Biomedical subjects

L D Fricker

Publications and source records attributed to L D Fricker.

At least 19 recordsLinked to original sources

PubMed1999

Purification and characterization of human metallocarboxypeptidase Z.

Carboxypeptidase Z (CPZ) is a recently discovered member of the metallocarboxypeptidase gene family that has an N-terminal domain related to the Wnt/wingless binding domain of frizzled receptors and other proteins. To further characterize the enzymatic properties of CPZ, the enzyme was purified using Arg- and heparin-affinity columns. CPZ has a neutral pH optimum, and is inhibited by chelating agents and several divalent cations (Zn2+, Mn2+, Cd2+, Cu2+, Hg2+). Active site-directed inhibitors of several other metallocarboxypeptidases also inhibit CPZ activity with moderate potency. CPZ cleaves substrates with C-terminal Arg residues, preferring peptides with an Ala in the penultimate position. No activity is detected toward substrates with an Ile-Arg or a Pro-Arg sequence. The Km for dansyl-Phe-Ala-Arg and dansyl-Pro-Ala-Arg are both approximately 2 mM. Taken together, these data suggests a selective role for CPZ in the processing of extracellular peptides or proteins.

Animals↗

PubMed1999

Cloning and expression of Aplysia carboxypeptidase D, a candidate prohormone-processing enzyme.

Many peptide hormones in a variety of species are produced from larger precursors by limited proteolysis at basic amino acid-containing sites. The marine mollusc Aplysia has homologs of mammalian peptide-processing enzymes, including furin, prohormone convertase 1 (PC1), PC2, and carboxypeptidase E (CPE). A novel neuronal Aplysia enzyme was recently identified that was most closely related to carboxypeptidase D (CPD; Fan and Nagle, DNA Cell Biol. 15, 937-945, 1996), a second carboxypeptidase thought to be present in the secretory pathway and to contribute to peptide hormone processing. We have identified and cloned multiple overlapping bag-cell neuron cDNAs that encode two proteins that are members of the CPD family. Sequence analyses demonstrate that the longer CPD protein (1446 residues) contains an N-terminal signal peptide and four carboxypeptidase-like domains; the third and fourth domains are not predicted to form active enzymes, as several critical residues are absent. The shorter CPD protein is predicted to contain two active carboxypeptidase-like domains. Northern blot analysis identified a major Aplysia CPD mRNA (5.3 kb) and several smaller minor transcripts in central nervous system tissue. The CPD was purified from Aplysia ovotestis using a method previously developed for mammalian CPD. The purified Aplysia CPD binds antisera raised against regions of the protein encoded by the Aplysia cDNA clone, as well as an antiserum raised against duck CPD. The enzymatic properties of purified Aplysia CPD are generally similar to those of mammalian CPD. Aplysia CPD is a candidate prohormone-processing enzyme that may play a role in the processing of Aplysia prohormones in the secretory pathway.

Amino Acid Sequence↗

PubMed1999

Identification of mouse CPX-1, a novel member of the metallocarboxypeptidase gene family with highest similarity to CPX-2.

The recent finding that Cpe(fat)/Cpe(fat) mice, which lack carboxypeptidase E (CPE) activity because of a point mutation, are still capable of a reduced amount of neuroendocrine peptide processing suggested that additional carboxypeptidases (CPs) participate in this processing reaction. Searches for novel members of the CPE gene family led to the discovery of CPD, CPZ, AEBP1, and CPX-2. In the present report, we describe mouse CPX-1, another novel member of this gene family. Like AEBP1 and CPX-2, CPX-1 contains an N-terminal region of 160 amino acids with sequence similarity to the discoidin domain of a variety of proteins. The 410-residue CP-like domain of CPX-1 has 54% to 62% amino acid sequence identity with AEBP1 and CPX-2 and 33% to 49% amino acid identity with other members of the CPE subfamily. However, several active-site residues that are important for catalytic activity of other CPs are not conserved in CPX-1. Furthermore, CPX-1 expressed in either the baculovirus system or the mouse AtT-20 cell line does not cleave standard CP substrates. Northern blot analysis showed the highest levels of CPX-1 mRNA in testis and spleen and lower levels in salivary gland, brain, heart, lung, and kidney. In situ hybridization of CPX-1 mRNA in embryonic and fetal mouse tissue showed expression throughout the head and thorax, with abundance in primordial cartilage and skeletal structures. In the head, high levels of CPX-1 mRNA were associated with the nasal mesenchyme, primordial cartilage structures in the ear, and the meninges. In the thorax, CPX-1 mRNA was expressed in multiple developing skeletal structures, including chondrocytes and perichondrial cells of the rib, vertebral, and long-bone primordia. Taken together, these findings suggest that it is unlikely that CPX-1 functions in the processing of neuroendocrine peptides. Instead, CPX-1 may have a role in development, possibly mediating cell interactions via its discoidin domain.

