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Neuropeptide-processing carboxypeptidases
Life Sciences 73 (2003) 655 – 662 www.elsevier.com/locate/lifescie
Neuropeptide-processing carboxypeptidases Suwen Wei, Yun Feng, Elena Kalinina, Lloyd D. Fricker * Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
Abstract Neuropeptides are generally produced from precursor proteins by selective cleavage at specific sites, usually involving basic amino acids. Enzymes such as the prohormone convertases and carboxypeptidase E are highly specific for these basic amino acid-containing sites. In addition to this ‘‘traditional’’ pathway, several neuropeptides are known to be cleaved at non-basic sites, and the enzymes responsible for these cleavages have not been conclusively identified. In a recent search for novel members of the metallocarboxypeptidase family, we found three human genes. One of these, named ‘‘CPA-5,’’ has a specificity for C-terminal hydrophobic amino acids and mRNA expression in brain, pituitary, and testis. To test whether CPA-5 protein has a distribution pattern in pituitary that is consistent with a role for this enzyme in the non-basic processing of proopiomelanocortin-derived peptides such as beta-endorphin and adrenocorticotropin, we examined the distribution of CPA-5 using immunocytochemistry. In the pituitary, CPA-5 is detected in the neurointermediate lobe and in scattered cells in the anterior lobe. In the AtT-20 corticotroph cell line, CPA-5 has a perinuclear distribution. Taken together, these results are consistent with a role for CPA-5 in the intracellular processing of proopiomelanocortin-derived peptides at non-basic sites. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Peptide processing; Carboxypeptidase E; Enkephalin convertase; Carboxypeptidase A-5; Metallocarboxypeptidase
Introduction The vast majority of neuropeptides are generated by selective cleavage of precursors at sites that contain one or more basic amino acids (Steiner, 1991). First, one or more endopeptidase cleaves the precursor to generate intermediates containing C-terminal basic residues (Zhou et al., 1999; Seidah and Chretien, 1997). Then, a carboxypeptidase removes these basic residues (Fricker, 1988). In some cases,
* Corresponding author. Tel.: +1-718-430-4225; fax: +1-718-430-8954. E-mail address: [email protected] (L.D. Fricker). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00386-2
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further processing occurs to generate a C-terminal amide residue (Eipper and Mains, 1988). Also, some peptides acquire other post-translational modifications such as acetylation, phosphorylation, sulfation and glycosylation (Bennett, 1991). In some cases, these post-translational modifications alter the biological activity or stability of the peptide. In addition to this ‘‘traditional’’ scheme involving proteolytic cleavage at basic amino acid-containing sites, some peptides undergo cleavage at non-basic sites. For example, neuropeptide FF is generated by two endoproteolytic cleavages, one involving the traditional pair of basic residues and the other involving cleavage between an Ala-Phe bond (Vilim et al., 1999). Other peptides have been found to undergo C-terminal processing at non-basic residues. Examples include human beta-endorphin 1–26, which is presumably generated from beta-endorphin 1–27 by removal of the C-terminal Tyr residue, and adrenocorticotrophin 1–38, which is generated from the full-length 39 residue adrenocorticotrophin (ACTH) by removal of the C-terminal Phe residue (Friedman et al., 1993). The proteolytic processing often has a dramatic impact on the biological activity of the peptide. A classic example of the importance of proteolytic processing is with ACTH and alpha-melanocytestimulating hormone (a-MSH), the latter representing the N-terminal 13 residues of ACTH (Eipper et al., 1986). These two peptides have substantially different biological activities, with ACTH stimulating the hormone secretion from the adrenal gland while a-MSH stimulates melanocytes and also appears to play a role in the control of feeding and energy balance (Eipper et al., 1986; Liotta and Krieger, 1983). The non-traditional cleavages also can impact on the resulting biological activity of the peptide. For example, h-endorphin 1–31 is an opiate agonist, 1–27 is an antagonist, and 1–26 is devoid of activity (Millington and Smith, 1991). The conversion of h-endorphin 1–27 from 1–31 involves the traditional pathway of cleavage at a pair of basic amino acids followed by C-terminal removal of these basic residues. However, as mentioned above, the conversion of h-endorphin 1–27 into 1–26 involves removal of an aromatic amino acid, which is not catalyzed by the same enzymes as in the traditional pathway. The enzymes in the traditional pathway have been well studied over the past 10–20 years. The major peptide-processing endopeptidases are prohormone convertase (PC) 1 and 2 (Zhou et al., 1999; Seidah and Chretien, 1998a,b). These enzymes efficiently cleave many precursors at sites containing two basic amino acids, either adjacent to each other (e.g. Lys-Arg, or Arg-Arg) or separated by 2, 4, or 6 other amino acids (e.g. Arg-Xaa-Xaa-Arg) (Seidah and Chretien, 1998a,b). Neither PC1 or PC2 are known to cleave precursors at non-basic residues. Following the action of PC1 or PC2, a carboxypeptidase is required to remove the C-terminal basic residues (Fricker, 1988, 1991). For many years carboxypeptidase E (CPE, also known as enkephalin convertase, carboxypeptidase H, and EC3.4.17.10) was thought to be the only carboxypeptidase involved in neuropeptide processing (Fricker, 1988, 1991). However, the discovery in 1995 that mice lacking CPE activity due to a genetic point mutation were still capable of producing low levels of correctly processed peptides raised the possibility that another enzyme participated in this processing reaction (Naggert et al., 1995). Because the other carboxypeptidases known at the time had distributions and/or substrate specificities that were not appropriate for intracellular neuropeptide-processing enzymes, a search for novel enzymes was initiated. A variety of techniques were used to screen for new members of the metallocarboxypeptidase family, including enzyme assays, polymerase chain reaction with oligonucleotides to conserved regions of carboxypeptidases, and more recently, database mining. Altogether, we identified 7 new members of the metallocarboxypeptidase family (Song and Fricker, 1995, 1997; Xin et al., 1998; Lei et al., 1999; Wei et al., 2002). Five of these appear to be functional as carboxypeptidases and were named carboxypeptidase D,
