cells (PMVECs) against the peroxidative stress of treatment with tert-butyl hydroperoxide (tBOOH).
Results: PMVEC from Prdx6 null mice showed increased lethality on tBOOH exposure (50-200 μM) compared with wild-type (WT) controls. Treatment with 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), a Prdx6 PLA(2) activity inhibitor, increased the sensitivity of WT cells to peroxidative stress, but did not further sensitize Prdx6 null cells. Lethality in Prdx6 null PMVEC was "rescued" by transfection with a construct leading to the expression of WT rat Prdx6. Expression of mutant Prdx6 with either peroxidase activity or PLA(2) activity alone each partially rescued the survival of Prdx6 null cells, while constructs with both active sites mutated failed to rescue. Co-transfection with two different constructs, each expressing one activity, rescued cells as well as the WT construct.
Innovation and conclusion: Contrary to the general assumption that the peroxidase activity is the main mechanism for Prdx6 antioxidant function, these results indicate that the PLA(2) activity also plays a substantial role in protecting cells against oxidant stress caused by an exogenous hydroperoxide.