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 g/ml and was Ca 2؉ -independent. Activity at pH 7.4 was ϳ50% 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.Pulmonary surfactant, a lipoprotein complex lining the lung surface, consists of phospholipids, specific proteins, and other lipid components. It is synthesized by alveolar type II epithelial cells, assembled in lamellar bodies, the intracellular surfactant storage organelle, and secreted into the alveolar space and terminal airways where it functions to reduce surface tension and stabilize alveoli (1). Dipalmitoylphosphatidylcholine (DPPC), 2 the major phospholipid of surfactant, is the critical component for the surface-tension-lowering function (2). DPPC is cleared from the alveolar space predominantly through endocytosis by type II cells with a minor contribution from alveolar macrophages. Under normal physiological conditions, clearance and secretion of DPPC appear to be coordinately regulated (3).Peroxiredoxins (Prdxs) are a recently described superfamily of Se-independent peroxidases that are distributed in all phyla (4). They are classified according to the number (1 or 2) of conserved cysteine (Cys) residues directly involved in peroxidase catalysis. Of the six mammalian peroxiredoxins, Prdx6 has a single conserved Cys, whereas Prdx1-5 are 2-Cys enzymes. Prdx6 is expressed in various tissues, but is especially enriched in lung and brain (5, 6). By immunocytochemistry, Prdx6 in lung is present in alveolar type II cells, alveolar macrophages, and bronchiolar epithelium (6), and subcellular fractionation of lungs has demonstrated the presence of this protein in lamellar body, lysosomal, and cytosolic...