Neutrophil p29 Peroxiredoxin VI/aiPLA2 (p29 Prdx VI) Enhances Superoxide Production by the NADPH Oxidase in a Manner Dependent On the PLA2 Active Site.
Authors: Ellison Michael , Thurman Gail , Ambruso Daniel R. 
Published: October 2019

Abstract 3592

Poster Board III-529

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. We have identified a 29 kDa protein from human neutrophils (p29) which binds to the NADPH oxidase component p67phox and enhances superoxide anion (O2−) production in a cell-free, NADPH activity oxidase system. The protein was identified as Prdx VI by sequence and the recombinant molecule was found to have Prdx activity. Interestingly, Prdx VI also has a calcium-independent PLA2 activity that is optimal at low pH (aiPLA2). Although Prdx VI is found in many tissues, its function in myeloid cells is not well established. p29 Prdx VI was knocked down in a myeloid cell line (PLB-985) and the effect on the respiratory burst of theses cells was investigated to determine its role in the respiratory burst. In addition, mutant or wild type p29 Prdx were introduced into the cell free system, 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 Prdx VI targeting shRNA or a negative control plasmid and stable transfectants were selected in puromycin containing media. Knockdown of p29 Prdx VI was confirmed by Western blot, but no change in the other known oxidase components was detected. After maturation of the knockdown and control cells by DMSO for 4 days, they were stimulated with fMLP (1μM) and O2− production was measured by luminescence of Diogenes probe. In separate experiments, neutrophil plasma membranes, recombinant p47phox, p67phox and constitutively active Rac1 were added to an assay mix. Addition of SDS assembled of the complex in this cell free system, NADPH was added and O2− production was measured as SOD inhibitable, cytochrome C reduction. O2− production when recombinant wild type p29 Prdx VI was added to the system was compared to activity caused by p29 Prdx VI mutated in the peroxiredoxin active site (C47S) or the aiPLA2 active site (D140A). Wild type and mutant p29 Prdx VI were expressed with tags in sf9 insect cells and purified by Ni2+ chelation chromatography.

Results

Using stable expression of shRNA, p29 Prdx VI protein was reduced to 31+/−18% (SD) of that in non-knockdown control cells but the levels of the other known oxidase components were unchanged. O2− production in response to fMLP was reduced to 51%+/−12% (SD). When added to the cell-free oxidase system, wild type p29 Prdx VI and p29 Prdx VI mutated in the peroxiredoxin active site increased the O2− production rate by similar amounts (to 323+/−94% and 417+/−125% of the assay rate in the absence of p29 Prdx VI respectively; numbers are mean ± SD for three separate experiments). Recombinant p29 mutated in the PLA2 active site however increased O2− production to only 178+/−33%.

Conclusion

These results indicate that p29 Prdx VI is required for optimal O2− production in intact myeloid cells. Data from the cell-free oxidase system suggest that the PLA2 activity of p29 Prdx VI, rather than its peroxiredoxin activity, account for its ability to stimulate O2- production. These studies document a specific biochemical role for p29 Prdx VI in the neutrophil respiratory burst and associated microbicidal activity. Furthermore, lipid modification by p29 Prdx VI may thus be a key step in maximal activation of the NADPH oxidase.

Disclosures:

No relevant conflicts of interest to declare.