| Summary: | 碩士 === 輔仁大學 === 基礎醫學研究所碩士班 === 95 === The alveolus is the major gas exchange unit in lungs. The alveolar epithelium is mainly composed of two types of cells, alveolar type I and type II cells. The major function of type II cells is to produce pulmonary surfactant. Pulmonary surfactant is secreted from alveolar type II cells to the air–liquid interface where it reduces surface tension and prevents atelectasis of alveoli. Pulmonary surfactant is composed of phospholipids and surfactant proteins. Phospholipids include phosphatidylcholines (PC), mainly dipalmitoylphosphatidylcholine (DPPC), and phosphatidylglycerols. DPPC is most responsible for the surface tension-lowering properties of pulmonary surfactant. Production of DPPC can be achieved by both de novo synthesis and the remodeling pathways. In the remodeling pathway, unsaturated acyl group of cellular phosphatidylcholines is removed by phospholipase A2 resulting in 1-palmitoyl-2-
lysophosphatidylcholines, followed by reacylation of 1-palmitoyl-2-lysophos-
phatidylcholines with palmitoyl-CoA via the catalysis of a lysophosphatidylcholine (lysoPC) acyltransferase (LPCAT). We identified a putative LPCAT whose expression is enriched in alveolar type II cells of Sprague Dawley (SD) rat. The cloned cDNA encodes a protein of 534 amino acids with an estimated molecular mass of 59 kDa. Amino acid sequences of this putative acyltransferase revealed a highly conserved domain similar to 1-acyl-sn-glycerol-3-phosphate acyltransferase (plsC) in Escherichia coli(E. coli). Using prokaryotic expression system, a maltose binding protein and 6x histidine tagged LPCAT was successfully expressed and purified using affinity chromatography. Purified proteins were injected into rabbits for specific anti-sera production. Anti-LPCAT antibodies were also purified from immunized sera by affinity chromatography. Using purified anti-LPCAT antibodies, we localized LPCAT protein to alveolar type II cells by immunohistochemistry, immunoflurosence and Western blotting. Using zonal centrifugation, LPCAT was localized to the microsomal organelle fraction. The result was also confirmed by confocal microscopy. Using eukaryotic 293T cell expression system in combination with zonal centrifugation, the microsomal organelle fractions from transfected cells
were tested for acyltransferase activity.
|
|---|
