P-22 Exploring the Role of Carboxypeptidase O in Chylomicron and Lipid Droplet Formation

Carboxypeptidase O (CPO) cleaves acidic and polar C-terminal amino acids of peptides and proteins; however, its biological function remains unknown. At the subcellular level, CPO is anchored to the inner leaflet of the endoplasmic reticulum (ER) membrane and associated with lipid droplets. The ER membrane is the site of lipid droplet and chylomicron formation, the latter of which occurs only in the intestine where CPO is expressed. This study aims to determine if CPO has a role in chylomicron formation, and to explore the intracellular function of CPO through the identification of its binding partners and substrates. To do so, we have made an inactive CPO mutant by site-directed mutagenesis. Transfection of human colon carcinoma (Caco-2) cells with plasmids expressing wild-type and mutant CPO, as well as GFP as control, has been successful. However, selection of cells stably-integrating this DNA is currently being optimized in preparation for immunocytochemistry to assess any differences in the amount of lipid droplets and chylomicrons that associate with CPO. Immunoprecipitation and tandem mass spectrometry will be used to identify binding partners and substrates.