Pulmonary surfactant is a lipid and protein complex present in the alveolus that greatly diminishes the work of breathing by reducing surface tension at athe air-liquid interface. The major components of surfactant are diplmitoylphosphatidylcholine and the surfactant associated proteins A, B, C and D (designated SP-A, SP-B SP-C and SP-D). SP-A interacts with dipalmitoylphosphatidylcholine, alveolar type 2 cells and macrophages and is an important contributor to surfactant homeostasis. SP-A belongs to the collection family and has four distinctive structural domains; 1) an amino terminus involved in interprotomeric disulfide formation, 2) a collagen domain, 3) an alpha helical forming neck region, and 4) a carbohydrate recognition domain (CRD). The CRD has been implicated as an important protein domain involved in lipid binding and high affinity interactions with type 2 cells. The neck region also plays a role in lipid binding. The collagen domain is involved in oligomer formation and probably high affinity binding to alveolar macrophages. The goals of this proposal are to define specific amino acids within each of these domains that are responsible for the functions of the protein. To achieve these goals, we will use site-directed mutagenesis of the collagen domain, the CRD, the known carbohydrate binding site and the neck region to elucidate the structural basis of the multiple functions of the protein. The mutant proteins will be examined for their activity as a ligand for the SP-A receptor on alveolar type 2 cells and an agent that promotes lipid uptake and negatively regulates surfactant secretion by these cells. In addition, the lipid binding and aggregating activity of the mutant proteins will also be investigated. These studies will also map the location of th high affinity binding site present on SP-A for macrophages. From these studies we will define specific structural elements of SP-A sequence that are involved in its multiple functions. Ultimately these findings should prove useful for developing mimetics and antagonists to SP-A function in vivo.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL045286-08
Application #
2735178
Study Section
Lung Biology and Pathology Study Section (LBPA)
Project Start
1990-07-01
Project End
2001-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
8
Fiscal Year
1998
Total Cost
Indirect Cost
Name
National Jewish Health
Department
Type
DUNS #
City
Denver
State
CO
Country
United States
Zip Code
80206
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