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Z, A-5, A-6, and O. In addition, two members of the family do not appear to encode enzymes that are functional carboxypeptidases and were named CPX-1 and CPX-2 (with ‘‘CP’’ standing for carboxypeptidase-like, based on the 40–50% amino acid sequence identity between CPE and these two proteins). CPX-1 and 2, as well as another family member discovered by another group and named AEBP-1, lack one or more critical active site residues and are inactive towards standard carboxypeptidase substrates (He et al., 1995; Song and Fricker, 1997; Xin et al., 1998; Lei et al., 1999). The physiological function of these carboxypeptidase-like proteins is not clear, but due to the strong conservation of these three proteins between human and rodents it is likely that they play an important role. Furthermore, even C.elegans and Drosophila melanogaster contain carboxypeptidase-like genes that are lacking one or more critical active site residues (Sidyelyeva and Fricker, 2002). Of the functional enzymes, only CPD has the correct distribution and enzyme properties to function in the intracellular processing of neuropeptides (Song and Fricker, 1995; Varlamov and Fricker, 1998; Dong et al., 1999; Novikova et al., 1999). This enzyme is broadly distributed throughout the body and is present in the trans Golgi network (Song and Fricker, 1995; Kuroki et al., 1995; Varlamov and Fricker, 1998; Dong et al., 1999). As with CPE, CPD is highly selective for C-terminal basic residues and is optimally active over the pH range from 5–7, consistent with the intravesicular pH of the late Golgi and secretory pathway (Song and Fricker, 1995; Novikova et al., 1999). The role of the other novel carboxypeptidases is not clear. CPZ may play a role in development, based on its broad distribution during embryogenesis (Novikova et al., 2001). Because this protein is primarily present in the extracellular matrix, it is possible that it participates in the breakdown of the matrix by removing Cterminal basic residues from intermediates generated after the action of matrix metalloproteases (Novikova et al., 2000). The recently discovered CPA-5 was predicted from modeling to have a specificity for aliphatic and aromatic amino acids (Wei et al., 2002). Upon expression in the baculovirus system, it was confirmed that this enzyme is capable of cleaving C-terminal Leu residues; additional substrates were not tested. Highest levels of CPA-5 mRNA were found in testis, with lower levels in brain and pituitary (Wei et al., 2002). In situ hybridization showed a distribution in testis that was consistent with expression in germ cells (Wei et al., 2002). In pituitary, CPA-5 mRNA was found in the anterior and intermediate lobes, with only background expression in the posterior lobe (Wei et al., 2002). Because a CPA-like enzyme is thought to be involved in the generation of the pituitary peptides hendorphin 1–26 and ACTH 1–38, it was of interest to explore the distribution of CPA-5 protein in pituitary, and to examine the intracellular distribution in the mouse corticotrophic AtT-20 cell line. The results of these analysis support the possibility that CPA-5 functions in the intracellular processing of pituitary peptides.
Materials and methods Immunohistochemistry of rat pituitary Pituitaries of Sprague-Dawley rats (Jackson Laboratory, Bar Harbor, ME, USA) were removed, fixed in 10% formalin, and embedded in paraffin. Each 5 Am thick section was cut and mounted to a poly-L-lysinecoated microscope slide. The slides were deparaffinize in two changes of xylene and rehydrated through a series of ethanol (95%, 80%, and 75%) and finally water. Following hydration, sections were depleted of
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endogenous peroxidase in 3% H2O2 for 20 min at room temperature. Tissues were then heated in a microwave in antigen retrieval solution (0.1 M sodium citrate, 0.1 M citric acid and 0.01% Triton X-100), cooled at 4 jC to allow the epitopes to reconfigure, and washed with phosphate buffered saline (PBS). An antiserum (AE658) was raised to the C-terminal 12 residues of mouse CPA-5 containing an additional Cys residue on the N-terminus (CQTIMKHTLNHPY) to permit coupling to maleimideactivated keyhole limpet hemocyanin (Pierce). The antiserum was diluted 1:1000 in 1% bovine serum albumin (BSA), 0.1% Tween-20 in PBS and incubated with the tissue for 1.5 h at room temperature. As a control, primary antiserum was replaced by preimmune serum from the same rabbit. Following the incubation, the tissues were washed in 0.2% Tween-20 in PBS and then processed for immunohistochemistry using the LSAB peroxidase Kit (DAKO Corp., Carpinteria, CA, USA). Basically, this involved reaction for 20 min in biotinylated anti-Rabbit immunoglobulins, washing with 0.2% Tween-20 in PBS, incubation for 20 min with streptavidin, washing again in the same buffer, and treatment with substrate-chromogen solution for 5 min. Tissue was counterstained with hematoxylin and mounted for microscopy. The slides were examined under a light microscope and images captured from an attached camera using Kodak Ektachrome 64T film. Immunofluorescence of AtT-20 cells Cells were grown on poly-L-lysine-coated glass cover slips (Fisher Scientific, Houston, TX) in 12well plates in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum. In some experiments, cells were treated with brefeldin A (5 Ag/ml) for 1 h or with cycloheximide (100 Ag/ ml) for 2 h at 37 jC before immunostaining. The cells were washed twice with DMEM and once with PBS, fixed for 10 min in 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS for 15 min, washed with PBS, and then blocked with 5% BSA in PBS for 1 h. Cells were first incubated for 1 h with primary antisera diluted 1:1000 in 5% BSA in PBS. The anti-CPA-5 antiserum is described above. The CPE (AE139) and CPD (AE142) antisera were previously described (Fricker et al., 1996; Song and Fricker, 1996). Unbound antibodies were removed by washing four times with 0.2% Tween-20 in PBS over 10 min. Then, the cells were incubated with secondary antibody fluorescein isothiocyanateconjugated goat anti-rabbit IgG diluted 1:200 in PBS containing 5% BSA in the dark for 1 h. Following this incubation, unbound antibodies were removed by washing as described above. The cover slips were mounted on glass slides with Prolong antifade reagent (Molecular Probes, Eugene, OR). Representative images were acquired using a Nikon Eclipse 400 fluorescence microscope with Nikon CFI Plan Apo 60 1.4 numerical aperture optics and a Cohu charge-coupled device camera linked to a Scion VG5 frame grabber. Figures were assembled using Adobe PhotoShop.
Results In the pituitary, CPA-5 immunoreactivity is enriched in the intermediate lobe (Fig. 1A). In addition, scattered cells in the anterior lobe show immunoreactivity. The posterior lobe shows less immunoreactivity for CPA-5 than the intermediate lobe, although scattered cells show weak staining (Fig. 1C). Controls performed with preimmune sera show only background levels of staining in all three lobes (Fig. 1B). Additional controls in which the antiserum was preabsorbed with an excess of the antigen (i.e. the C-terminal CPA-5 peptide) also show no staining above background (data not shown).
