This project will evaluate the properties and physiological role of a novel phospholipase A2 (aiPLA2) that was identified during the preceding period of grant support. We have isolated the protein from rat and bovine lungs, have identified full length cDNA clones for the human, rat, mouse, and bovine enzyme, and have generated a panel of antibodies. This unique PLA2 is Ca++-independent and shows maximal activity at pH4 and thus internalized alveolar DPPC and also provides substrate for the reacylation pathway of DPPC synthesis. This study will utilize native and recombinant aiPLA2 mutant aiPLA2 proteins, isolated alveolar type II cells, several cell lines, isolated rat and mouse lungs, and intact animals, to study activity and regulation of aiPLA2 and its physiological role in lung surfactant metabolism.
Specific Aim 1 will investigate the regulation of aiPLA2 activity. We will utilize co-immunoprecipitation and surface plasmon resonance techniques to evaluate interactions between aiPLA2 and SP-A, which we propose is a physiological regulatory of enzyme activity. We will evaluate possible glutathiolation and phosphorylation as additional regulatory mechanisms.
Specific Aim 2 will evaluate structure-function relationships for aiPLA2 activity utilizing site directed mutagenesis and fluorescence resonance energy transfer measurements to determine binding of substrate to protein and to determine the amino acid residues required for activity.
Specific Aim 3 will investigate the regulation of aiPLA2 expression with emphasis on developmental expression using the timed pregnant rat and human lung samples. We will also evaluate a role for corticosteroids, cAMP, and KGF in regulation of aiPLA2 expression.
Specific Aim 4 will evaluate subcellular localization of the enzyme utilizing fluorescence and electron microscopy, subcellular fractionation techniques, and analysis of subcellular targeting signals. We postulate that the protein is targeted to the lysosomal compartment for PLA2 activity.
Specific Aim 5 will develop several models of under-and over-expression of aiPLA2 and evaluate the resultant effects of DPPC degradation and synthesis using isolated cells, the isolated perfused lung model, and in vivo studies. These studies will provide important insights into a novel enzyme of major importance to lung surfactant metabolism and will provide the basis for further in depth characterization of lysosomal PLA2 activity.
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