P-23 Carboxypeptidase O Is Able to Cleave both Acidic and Polar Amino Acids from Substrate Proteins within the Early Secretory Pathway

Buller Hall

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 hence function. The intracellular distribution of CPO, expressed in MDCK cells, was analyzed by immunofluorescence microscopy. CPO was detected on the endoplasmic reticulum in a punctate, circular, or reticular pattern that was poorly associated with lipid raft markers, although modulated by cholesterol. Cholesterol also modified the enzymatic activity of CPO in vitro; no effect was observed in vivo. The ability of CPO to cleave C-terminal amino acids within the early secretory pathway was examined using Gaussia luciferase as a substrate, C-terminally tagged with variants of an ER retention signal. These data show that CPO functions in the ER or Golgi to remove C-terminal glutamates and aspartates, as well as a number of polar amino acids. Following bioinformatics analysis to determine candidate substrates, Torsin 1A, an ER protein known for its role in early-onset torsion dystonia, was confirmed as a substrate of CPO.