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Fig. 1. Distribution of CPA-5 immunoreactivity in rat pituitary. A: CPA-5 is highly expressed in the intermediate lobe (IL) and moderately expressed in the anterior lobe (AL) and posterior lobe (PL). B: Background staining detected with the preimmune serum. C: High magnification of CPA-5 immunostaining of panel A. Scale bars: 500 Am in panels A and B, 25 Am in panel C.
The AtT-20 mouse corticotrophic cell line was previously found to express CPA-5 mRNA (Wei et al., 2002). The distribution of CPA-5 in the AtT-20 cell line was examined by immunofluorescence. In this cell line, CPA-5 is detected in a perinuclear compartment that appears to be located in the same position as the CPD-containing compartment (Fig. 2, top). Previously, CPD has been localized to the trans Golgi network and immature secretory vesicles (Varlamov and Fricker, 1998; Varlamov et al., 1999). In contrast, CPE is present in both the trans Golgi network and in the secretory vesicles, which are enriched in the tips of the cells (Varlamov and Fricker, 1998; Varlamov et al., 1999). To distinguish Golgi from trans Golgi network, the cells were treated with brefeldin A (BFA) for 1 hour.
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Fig. 2. Immunofluorescence of CPA-5, CPD, and CPE in AtT-20 cells. The top panel shows cells without drug treatment, the middle panel represents cells treated with 5 Ag/ml brefeldin A (BFA), and the bottom panel shows cells treated with 100 Ag/ml cycloheximide (CHX). Cells were stained with anti-CPA-5 (left), anti-CPD (middle), or anti-CPE (right).
This treatment causes the endoplasmic reticulum and the Golgi to fuse, while the trans Golgi compartment remains intact (Varlamov and Fricker, 1998). The CPA-5 immunoreactivity shows the same perinuclear distribution following the BFA treatment, as does CPD, indicating that the perinuclear compartment(s) containing these proteins are not Golgi. To distinguish proteins that are resident in the trans Golgi network or other post-Golgi compartment with proteins like CPE that are only transiently expressed in this compartment on their way to the secretory vesicles, the cells were treated with cycloheximide (CHX) for 2 hours. This treatment blocks protein synthesis, and eliminates CPE staining in the perinuclear compartment but not in the tips of the cells. However, the perinuclear CPA-5 staining, like that of CPD, is not altered by the treatment. This result suggests that CPA-5 resides in a post-Golgi perinuclear compartment.
Discussion The distribution of CPA-5 is consistent with a role for this enzyme in the processing of secretory pathway proteins. Specifically, based on the presence of CPA-5 in the pituitary intermediate lobe and in a mouse corticotrophic cell line, it is possible that this enzyme processes proopiomelanocortin-derived peptides. In addition, the distribution of CPA-5 in the anterior lobe of the pituitary is suggestive of the
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presence in corticotrophs, but further studies using immunofluorescence and double labeling are needed to confirm this putative co-localization. Previously, in situ hybridization detected CPA-5 mRNA in the pituitary intermediate lobe and throughout the anterior lobe (Wei et al., 2002). Levels of CPA-5 mRNA appeared roughly comparable in these two lobes. However, at the protein level there is more CPA-5 immunoreactivity in the intermediate lobe than in the anterior lobe. Thus, the levels of CPA-5 mRNA do not accurately reflect the corresponding levels of the protein. A similar result is seen in the testis. Although levels of CPA-5 mRNA are fairly high in this tissue, there are much lower levels of CPA-5 protein as detected either by Western blot analysis or immunohistochemistry (not shown). Taken together, these results are consistent with a role for CPA-5 in the processing of proopiomelanocortin-derived peptides such as ACTH 1-38, and in humans, h-endorphin 1–26 (unlike human hendorphin 1–26, which requires removal of a C-terminal Tyr, rat h-endorphin 1–26 requires removal of a C-terminal His, and would therefore not be a likely substrate for CPA-5). Previously, Peng Loh and colleagues detected a carboxypeptidase that cleaved the C-terminal Phe from ACTH to generate ACTH 1-38 (Friedman et al., 1993), but the properties of this enzyme are considerably different than those of CPA-5. The enzyme reported by Loh et al appeared to be a serine carboxypeptidase, rather than a member of the CPA metallocarboxypeptidase family (Friedman et al., 1993). However, it was not clear if the serine carboxypeptidase was enriched in secretory vesicles, or merely present as lysosomal contamination. Because the authors detected the lysosomal enzyme h-glucuronidase in their preparation of secretory vesicles, it was clear that they were contaminated with lysosomes (Friedman et al., 1993). The reported serine protease was maximally active at pH 5.5, with < 15% maximal activity at pH 6.5; this is consistent with either a lysosomal or secretory vesicle distribution (Friedman et al., 1993). In contrast, CPA-5 is slightly more active at neutral pH than at acidic pH and would therefore be most likely to function in the trans Golgi network or another compartment with a neutral or slightly acidic internal pH. Further studies are needed to determine the precise intracellular compartment in which CPA5 is localized, and to examine the substrate specificity with various biological peptides. Acknowledgements This work was supported in part by National Institutes of Health grant R01 DA-04494 and Research Scientist Development Award DA-00194 (L.D.F.). Photography was performed in the Analytical Imaging Facility and in the laboratory of Dr. Jonathan Backer, Albert Einstein College of Medicine. References Bennett, H.P.J., 1991. Glycosylation, phosphorylation, and sulfation of peptide hormones and their precursors. In: Fricker, L.D. (Ed.), Peptide Biosynthesis and Processing. CRC Press, Boca Raton, pp. 111 – 140. Dong, W., Fricker, L.D., Day, R., 1999. Carboxypeptidase D is a potential candidate to carry out redundant processing functions of carboxypeptidase E based on comparative distribution studies in the rat central nervous system. Neurosci. 89, 1301 – 1317. Eipper, B.A., Mains, R.E., 1988. Peptide alpha-amidation. Ann. Rev. Physiol. 50, 333 – 344. Eipper, B.A., Mains, R.E., Herbert, E., 1986. Peptides in the nervous system. Trends Neurosci. 9, 463 – 468. Fricker, L.D., 1988. Carboxypeptidase E. Ann. Rev. Physiol. 50, 309 – 321. Fricker, L.D., 1991. Peptide processing exopeptidases: Amino- and carboxypeptidases involved with peptide biosynthesis. In: Fricker, L.D. (Ed.), Peptide Biosynthesis and Processing. CRC Press, Boca Raton, pp. 199 – 230.
