Interaction of Surfactant Protein A with Peroxiredoxin 6 Regulates Phospholipase A2 Activity

Yong-Zheng Wu,Yefim Manevich,James L Baldwin,Chandra Dodia,Kevin Yu,Sheldon I Feinstein,Aron B Fisher

doi:10.1074/jbc.m504525200

Yong-Zheng Wu, Yefim Manevich + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m504525200

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Abstract

Peroxiredoxin 6 (Prdx6) is a "moonlighting" protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA(2) activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA(2) activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA(2) activity) showed K(m)0.35 mm and V(max) 138 nmol/min/mg of protein. SP-A inhibited PLA(2) activity non-competitively with K(i) 10 mug/ml and was Ca(2+) -independent. Activity at pH 7.4 was approximately 50% less, and inhibition by SP-A was partially dependent on Ca(2+). Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni(2) -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca(2+) dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA(2) activity of Prdx6 by SP-A.

Highlights

Peroxiredoxins (Prdxs) are a recently described superfamily of Se-independent peroxidases that are distributed in all phyla [4]
We have reported previously that acidic calciumindependent PLA2 (aiPLA2) activity is trivial at pH Ͼ6 (6 – 8), we have subsequently found activity of recombinant protein after the addition of GSH to the incubation medium
In the presence of 50 ␮g/ml surfactant protein A (SP-A), aiPLA2 activity was inhibited by 80% under acidic conditions, whereas with alkaline conditions, inhibition was 70% in the presence of Ca2ϩ and 50% in its absence

Summary

Introduction

Peroxiredoxins (Prdxs) are a recently described superfamily of Se-independent peroxidases that are distributed in all phyla [4]. Addition of SP-A to rat lung homogenate, isolated lamellar bodies, or isolated rat alveolar type II cells inhibited aiPLA2 activity and the degradation of DPPC [3]. Incubation of Prdx6 with SP-A and anti-SP-A pAb followed by addition of protein-A-agarose beads resulted in precipitation of both SP-A (Fig. 6A, bottom panel) and Prdx6 (Fig. 6A, upper panel).

Results

Conclusion