Abstract
The first metallocarboxypeptidase (CP) was identified in pancreatic extracts more than 80 years ago and named carboxypeptidase A (CPA; now known as CPA1). Since that time, seven additional mammalian members of the CPA subfamily have been described, all of which are initially produced as proenzymes, are activated by endoproteases, and remove either C-terminal hydrophobic or basic amino acids from peptides. Here we describe the enzymatic and structural properties of carboxypeptidase O (CPO), a previously uncharacterized and unique member of the CPA subfamily. Whereas all other members of the CPA subfamily contain an N-terminal prodomain necessary for folding, bioinformatics and expression of both human and zebrafish CPO orthologs revealed that CPO does not require a prodomain. CPO was purified by affinity chromatography, and the purified enzyme was able to cleave proteins and synthetic peptides with greatest activity toward acidic C-terminal amino acids unlike other CPA-like enzymes. CPO displayed a neutral pH optimum and was inhibited by common metallocarboxypeptidase inhibitors as well as citrate. CPO was modified by attachment of a glycosylphosphatidylinositol membrane anchor to the C terminus of the protein. Immunocytochemistry of Madin-Darby canine kidney cells stably expressing CPO showed localization to vesicular membranes in subconfluent cells and to the plasma membrane in differentiated cells. CPO is highly expressed in intestinal epithelial cells in both zebrafish and human. These results suggest that CPO cleaves acidic amino acids from dietary proteins and peptides, thus complementing the actions of well known digestive carboxypeptidases CPA and CPB.
Highlights
All previously characterized metallocarboxypeptidases of the A/B subfamily are secreted enzymes that cleave aliphatic or basic residues and are initially produced as inactive proenzymes
These results suggest that carboxypeptidase O (CPO) cleaves acidic amino acids from dietary proteins and peptides, complementing the actions of well known digestive carboxypeptidases carboxypeptidase A (CPA) and CPB
We propose that CPO completes the complement of digestive enzymes within the intestine: CPA1 and CPA2 cleave aliphatic/aromatic amino acids, CPB1 cleaves basic amino acids, and CPO cleaves acidic amino acids
Summary
All previously characterized metallocarboxypeptidases of the A/B subfamily are secreted enzymes that cleave aliphatic or basic residues and are initially produced as inactive proenzymes. CPO is highly expressed in intestinal epithelial cells in both zebrafish and human These results suggest that CPO cleaves acidic amino acids from dietary proteins and peptides, complementing the actions of well known digestive carboxypeptidases CPA and CPB. This enzyme is known as CPA1 because an additional pancreatic enzyme was discovered and named CPA2 [8] None of these enzymes cleave acidic C-terminal amino acids, raising the question of how peptides and proteins containing C-terminal aspartate and glutamate are digested in the intestine. No previously characterized mammalian A/B or N/E CPs have been shown to cleave acidic residues, a number of members of the cytosolic carboxypeptidase (CCP) [19] and aminoacylase [20] subfamilies are able to cleave substrates with C-terminal aspartate or glutamate residues. We propose that CPO completes the complement of digestive enzymes within the intestine: CPA1 and CPA2 cleave aliphatic/aromatic amino acids, CPB1 cleaves basic amino acids, and CPO cleaves acidic amino acids
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