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Fricker, L.D., Berman, Y.L., Leiter, E.H., Devi, L.A., 1996. Carboxypeptidase E activity is deficient in mice with the fat mutation: Effect on peptide processing. J. Biol. Chem. 271, 30619 – 30624. Friedman, T.C., Chen, H., Loh, Y.P., 1993. Generation of 1 – 37 and 1 – 38 forms of adrenocorticotropin by mono- and dipeptidyl serine carboxypeptidase activities in bovine pituitary secretory vesicles. Endocrinol. 133, 2951 – 2961. He, G.P., Muise, A., Li, A.W., Ro, H.S., 1995. A eukaryotic transcriptional repressor with carboxypeptidase activity. Nature 378, 92 – 96. Kuroki, K., Eng, F., Ishikawa, T., Turck, C., Harada, F., Ganem, D., 1995. gp180, a host cell glycoprotein that binds duck hepatitis B virus particles, is encoded by a member of the carboxypeptidase gene family. J. Biol. Chem. 270, 15022 – 15028. Lei, Y., Xin, X., Morgan, D., Pintar, J.E., Fricker, L.D., 1999. Identification of mouse CPX-1, a novel member of the metallocarboxypeptidase gene family with highest similarity to CPX-2. DNA Cell Biol. 18, 175 – 185. Liotta, A.S., Krieger, D.T., 1983. Pro-opiomelanocortin-related and other pituitary hormones in the central nervous system. In: Krieger, D.T., Brownstein, M.J., Martin, J.B. (Eds.), Brain Peptides. John Wiley and Sons, New York, pp. 615 – 660. Millington, W.R., Smith, D.L., 1991. The posttranslational processing of beta-endorphin in human hypothalamus. J. Neurochem. 57, 775 – 781. Naggert, J.K., Fricker, L.D., Varlamov, O., Nishina, P.M., Rouille, Y., Steiner, D.F., Carroll, R.J., Paigen, B.J., Leiter, E.H., 1995. Hyperproinsulinemia in obese fat/fat mice associated with a point mutation in the carboxypeptidase E gene and reduced carboxypeptidase E activity in the pancreatic islets. Nature Genetics 10, 135 – 142. Novikova, E.G., Eng, F.J., Yan, L., Qian, Y., Fricker, L.D., 1999. Characterization of the enzymatic properties of the first and second domains of metallocarboxypeptidase D. J. Biol. Chem. 274, 28887 – 28892. Novikova, E.G., Reznik, S.E., Varlamov, O., Fricker, L.D., 2000. Carboxypeptidase Z is present in the regulated secretory pathway and extracellular matrix in cultured cells and in human tissues. J. Biol. Chem. 275, 4865 – 4870. Novikova, E.G., Fricker, L.D., Reznik, S.E., 2001. Carboxypeptidase Z is dynamically expressed in mouse development. Mech. Develop. 102, 259 – 262. Seidah, N.G., Chretien, M., 1997. Eukaryotic protein processing: endoproteolysis of precursor proteins. Curr. Opin. Biotechnol. 8, 602 – 607. Seidah, N.G., Chretien, M., 1998a. Proprotein convertase I. In: Barrett, A.J., Rawlings, N.D., Woessner, J.F. (Eds.), Handbook of Proteolytic Enzymes. Academic Press, San Diego, pp. 349 – 353. Seidah, N.G., Chretien, M., 1998b. Prohormone convertase 2. In: Barrett, A.J., Rawlings, N.D., Woessner, J.F. (Eds.), Handbook of Proteolytic Enzymes. Academic Press, San Diego, pp. 345 – 357. Sidyelyeva, G., Fricker, L.D., 2002. Characterization of Drosophila carboxypeptidase D. J. Biol. Chem. 277, 49613 – 49620. Song, L., Fricker, L.D., 1995. Purification and characterization of carboxypeptidase D, a novel carboxypeptidase E-like enzyme, from bovine pituitary. J. Biol. Chem. 270, 25007 – 25013. Song, L., Fricker, L.D., 1996. Tissue distribution and characterization of soluble and membrane-bound forms of metallocarboxypeptidase D. J. Biol. Chem. 271, 28884 – 28889. Song, L., Fricker, L.D., 1997. Cloning and expression of human carboxypeptidase Z, a novel metallocarboxypeptidase. J. Biol. Chem. 272, 10543 – 10550. Steiner, D.F., 1991. The biosynthesis of biologically active peptides: A perspective. In: Fricker, L.D. (Ed.), Peptide Biosynthesis and Processing. CRC Press, Boca Raton, pp. 1 – 16. Varlamov, O., Fricker, L.D., 1998. Intracellular trafficking of metallocarboxypeptidase D in AtT-20 cells: Localization to the trans-Golgi network and recycling from the cell surface. J. Cell. Sci. 111, 877 – 885. Varlamov, O., Eng, F.J., Novikova, E.G., Fricker, L.D., 1999. Localization of metallocarboxypeptidase D in AtT-20 cells: Potential role in prohormone processing. J. Biol. Chem. 274, 14759 – 14767. Vilim, F.S., Aarnisalo, A.A., Nieminen, M., Lintunen, M., Karlstedt, K., Kontinen, V.K., Kalso, E., States, B., Panula, P., Ziff, E., 1999. Gene for pain modulatory neuropeptide NPFF: Induction in spinal cord by noxious stimuli. Mol. Pharm. 55, 804 – 811. Wei, S., Segura, S., Vendrell, J., Aviles, F.X., Lanoue, E., Day, R., Feng, Y., Fricker, L.D., 2002. Identification and characterization of three members of the human metallocarboxypeptidase gene family. J. Biol. Chem. 277, 14954 – 14964. Xin, X., Day, R., Dong, W., Lei, Y., Fricker, L.D., 1998. Identification of mouse CPX-2, a novel member of the metallocarboxypeptidase gene family: cDNA cloning, mRNA distribution, and protein expression and characterization. DNA Cell Biol. 17, 897 – 909. Zhou, A., Webb, G., Zhu, X., Steiner, D.F., 1999. Proteolytic processing in the secretory pathway. J. Biol. Chem. 274, 20745 – 20748.
