Interaction of surfactant protein A with peroxiredoxin 6 regulates phospholipase A2 activity.
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.
Wu
Yong-Zheng
YZ
Institute for Environmental Medicine, University of Pennsylvania, One John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6068, USA.
Manevich
Yefim
Y
Baldwin
James L
JL
Dodia
Chandra
C
Yu
Kevin
K
Feinstein
Sheldon I
SI
Fisher
Aron B
AB
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002845:Chromatography; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005979:Glutathione Peroxidase / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D047468:Immunoprecipitation; D007700:Kinetics; D008027:Light; D008168:Lung / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009532:Nickel / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry*; D054465:Peroxiredoxin VI; D054464:Peroxiredoxins; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D037662:Pulmonary Surfactant-Associated Protein A / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D012685:Sepharose / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013997:Time Factors
D003432:Cross-Linking Reagents; D010743:Phospholipids; D037662:Pulmonary Surfactant-Associated Protein A; D011994:Recombinant Proteins; D015060:1,2-Dipalmitoylphosphatidylcholine; D009532:Nickel; D012685:Sepharose; D010544:Peroxidases; C515977:PRDX6 protein, human; D054465:Peroxiredoxin VI; D054464:Peroxiredoxins; C515983:Prdx6 protein, mouse; C515984:Prdx6 protein, rat; D005979:Glutathione Peroxidase; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium