Carboxypeptidase O is a glycosylphosphatidylinositol-anchored intestinal peptidase with acidic amino acid specificity

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.

FIGURE 1.

Comparison of domain structure of CP subfamilies. A, all M14 CPs have a 300-residue catalytic domain (light gray). Members of the A/B and cytosolic CCP subfamilies usually have an N-terminal β-sheet-rich domain (dark gray), whereas members of the N/E and aminoacylase (AA) subfamilies have a C-terminal β-sheet-rich domain (dark gray). Some CPs have signal peptides (black) and additional domains with uncharacterized structure (white). The CCPs vary in length (indicated by “ … ”). B, CPO consists of an N-terminal signal peptide, a catalytic domain, and a short C-terminal sequence. In mammals and birds, CPO also has a short peptide between the signal peptide and the CP domain; this sequence is not present in fish CPO.

FIGURE 2.

CPO protein sequences from representative species. Genomic and cDNA databases (Ensembl and NCBI) were searched for CPO sequences, which were aligned with each other and with human CPA1 using ClustalW. Signal peptide, N-terminal domain/prodomain, and C-terminal domain sequences are indicated as well as all critical active site residues necessary for zinc binding (His-69, Glu-72, and His-196), substrate binding (Arg-127, Arg-145, Tyr-248, and Arg/Ile-255), and catalytic activity (Glu-270; numbering system based on the position of residues in bovine CPA by convention). Greater sequence similarity is indicated by darker shading. Sequences shown are from the following database entries: human CPO, ENSP00000272852; cow CPO, ENSBTAP00000025539; dog CPO, ENSCAFP00000019833; rabbit CPO, ENSOCUP00000008449; chicken CPO, gi|118093691:4613825–4669825; finch CPO, NW_002198919.1|Tgu7_WGA826_1; zebrafish CPO, NM_001145629; stickleback CPO, ENSGACP00000018671; and human CPA1, NP_001859.

FIGURE 3.

Specificity and inhibition of CPO enzyme activity. A, zebrafish CPO (0.4 ng/μl) was incubated with a variety of commercially available synthetic substrates at 0.5 mm concentrations. Reaction rates were determined by measuring the change in absorption at 340 nm. B, zebrafish CPO (0.4 ng/μl) was incubated with its optimal substrate, fa-EE, at a concentration of 0.5 mm and at a range of pH values to determine the pH optimum of CPO. C, citrate was found to inhibit the enzymatic activity of CPO assayed with 0.5 mm fa-EE, pH 7.5, although inhibition was less potent toward the human enzyme than the zebrafish enzyme. Human enzyme was incubated at a concentration of 4.0 ng/μl, whereas zebrafish CPO was incubated at 0.4 ng/μl. For all panels, error bars show S.E. for triplicate determinations.

FIGURE 4.

CPO cleaves C-terminal glutamate from protein substrates. Purified human and zebrafish CPO enzymes were incubated with 5 μg of purified porcine tubulin. Proteins were resolved by SDS-PAGE, and tubulin C-terminal and side-chain modifications were analyzed by Western blotting. All bands were quantified, normalized to total α-tubulin, and expressed as a percentage of tubulin not incubated with CPO enzyme. B, schematic showing the C terminus of α-tubulin and the antibodies that recognize the various modifications. Tyr, tyrosinated; DeTyr, detyrosinated.

FIGURE 5.

CPO is GPI-anchored. A, the most likely GPI modification sites predicted by the big-PI program are indicated with black squares. For mammalian CPO and chicken CPO, the program predicted a high likelihood of GPI modification (p < 0.05). For finch, zebrafish, and stickleback fish, the prediction was less confident (p > 0.05). B, proteins from two different clones (c1 and c2) of differentiated MDCK cells stably transfected with human CPO (+) or empty vector (−) were extracted with Triton X-114, and detergent and aqueous phases were separated. CPO was found primarily in the detergent phase by Western blotting with a CPO-specific antibody. C, detergent-soluble proteins from control (−hCPO) or hCPO-expressing (+hCPO) MDCK cells were incubated with (+) or without (−) PI-PLC followed by a second phase extraction into Triton X114. Most CPO (detected by Western blotting) transitioned from the detergent to aqueous phase following incubation with PI-PLC, indicating the presence of GPI modification.

FIGURE 6.

Stably expressed CPO is localized to intracellular vesicular membranes as well as plasma membrane of MDCK cells. A, MDCK cells stably expressing human CPO were fixed at a subconfluent state. CPO was identified by immunocytochemistry in vesicular structures (see inset). No colocalization of CPO was found with markers of either early (EEA1) or late (LAMP2) endosomes. Nuclei (blue) were stained with DAPI. B, when the above MDCK cells were differentiated into a polarized epithelial monolayer, CPO was more prominently seen at the plasma membrane as indicated by colocalization with Na⁺/K⁺-ATPase, a marker of the basolateral membrane. Scale bars, 10 μm.

FIGURE 7.

CPO is expressed in intestinal epithelial cells. A, a database search of expressed sequence tags (ESTs) for CPO mRNA from all species revealed 19 sequences from intestinal tissues and four or fewer from other tissues. B, in situ hybridization (ISH) of 4-dpf zebrafish indicated that CPO mRNA was found predominantly in intestinal tissues. Both lateral and dorsal views are shown. C, the above fish stained for CPO by in situ hybridization were paraffin-embedded, sectioned sagittally, and counterstained with methyl green. Blue in situ hybridization stain showed epithelial expression of CPO mRNA. D, CPO mRNA abundance was determined by quantitative PCR, which showed expression after 3 dpf in both fed and starved states. Error bars indicate S.E. for three biological replicates, each assayed in triplicate. E, frozen sections of human ileum were stained by immunohistochemical techniques with an unrelated control antibody and two different antibodies, one raised against the N-terminal region of CPO and the other raised against the C-terminal region of CPO (shown in “red”). Nuclei were stained with DAPI (blue). Both low (left) and high (right) magnification indicated CPO staining in the apical region of enterocytes. Scale bars in E, 100 μm (left) and 10 μm (right).