Neuropeptide-processing carboxypeptidases Suwen Wei, Yun Feng, Elena Kalinina, Lloyd D. Fricker * Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
Abstract Neuropeptides are generally produced from precursor proteins by selective cleavage at specific sites, usually involving basic amino acids. Enzymes such as the prohormone convertases and carboxypeptidase E are highly specific for these basic amino acid-containing sites. In addition to this ‘‘traditional’’ pathway, several neuropeptides are known to be cleaved at non-basic sites, and the enzymes responsible for these cleavages have not been conclusively identified. In a recent search for novel members of the metallocarboxypeptidase family, we found three human genes. One of these, named ‘‘CPA-5,’’ has a specificity for C-terminal hydrophobic amino acids and mRNA expression in brain, pituitary, and testis. To test whether CPA-5 protein has a distribution pattern in pituitary that is consistent with a role for this enzyme in the non-basic processing of proopiomelanocortin-derived peptides such as beta-endorphin and adrenocorticotropin, we examined the distribution of CPA-5 using immunocytochemistry. In the pituitary, CPA-5 is detected in the neurointermediate lobe and in scattered cells in the anterior lobe. In the AtT-20 corticotroph cell line, CPA-5 has a perinuclear distribution. Taken together, these results are consistent with a role for CPA-5 in the intracellular processing of proopiomelanocortin-derived peptides at non-basic sites. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Peptide processing; Carboxypeptidase E; Enkephalin convertase; Carboxypeptidase A-5; Metallocarboxypeptidase
Introduction The vast majority of neuropeptides are generated by selective cleavage of precursors at sites that contain one or more basic amino acids (Steiner, 1991). First, one or more endopeptidase cleaves the precursor to generate intermediates containing C-terminal basic residues (Zhou et al., 1999; Seidah and Chretien, 1997). Then, a carboxypeptidase removes these basic residues (Fricker, 1988). In some cases,
* Corresponding author. Tel.: +1-718-430-4225; fax: +1-718-430-8954. E-mail address: [email protected] (L.D. Fricker). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00386-2
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further processing occurs to generate a C-terminal amide residue (Eipper and Mains, 1988). Also, some peptides acquire other post-translational modifications such as acetylation, phosphorylation, sulfation and glycosylation (Bennett, 1991). In some cases, these post-translational modifications alter the biological activity or stability of the peptide. In addition to this ‘‘traditional’’ scheme involving proteolytic cleavage at basic amino acid-containing sites, some peptides undergo cleavage at non-basic sites. For example, neuropeptide FF is generated by two endoproteolytic cleavages, one involving the traditional pair of basic residues and the other involving cleavage between an Ala-Phe bond (Vilim et al., 1999). Other peptides have been found to undergo C-terminal processing at non-basic residues. Examples include human beta-endorphin 1–26, which is presumably generated from beta-endorphin 1–27 by removal of the C-terminal Tyr residue, and adrenocorticotrophin 1–38, which is generated from the full-length 39 residue adrenocorticotrophin (ACTH) by removal of the C-terminal Phe residue (Friedman et al., 1993). The proteolytic processing often has a dramatic impact on the biological activity of the peptide. A classic example of the importance of proteolytic processing is with ACTH and alpha-melanocytestimulating hormone (a-MSH), the latter representing the N-terminal 13 residues of ACTH (Eipper et al., 1986). These two peptides have substantially different biological activities, with ACTH stimulating the hormone secretion from the adrenal gland while a-MSH stimulates melanocytes and also appears to play a role in the control of feeding and energy balance (Eipper et al., 1986; Liotta and Krieger, 1983). The non-traditional cleavages also can impact on the resulting biological activity of the peptide. For example, h-endorphin 1–31 is an opiate agonist, 1–27 is an antagonist, and 1–26 is devoid of activity (Millington and Smith, 1991). The conversion of h-endorphin 1–27 from 1–31 involves the traditional pathway of cleavage at a pair of basic amino acids followed by C-terminal removal of these basic residues. However, as mentioned above, the conversion of h-endorphin 1–27 into 1–26 involves removal of an aromatic amino acid, which is not catalyzed by the same enzymes as in the traditional pathway. The enzymes in the traditional pathway have been well studied over the past 10–20 years. The major peptide-processing endopeptidases are prohormone convertase (PC) 1 and 2 (Zhou et al., 1999; Seidah and Chretien, 1998a,b). These enzymes efficiently cleave many precursors at sites containing two basic amino acids, either adjacent to each other (e.g. Lys-Arg, or Arg-Arg) or separated by 2, 4, or 6 other amino acids (e.g. Arg-Xaa-Xaa-Arg) (Seidah and Chretien, 1998a,b). Neither PC1 or PC2 are known to cleave precursors at non-basic residues. Following the action of PC1 or PC2, a carboxypeptidase is required to remove the C-terminal basic residues (Fricker, 1988, 1991). For many years carboxypeptidase E (CPE, also known as enkephalin convertase, carboxypeptidase H, and EC3.4.17.10) was thought to be the only carboxypeptidase involved in neuropeptide processing (Fricker, 1988, 1991). However, the discovery in 1995 that mice lacking CPE activity due to a genetic point mutation were still capable of producing low levels of correctly processed peptides raised the possibility that another enzyme participated in this processing reaction (Naggert et al., 1995). Because the other carboxypeptidases known at the time had distributions and/or substrate specificities that were not appropriate for intracellular neuropeptide-processing enzymes, a search for novel enzymes was initiated. A variety of techniques were used to screen for new members of the metallocarboxypeptidase family, including enzyme assays, polymerase chain reaction with oligonucleotides to conserved regions of carboxypeptidases, and more recently, database mining. Altogether, we identified 7 new members of the metallocarboxypeptidase family (Song and Fricker, 1995, 1997; Xin et al., 1998; Lei et al., 1999; Wei et al., 2002). Five of these appear to be functional as carboxypeptidases and were named carboxypeptidase D,