Amino Acid Sequence↗

PubMed1999

Sequences within the cytoplasmic domain of gp180/carboxypeptidase D mediate localization to the trans-Golgi network.

Gp180, a duck protein that was proposed to be a cell surface receptor for duck hepatitis B virus, is the homolog of metallocarboxypeptidase D, a mammalian protein thought to function in the trans-Golgi network (TGN) in the processing of proteins that transit the secretory pathway. Both gp180 and mammalian metallocarboxypeptidase D are type I integral membrane proteins that contain a 58-residue cytosolic C-terminal tail that is highly conserved between duck and rat. To investigate the regions of the gp180 tail involved with TGN retention and intracellular trafficking, gp180 and various deletion and point mutations were expressed in the AtT-20 mouse pituitary corticotroph cell line. Full length gp180 is enriched in the TGN and also cycles to the cell surface. Truncation of the C-terminal 56 residues of the cytosolic tail eliminates the enrichment in the TGN and the retrieval from the cell surface. Truncation of 12-43 residues of the tail reduced retention in the TGN and greatly accelerated the turnover of the protein. In contrast, deletion of the C-terminal 45 residues, which truncates a potential YxxL-like sequence (FxxL), reduced the protein turnover and caused accumulation of the protein on the cell surface. A point mutation of the FxxL sequence to AxxL slowed internalization, showing that this element is important for retrieval from the cell surface. Mutation of a pair of casein kinase II sites within an acidic cluster showed that they are also important for trafficking. The present study demonstrates that multiple sequence elements within the cytoplasmic tail of gp180 participate in TGN localization.

Amino Acid Sequence↗

PubMed1998

gp180, a protein that binds duck hepatitis B virus particles, has metallocarboxypeptidase D-like enzymatic activity.

Duck gp180 was previously identified by its ability to bind to the preS envelope protein of duck hepatitis B virus particles (Kuroki, K. , Cheung, R., Marion, P. L., and Ganem, D. (1994) J. Virol. 68, 2091-2096). Cloning and sequencing of gp180 cDNA revealed that it is a polyprotein with three carboxypeptidase-like domains (Kuroki, K., Eng, F., Ishikawa, T., Turck, C., Harada, F., and Ganem, D. (1995) J. Biol. Chem. 270, 15022-15028). To evaluate enzymatic properties of this protein, a soluble 170-kDa form of the protein (gp170) lacking the C-terminal transmembrane domain and cytoplasmic tail was expressed in a baculovirus system. The purified 170-kDa protein cleaved 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Phe-Ala-Arg with a pH optimum of 5.5-6.5. With this substrate at pH 5.5, the 170-kDa protein displayed a Km of 12 microM and a Kcat of 57 s-1. Dansyl-Pro-Ala-Arg and dansyl-Phe-Phe-Arg were cleaved with Km values of 17 and 21 microM, and Kcat values of 57 and 17 s-1, respectively. Constructs containing only the first or second carboxypeptidase domains also showed enzymatic activity. The effects of inhibitors and ions on enzyme activity of gp170 were generally similar to the effects of these compounds on purified bovine carboxypeptidase D. To evaluate the regions within gp180 necessary for binding preS, a series of deletion mutants were expressed in the 293T human kidney cell line. Deletions of the first and second domains, leaving the third domain intact, eliminated carboxypeptidase activity but retained preS binding. Deletion of the third domain eliminated preS binding but not carboxypeptidase activity. These results indicate that the third domain is responsible for preS binding, and this binding does not require carboxypeptidase activity.

