Neutrophil p29 Peroxiredoxin 6/aiPLA2 (p29 Prdx6) Enhances Superoxide Anion (O₂^-) Production by Nox2: Role for the PLA2 Active Site

Abstract 926

Introduction: An intact respiratory burst is critical to the microbial activity of neutrophils. The respiratory burst is initiated by assembly and activation of the NADPH oxidase, Nox2. We have identified a 29 kDa protein from human neutrophils (p29) which binds to the oxidase component p67phox and enhances superoxide anion (O₂^-) production in a cell-free, NADPH activity oxidase system. Identified as Prdx6 by sequence and was found to have Prdx activity by the recombinant protein. p29 Prdx6 also has a calcium-independent PLA2 activity that is optimal at low pH (aiPLA2). Although Prdx6 is found in many tissues, its function in myeloid cells is not well established. p29 Prdx6 was knocked down in a myeloid cell line (PLB-985) and the effect on the Nox2 activity of theses cells was investigated. In addition, wild type p29 Prdx6 or mutants with altered Prdx and PLA2 active sites were re-expressed in the knockdown cells, to explore the possibility that at least one of the catalytic activities is involved in enhancing the oxidase. Methods: PLB-985 cells were transfected with a plasmid encoding a p29 Prdx6 targeting shRNA or a negative control plasmid and stable transfectants were selected in puromycin containing media. Knockdown of p29 Prdx6 was confirmed by Western blot, but no change in the other known oxidase components was detected. cDNAs for wild type (wt) p29 Prdx6 and known mutants for the Prdx activity site (C47S) and PLA2 active site (H26A and S32A) were cloned into pcDNA3.1 producing plasmids generating mRNAs resistant to shRNA. Knockdown cells transfected with these plasmids were cultured with both puromycin and G418 with re-expression of wt or mutant Prdx6 to comparable levels in PLB985 cells, documented by Western blot. After maturation of the knockdown, Prdx re-expressing, and control cells with DMSO for 4 days, fMLP (1μM) stimulated O₂^- production was measured by luminescence of Diogenes probe. Results: Using stable expression of shRNA, p29 Prdx6 protein was reduced to 31±8% (mean ± SEM, n=5) of that in non-knockdown control PLB985 cells. O₂^- production in response to fMLP was reduced (Student's t-test, p<0.05) to 51±4% (mean ± SEM, n=25). Re-expression of wt p29 Prdx6 reestablished full Nox2 activity (100±5%, n=6). For comparison, empty re-expression vector control was 52±6%, n=22). When the Prdx activity (C47S) mutant was expressed, Nox2 activity was slightly increased (133±5%, n=6). In contrast, expression of mutants at PLA2 active site had no effect on recovery of Nox2 activity (H26A, 60±2%, n=5; S32A, 58±9%, n=10, different from non-knockdown and C47S, p<0.05). Conclusion: These results indicate that p29 Prdx VI is required for optimal O₂^- production in intact myeloid cells confirming our data from the cell-free oxidase system. Furthermore, studies evaluating expression of p29 Prdx protein mutated at sites associated with Prdx and PLA2 suggest the latter is responsible for Nox2 activity in intact cells. These studies document a specific biochemical role and mechanism for p29 Prdx6 in the neutrophil respiratory burst and associated microbicidal activity and define a molecular target for modulating Nox2 activity.