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Z, A-5, A-6, and O. In addition, two members of the family do not appear to encode enzymes that are functional carboxypeptidases and were named CPX-1 and CPX-2 (with ‘‘CP’’ standing for carboxypeptidase-like, based on the 40–50% amino acid sequence identity between CPE and these two proteins). CPX-1 and 2, as well as another family member discovered by another group and named AEBP-1, lack one or more critical active site residues and are inactive towards standard carboxypeptidase substrates (He et al., 1995; Song and Fricker, 1997; Xin et al., 1998; Lei et al., 1999). The physiological function of these carboxypeptidase-like proteins is not clear, but due to the strong conservation of these three proteins between human and rodents it is likely that they play an important role. Furthermore, even C.elegans and Drosophila melanogaster contain carboxypeptidase-like genes that are lacking one or more critical active site residues (Sidyelyeva and Fricker, 2002). Of the functional enzymes, only CPD has the correct distribution and enzyme properties to function in the intracellular processing of neuropeptides (Song and Fricker, 1995; Varlamov and Fricker, 1998; Dong et al., 1999; Novikova et al., 1999). This enzyme is broadly distributed throughout the body and is present in the trans Golgi network (Song and Fricker, 1995; Kuroki et al., 1995; Varlamov and Fricker, 1998; Dong et al., 1999). As with CPE, CPD is highly selective for C-terminal basic residues and is optimally active over the pH range from 5–7, consistent with the intravesicular pH of the late Golgi and secretory pathway (Song and Fricker, 1995; Novikova et al., 1999). The role of the other novel carboxypeptidases is not clear. CPZ may play a role in development, based on its broad distribution during embryogenesis (Novikova et al., 2001). Because this protein is primarily present in the extracellular matrix, it is possible that it participates in the breakdown of the matrix by removing Cterminal basic residues from intermediates generated after the action of matrix metalloproteases (Novikova et al., 2000). The recently discovered CPA-5 was predicted from modeling to have a specificity for aliphatic and aromatic amino acids (Wei et al., 2002). Upon expression in the baculovirus system, it was confirmed that this enzyme is capable of cleaving C-terminal Leu residues; additional substrates were not tested. Highest levels of CPA-5 mRNA were found in testis, with lower levels in brain and pituitary (Wei et al., 2002). In situ hybridization showed a distribution in testis that was consistent with expression in germ cells (Wei et al., 2002). In pituitary, CPA-5 mRNA was found in the anterior and intermediate lobes, with only background expression in the posterior lobe (Wei et al., 2002). Because a CPA-like enzyme is thought to be involved in the generation of the pituitary peptides hendorphin 1–26 and ACTH 1–38, it was of interest to explore the distribution of CPA-5 protein in pituitary, and to examine the intracellular distribution in the mouse corticotrophic AtT-20 cell line. The results of these analysis support the possibility that CPA-5 functions in the intracellular processing of pituitary peptides.
Materials and methods Immunohistochemistry of rat pituitary Pituitaries of Sprague-Dawley rats (Jackson Laboratory, Bar Harbor, ME, USA) were removed, fixed in 10% formalin, and embedded in paraffin. Each 5 Am thick section was cut and mounted to a poly-L-lysinecoated microscope slide. The slides were deparaffinize in two changes of xylene and rehydrated through a series of ethanol (95%, 80%, and 75%) and finally water. Following hydration, sections were depleted of
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endogenous peroxidase in 3% H2O2 for 20 min at room temperature. Tissues were then heated in a microwave in antigen retrieval solution (0.1 M sodium citrate, 0.1 M citric acid and 0.01% Triton X-100), cooled at 4 jC to allow the epitopes to reconfigure, and washed with phosphate buffered saline (PBS). An antiserum (AE658) was raised to the C-terminal 12 residues of mouse CPA-5 containing an additional Cys residue on the N-terminus (CQTIMKHTLNHPY) to permit coupling to maleimideactivated keyhole limpet hemocyanin (Pierce). The antiserum was diluted 1:1000 in 1% bovine serum albumin (BSA), 0.1% Tween-20 in PBS and incubated with the tissue for 1.5 h at room temperature. As a control, primary antiserum was replaced by preimmune serum from the same rabbit. Following the incubation, the tissues were washed in 0.2% Tween-20 in PBS and then processed for immunohistochemistry using the LSAB peroxidase Kit (DAKO Corp., Carpinteria, CA, USA). Basically, this involved reaction for 20 min in biotinylated anti-Rabbit immunoglobulins, washing with 0.2% Tween-20 in PBS, incubation for 20 min with streptavidin, washing again in the same buffer, and treatment with substrate-chromogen solution for 5 min. Tissue was counterstained with hematoxylin and mounted for microscopy. The slides were examined under a light microscope and images captured from an attached camera using Kodak Ektachrome 64T film. Immunofluorescence of AtT-20 cells Cells were grown on poly-L-lysine-coated glass cover slips (Fisher Scientific, Houston, TX) in 12well plates in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum. In some experiments, cells were treated with brefeldin A (5 Ag/ml) for 1 h or with cycloheximide (100 Ag/ ml) for 2 h at 37 jC before immunostaining. The cells were washed twice with DMEM and once with PBS, fixed for 10 min in 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS for 15 min, washed with PBS, and then blocked with 5% BSA in PBS for 1 h. Cells were first incubated for 1 h with primary antisera diluted 1:1000 in 5% BSA in PBS. The anti-CPA-5 antiserum is described above. The CPE (AE139) and CPD (AE142) antisera were previously described (Fricker et al., 1996; Song and Fricker, 1996). Unbound antibodies were removed by washing four times with 0.2% Tween-20 in PBS over 10 min. Then, the cells were incubated with secondary antibody fluorescein isothiocyanateconjugated goat anti-rabbit IgG diluted 1:200 in PBS containing 5% BSA in the dark for 1 h. Following this incubation, unbound antibodies were removed by washing as described above. The cover slips were mounted on glass slides with Prolong antifade reagent (Molecular Probes, Eugene, OR). Representative images were acquired using a Nikon Eclipse 400 fluorescence microscope with Nikon CFI Plan Apo 60 1.4 numerical aperture optics and a Cohu charge-coupled device camera linked to a Scion VG5 frame grabber. Figures were assembled using Adobe PhotoShop.
Results In the pituitary, CPA-5 immunoreactivity is enriched in the intermediate lobe (Fig. 1A). In addition, scattered cells in the anterior lobe show immunoreactivity. The posterior lobe shows less immunoreactivity for CPA-5 than the intermediate lobe, although scattered cells show weak staining (Fig. 1C). Controls performed with preimmune sera show only background levels of staining in all three lobes (Fig. 1B). Additional controls in which the antiserum was preabsorbed with an excess of the antigen (i.e. the C-terminal CPA-5 peptide) also show no staining above background (data not shown).
