Pulmonary surfactant plays a crucial role in maintaining the integrity of the alveoli at reduced lung volumes. The regulation of the secretion and metabolism of surfactant is consequently an integral component of normal gas exchange in the lungs. Surfactant protein A (SP-A) is the major protein component of pulmonary surfactant. The SP-A has recently been identified as a potent negative regulator of surfactant lipid secretion by alveolar type II cells in vitro. In addition, SP-A has also been shown to recognize a high affinity receptor on the alveolar type II cell and facilitate the uptake of phospholipid by these cells. An emerging view of SP-A is that the protein plays a central role in research described in this proposal will investigate the structural elements of rat SP-A that are responsible for these biological activities. The specific structural elements of SP-A that will be studied are: 1) the oligosaccharide moiety, 2) disulfide bridges, 3) the collagen-like region, 4) the conserved interruption in the collagen-like region, 5) the phospholipid binding domain, and 6) selected conserved (across rat, human, dog and rabbit) basic amino acids. The role of discrete structural elements of SP-A in function will be examined using site directed mutagenesis. Modified forms of the SP-A will be constructed utilizing oligonucleotide directed mutagenesis of the cDNA for the protein and subsequent expression in a heterologous eukaryotic cell. The mutant forms of SP-A will be isolated by carbohydrate affinity and antibody affinity chromatography. The effects of specific receptors on isolated membranes, c) inhibit surfactant lipid secretion by alveolar type II cells, d) mediate phospholipid uptake, and e) bind specific carbohydrate moieties will be examined. Furthermore, the effects of these mutations upon the physical properties of the protein will also be investigated. These studies will provide detailed information about structural aspects of SP-A that are required for biological activities and should provide insights into some of the mechanisms of regulating surfactant homeostasis in the alveolar spaces of the lung. A clear understanding of the structural elements of SP-A that are necessary for its biological activities will form the basis of developing agents that can mimic, enhance or antagonize the effects of SP-A as a regular of pulmonary surfactant secretion.
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