Pulmonary surfactant is a complex mixture of phospholipid and protein which is essential to the maintenance of alveolar stability. The surfactant-associated proteins, surfactant protein A(SP-A), B(SP-B) and C(SP-C) have received increasing attention as important biophysical and regulatory mediators of surfactant function and metabolism. The most abundant surfactant associated protein, SP-A, is a 26,000-38,000 MW secretory product of the alveolar type II call. SP-A has been shown to enhance the adsorption of phospholipid to the air-liquid interface, to promote the uptake of liposomal phospholipid and to inhibit the secretion of phospholipid from type II cells. These in vitro observations suggest a central role for SP-A in surfactant homeostasis in the functioning alveolus. The functions of SP-A are thought to be mediated, in part, through a high affinity cell surface receptor for SP-A. A tentative functional map of the structural moieties of SP-A responsible for receptor binding and biologic function has been provided from studies of partial proteolytic digestion, monoclonal antibody binding and biochemical modification of the apoprotein. We propose to employ the technique of site directed mutagenesis to more precisely address the structure-function relationships of SP-A.
The specific aims are to: 1) express recombinant rat SP-A in a heterologous eukaryotic cell line and characterize the recombinant product physically and functionally, 2) produce mutant SP-A cDNA's encoding for alterations in specific structural domains of SP-A 3) express these mutants in a heterologous cell line, isolate the mutant recombinant SP-A's and characterize the protein physically and functionally in assays of biologic function. The cDNA for rat SP-A has been isolated and sequenced. The production of mutant cDNA's will be accomplished through site directed mutagenesis using mutagenic oligonucleotides hybridized with single stranded plasmid template containing the cDNA for SP-A. The plasmid containing the mutant SP-A cDNA is then used to transform a eukaryotic host. The secretion of the mature post translationally modified protein is determined using an ELISA assay and the recombinant product is isolated by affinity chromatography on mannose sepharose or IgG (anti-SP-A) columns. The effects of these mutations to SP-A upon: 1) binding to the type II cell surface receptor, 2) binding to isolated plasma membrane from type II cells, 3) entry into the intracellular pool, 4) inhibition of secretion of phospholipid from type II cells 5) the uptake of liposomal phospholipid by isolated alveolar type II cells will be determined. The significance of this project is that it will provide a detailed functional map of the SP-A molecule and in doing so may clarify the factors involved in surfactant homeostasis. Ultimately this study will provide information necessary to the development of agents which mimic or antagonize the activity of SP-A and may suggest potential treatment strategies for diseases of surfactant deficiency or excess.