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Fig. 1. Distribution of CPA-5 immunoreactivity in rat pituitary. A: CPA-5 is highly expressed in the intermediate lobe (IL) and moderately expressed in the anterior lobe (AL) and posterior lobe (PL). B: Background staining detected with the preimmune serum. C: High magnification of CPA-5 immunostaining of panel A. Scale bars: 500 Am in panels A and B, 25 Am in panel C.
The AtT-20 mouse corticotrophic cell line was previously found to express CPA-5 mRNA (Wei et al., 2002). The distribution of CPA-5 in the AtT-20 cell line was examined by immunofluorescence. In this cell line, CPA-5 is detected in a perinuclear compartment that appears to be located in the same position as the CPD-containing compartment (Fig. 2, top). Previously, CPD has been localized to the trans Golgi network and immature secretory vesicles (Varlamov and Fricker, 1998; Varlamov et al., 1999). In contrast, CPE is present in both the trans Golgi network and in the secretory vesicles, which are enriched in the tips of the cells (Varlamov and Fricker, 1998; Varlamov et al., 1999). To distinguish Golgi from trans Golgi network, the cells were treated with brefeldin A (BFA) for 1 hour.
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Fig. 2. Immunofluorescence of CPA-5, CPD, and CPE in AtT-20 cells. The top panel shows cells without drug treatment, the middle panel represents cells treated with 5 Ag/ml brefeldin A (BFA), and the bottom panel shows cells treated with 100 Ag/ml cycloheximide (CHX). Cells were stained with anti-CPA-5 (left), anti-CPD (middle), or anti-CPE (right).
This treatment causes the endoplasmic reticulum and the Golgi to fuse, while the trans Golgi compartment remains intact (Varlamov and Fricker, 1998). The CPA-5 immunoreactivity shows the same perinuclear distribution following the BFA treatment, as does CPD, indicating that the perinuclear compartment(s) containing these proteins are not Golgi. To distinguish proteins that are resident in the trans Golgi network or other post-Golgi compartment with proteins like CPE that are only transiently expressed in this compartment on their way to the secretory vesicles, the cells were treated with cycloheximide (CHX) for 2 hours. This treatment blocks protein synthesis, and eliminates CPE staining in the perinuclear compartment but not in the tips of the cells. However, the perinuclear CPA-5 staining, like that of CPD, is not altered by the treatment. This result suggests that CPA-5 resides in a post-Golgi perinuclear compartment.
Discussion The distribution of CPA-5 is consistent with a role for this enzyme in the processing of secretory pathway proteins. Specifically, based on the presence of CPA-5 in the pituitary intermediate lobe and in a mouse corticotrophic cell line, it is possible that this enzyme processes proopiomelanocortin-derived peptides. In addition, the distribution of CPA-5 in the anterior lobe of the pituitary is suggestive of the
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presence in corticotrophs, but further studies using immunofluorescence and double labeling are needed to confirm this putative co-localization. Previously, in situ hybridization detected CPA-5 mRNA in the pituitary intermediate lobe and throughout the anterior lobe (Wei et al., 2002). Levels of CPA-5 mRNA appeared roughly comparable in these two lobes. However, at the protein level there is more CPA-5 immunoreactivity in the intermediate lobe than in the anterior lobe. Thus, the levels of CPA-5 mRNA do not accurately reflect the corresponding levels of the protein. A similar result is seen in the testis. Although levels of CPA-5 mRNA are fairly high in this tissue, there are much lower levels of CPA-5 protein as detected either by Western blot analysis or immunohistochemistry (not shown). Taken together, these results are consistent with a role for CPA-5 in the processing of proopiomelanocortin-derived peptides such as ACTH 1-38, and in humans, h-endorphin 1–26 (unlike human hendorphin 1–26, which requires removal of a C-terminal Tyr, rat h-endorphin 1–26 requires removal of a C-terminal His, and would therefore not be a likely substrate for CPA-5). Previously, Peng Loh and colleagues detected a carboxypeptidase that cleaved the C-terminal Phe from ACTH to generate ACTH 1-38 (Friedman et al., 1993), but the properties of this enzyme are considerably different than those of CPA-5. The enzyme reported by Loh et al appeared to be a serine carboxypeptidase, rather than a member of the CPA metallocarboxypeptidase family (Friedman et al., 1993). However, it was not clear if the serine carboxypeptidase was enriched in secretory vesicles, or merely present as lysosomal contamination. Because the authors detected the lysosomal enzyme h-glucuronidase in their preparation of secretory vesicles, it was clear that they were contaminated with lysosomes (Friedman et al., 1993). The reported serine protease was maximally active at pH 5.5, with < 15% maximal activity at pH 6.5; this is consistent with either a lysosomal or secretory vesicle distribution (Friedman et al., 1993). In contrast, CPA-5 is slightly more active at neutral pH than at acidic pH and would therefore be most likely to function in the trans Golgi network or another compartment with a neutral or slightly acidic internal pH. Further studies are needed to determine the precise intracellular compartment in which CPA5 is localized, and to examine the substrate specificity with various biological peptides. Acknowledgements This work was supported in part by National Institutes of Health grant R01 DA-04494 and Research Scientist Development Award DA-00194 (L.D.F.). Photography was performed in the Analytical Imaging Facility and in the laboratory of Dr. Jonathan Backer, Albert Einstein College of Medicine. References Bennett, H.P.J., 1991. Glycosylation, phosphorylation, and sulfation of peptide hormones and their precursors. In: Fricker, L.D. (Ed.), Peptide Biosynthesis and Processing. CRC Press, Boca Raton, pp. 111 – 140. Dong, W., Fricker, L.D., Day, R., 1999. Carboxypeptidase D is a potential candidate to carry out redundant processing functions of carboxypeptidase E based on comparative distribution studies in the rat central nervous system. Neurosci. 89, 1301 – 1317. Eipper, B.A., Mains, R.E., 1988. Peptide alpha-amidation. Ann. Rev. Physiol. 50, 333 – 344. Eipper, B.A., Mains, R.E., Herbert, E., 1986. Peptides in the nervous system. Trends Neurosci. 9, 463 – 468. Fricker, L.D., 1988. Carboxypeptidase E. Ann. Rev. Physiol. 50, 309 – 321. Fricker, L.D., 1991. Peptide processing exopeptidases: Amino- and carboxypeptidases involved with peptide biosynthesis. In: Fricker, L.D. (Ed.), Peptide Biosynthesis and Processing. CRC Press, Boca Raton, pp. 199 – 230.
