Carboxypeptidase O is a Lipid Droplet-associated Enzyme Able to Cleave both Acidic and Polar C-terminal Amino Acids

Carboxypeptidase O (CPO) is a member of the M14 family of metallocarboxypeptidases with a preference for the cleavage of C-terminal acidic amino acids. CPO is largely expressed in the small intestine, although it has been detected in other tissues such as the brain and ovaries. CPO does not contain a prodomain, nor is it strongly regulated by pH, and hence appears to exist as a constitutively active enzyme. The goal of this study was to investigate the intracellular distribution and activity of CPO in order to predict physiological substrates and function. The distribution of CPO, when expressed in MDCK cells, was analyzed by immunofluorescence microscopy. Soon after addition of nutrient-rich media, CPO was found to associate with lipid droplets, causing an increase in lipid droplet quantity. As media became depleted, CPO moved to a broader ER distribution, no longer impacting lipid droplet numbers. Membrane cholesterol levels played a role in the distribution and in vitro enzymatic activity of CPO, with cholesterol enrichment leading to decreased lipid droplet association and enzymatic activity. The ability of CPO to cleave C-terminal amino acids within the early secretory pathway (in vivo) was examined using Gaussia luciferase as a substrate, C-terminally tagged with variants of an ER retention signal. While no effect of cholesterol was observed, these data show that CPO does function as an active enzyme within the ER where it removes C-terminal glutamates and aspartates, as well as a number of polar amino acids

Subcellular Distribution of Carboxypeptidase O Affected by Nutrient Availability (933.1)

Carboxypeptidase O (CPO) is a member of the M14 family of proteolytic enzymes. Many members of this family are secreted from cells and are involved in degradation of extracellular peptides; other carboxypeptidases remain within the secretory pathway where they are involved in the maturation of bioactive peptides. In order to determine the function of CPO, the subcellular localization of CPO was determined by immunofluorescence analysis. CPO, stably expressed in Madin-Darby canine kidney cells, was found in a punctate pattern partially associated with lipid droplets. When cells were serum-starved the structures associated with CPO became more numerous and often clustered. Addition of oleic acid to the medium caused CPO to be redistributed in a diffuse pattern with some concentration on the nuclear envelope. Western blot analysis of CPO following oleate incubation indicated an increase in the amount of a CPO isoform with lower mobility as seen by SDS-PAGE, suggestive of a post-translational modification. These changes in CPO size and distribution, dependent on nutrient availability, suggest a possible role for CPO in autophagy-related processes