Carboxypeptidase O is an intestinal peptidase with unique substrate specificity

Metallocarboxypeptidase (CP) enzymes were first identified in pancreatic extracts more than 80 years ago. Since that time thirteen active mammalian CPs have been described all having substrate specificity toward either C‐terminal hydrophobic or basic amino acids. Here we describe another carboxypeptidase, CPO, with unique substrate specificity toward acidic C‐terminal amino acids. While CPO is a pseudogene in the mouse and has not been identified in the rat, a number of intestinally‐derived CPO ESTs were identified for many other mammalian and fish species. In situ hybridization showed expression of zebrafish CPO restricted to the intestinal epithelia. CPO was predicted to enter the secretory pathway, and this was confirmed by immunocytochemistry showing HA‐tagged CPO co‐localization with an ER marker. Structurally, CPO contains a unique 25 amino acid C‐terminal segment and lacks the N‐terminal propeptide found in all other related CPs. Using a panel of synthetic substrates it was shown that purified zebrafish CPO exhibited specificity for acidic C‐terminal amino acids and a neutral pH optimum consistent with an extracellular role in dietary protein degradation. These results suggest that CPO is an intestinal digestive enzyme which complements the activity of other digestive CPs through its ability to cleave acidic C‐terminal amino acids.