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Fricker, L.D., Berman, Y.L., Leiter, E.H., Devi, L.A., 1996. Carboxypeptidase E activity is deficient in mice with the fat mutation: Effect on peptide processing. J. Biol. Chem. 271, 30619 – 30624. Friedman, T.C., Chen, H., Loh, Y.P., 1993. Generation of 1 – 37 and 1 – 38 forms of adrenocorticotropin by mono- and dipeptidyl serine carboxypeptidase activities in bovine pituitary secretory vesicles. Endocrinol. 133, 2951 – 2961. He, G.P., Muise, A., Li, A.W., Ro, H.S., 1995. A eukaryotic transcriptional repressor with carboxypeptidase activity. Nature 378, 92 – 96. Kuroki, K., Eng, F., Ishikawa, T., Turck, C., Harada, F., Ganem, D., 1995. gp180, a host cell glycoprotein that binds duck hepatitis B virus particles, is encoded by a member of the carboxypeptidase gene family. J. Biol. Chem. 270, 15022 – 15028. Lei, Y., Xin, X., Morgan, D., Pintar, J.E., Fricker, L.D., 1999. Identification of mouse CPX-1, a novel member of the metallocarboxypeptidase gene family with highest similarity to CPX-2. DNA Cell Biol. 18, 175 – 185. Liotta, A.S., Krieger, D.T., 1983. Pro-opiomelanocortin-related and other pituitary hormones in the central nervous system. In: Krieger, D.T., Brownstein, M.J., Martin, J.B. (Eds.), Brain Peptides. John Wiley and Sons, New York, pp. 615 – 660. Millington, W.R., Smith, D.L., 1991. The posttranslational processing of beta-endorphin in human hypothalamus. J. Neurochem. 57, 775 – 781. Naggert, J.K., Fricker, L.D., Varlamov, O., Nishina, P.M., Rouille, Y., Steiner, D.F., Carroll, R.J., Paigen, B.J., Leiter, E.H., 1995. Hyperproinsulinemia in obese fat/fat mice associated with a point mutation in the carboxypeptidase E gene and reduced carboxypeptidase E activity in the pancreatic islets. Nature Genetics 10, 135 – 142. Novikova, E.G., Eng, F.J., Yan, L., Qian, Y., Fricker, L.D., 1999. Characterization of the enzymatic properties of the first and second domains of metallocarboxypeptidase D. J. Biol. Chem. 274, 28887 – 28892. Novikova, E.G., Reznik, S.E., Varlamov, O., Fricker, L.D., 2000. Carboxypeptidase Z is present in the regulated secretory pathway and extracellular matrix in cultured cells and in human tissues. J. Biol. Chem. 275, 4865 – 4870. Novikova, E.G., Fricker, L.D., Reznik, S.E., 2001. Carboxypeptidase Z is dynamically expressed in mouse development. Mech. Develop. 102, 259 – 262. Seidah, N.G., Chretien, M., 1997. Eukaryotic protein processing: endoproteolysis of precursor proteins. Curr. Opin. Biotechnol. 8, 602 – 607. Seidah, N.G., Chretien, M., 1998a. Proprotein convertase I. In: Barrett, A.J., Rawlings, N.D., Woessner, J.F. (Eds.), Handbook of Proteolytic Enzymes. Academic Press, San Diego, pp. 349 – 353. Seidah, N.G., Chretien, M., 1998b. Prohormone convertase 2. In: Barrett, A.J., Rawlings, N.D., Woessner, J.F. (Eds.), Handbook of Proteolytic Enzymes. Academic Press, San Diego, pp. 345 – 357. Sidyelyeva, G., Fricker, L.D., 2002. Characterization of Drosophila carboxypeptidase D. J. Biol. Chem. 277, 49613 – 49620. Song, L., Fricker, L.D., 1995. Purification and characterization of carboxypeptidase D, a novel carboxypeptidase E-like enzyme, from bovine pituitary. J. Biol. Chem. 270, 25007 – 25013. Song, L., Fricker, L.D., 1996. Tissue distribution and characterization of soluble and membrane-bound forms of metallocarboxypeptidase D. J. Biol. Chem. 271, 28884 – 28889. Song, L., Fricker, L.D., 1997. Cloning and expression of human carboxypeptidase Z, a novel metallocarboxypeptidase. J. Biol. Chem. 272, 10543 – 10550. Steiner, D.F., 1991. The biosynthesis of biologically active peptides: A perspective. In: Fricker, L.D. (Ed.), Peptide Biosynthesis and Processing. CRC Press, Boca Raton, pp. 1 – 16. Varlamov, O., Fricker, L.D., 1998. Intracellular trafficking of metallocarboxypeptidase D in AtT-20 cells: Localization to the trans-Golgi network and recycling from the cell surface. J. Cell. Sci. 111, 877 – 885. Varlamov, O., Eng, F.J., Novikova, E.G., Fricker, L.D., 1999. Localization of metallocarboxypeptidase D in AtT-20 cells: Potential role in prohormone processing. J. Biol. Chem. 274, 14759 – 14767. Vilim, F.S., Aarnisalo, A.A., Nieminen, M., Lintunen, M., Karlstedt, K., Kontinen, V.K., Kalso, E., States, B., Panula, P., Ziff, E., 1999. Gene for pain modulatory neuropeptide NPFF: Induction in spinal cord by noxious stimuli. Mol. Pharm. 55, 804 – 811. Wei, S., Segura, S., Vendrell, J., Aviles, F.X., Lanoue, E., Day, R., Feng, Y., Fricker, L.D., 2002. Identification and characterization of three members of the human metallocarboxypeptidase gene family. J. Biol. Chem. 277, 14954 – 14964. Xin, X., Day, R., Dong, W., Lei, Y., Fricker, L.D., 1998. Identification of mouse CPX-2, a novel member of the metallocarboxypeptidase gene family: cDNA cloning, mRNA distribution, and protein expression and characterization. DNA Cell Biol. 17, 897 – 909. Zhou, A., Webb, G., Zhu, X., Steiner, D.F., 1999. Proteolytic processing in the secretory pathway. J. Biol. Chem. 274, 20745 – 20748.