Lipid peroxidation (LPO) of pulmonary surfactant, induced by ambient or supplemental oxygen, inflammation or airborne oxidants, compromises the biophysical properties of the surfactant membrane and contributes to respiratory failure in diseases such as adult respiratory distress syndrome. We have recently reported that the structurally related surfactant proteins A (SP-A) and D (SP-D) are potent antioxidants. Both proteins block accumulation of thiobarbituric acid reactive substances (TBARS) and conjugated dienes during metal-induced oxidation of surfactant phospholipids or low density lipoprotein particles, and protect macrophages from oxidant-induced cell death. SP-A and SP-D arrest LPO in progress by a novel mechanism that involves interference with the initiation or propagation of free radicals, but not protein mediated copper chelation or direct oxidative modification of the proteins. The antioxidant function was mapped to the C-terminal domains of SP-A, in the region which contains the C-type lectin domain (CLD) and the phospholipid binding site. The objective of this proposal is to examine the mechanisms of antioxidant protection by SP-A and SP-D by completing four specific aims.
In specific aim #1, we will define the boundaries of the antioxidant activity of SP-A and SP-D by characterizing LPO inhibition by recombinant SP-A and SP-D proteins containing telescoping replacement of C-terminal regions with sequences from the structurally related, but nonprotective, mannose binding protein (MBP), and then more finely map the activity using point mutations and synthetic peptides.
In specific aim #2, we will define the three dimensional structure of SP-A complexed with carbohydrate and surfactant ligands, including saturated, unsaturated and oxidized phosphatidylcholines.
In specific aim #3, we will examine the role of protein/lipid interactions in the antioxidant functions of SP-A and SP-D.
In specific aim #4, we will test the physiological relevance of the antioxidant functions of SP-A and SP-D using ozone and oxygen exposed transgenic animal models of surfactant protein excess and deficiency. The results will provide important insights into the roles of the surfactant proteins in preventing oxidative lung disease and may be useful for the design of novel recombinant proteins and peptides of therapeutic value.
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