Animals↗

PubMed1998

Cloning, sequence analysis, and distribution of rat metallocarboxypeptidase Z.

A cDNA encoding human carboxypeptidase Z (CPZ), a novel metallocarboxypeptidase, was recently cloned (Song and Fricker, J. Biol. Chem., 272, 1054, 1997). In the present study, a cDNA encoding the rat homolog of CPZ was identified. As with the human form, rat CPZ contains an N-terminal domain of 120 amino acids that has 20% to 30% amino acid identity with the "frizzled" domain found on proteins that interact with Wnt, a protein involved in tissue polarity in early embryogenesis. Sequence analysis showed rat and human CPZ to be highly conserved within the frizzled domain (77% amino acid identity), the carboxypeptidase domain (91%), and the C-terminal 28 residues (78%). The entire rat CPZ protein has high sequence similarity with human CPZ (81% amino acid identity), moderate sequence similarity to human carboxypeptidase N (45%), human carboxypeptidase E (41%), and human carboxypeptidase M (33%), and less sequence similarity with other metallocarboxypeptidases. Northern blot analysis showed rat CPZ mRNA to be abundant in the placenta, with low to moderate levels in the brain, lung, thymus, and kidney. The BRL3A rat liver cell line and the PC12 rat adrenal cell line express high levels of CPZ mRNA. In situ hybridization analysis indicated that CPZ is expressed only in specific cell types. For example, in the brain, CPZ mRNA is present in leptomeningeal cells, but not in the majority of other cell types. This distribution in leptomeningeal cells is shared by AEBP1, a recently reported member of the metallocarboxypeptidase gene family. However, the distribution of CPZ and AEBP1 differ in pituitary and thyroid. Taken together, these studies suggest that CPZ functions in a range of cell types.

Amino Acid Sequence↗

PubMed1998

Identification of mouse CPX-2, a novel member of the metallocarboxypeptidase gene family: cDNA cloning, mRNA distribution, and protein expression and characterization.

A novel member of the metallocarboxypeptidase gene family was identified from its homology with carboxypeptidase E and has been designated CPX-2. The cDNA of 2500 nucleotides encodes a protein of 764 amino acids that contains an N-terminal signal peptide-like sequence, a 158-residue discoidin domain, and a 400-residue carboxypeptidase domain. The 400-residue metallocarboxypeptidase domain has 59% amino acid identity with a protein designated AEBP-1; 44% to 46% identity with carboxypeptidases E, N, and Z; and lower homology with other members of the metallocarboxypeptidase gene family. The discoidin domain of CPX-2 has 22% amino acid identity with the carbohydrate-binding domain of discoideum-I, 29% to 34% identity with the phospholipid-binding domain of human factors V and VIII, and 59% identity with the discoidin-like domain on AEBP-1. CPX-2 is missing several of the predicted active-site residues that are conserved in most other members of the metallocarboxypeptidase gene family and which are thought to be required for enzyme activity. Expression of CPX-2 using the baculovirus system produced several forms of protein, from 80 to 105 kDa, but no detectable activity toward a variety of carboxypeptidase substrates. A shorter 50-kDa form of CPX-2, which contains the carboxypeptidase domain but not the discoidin domain, was also inactive when expressed in the baculovirus system. CPX-2 is able to bind to Sepharose-Arg; this binding is blocked by 10 mM Arg. Northern blot analysis showed CPX-2 mRNA in mouse brain, liver, kidney, and lung. In situ hybridization analysis of brain revealed a broad distribution. Areas that are enriched in CPX-2 include the hippocampus, cerebral cortex, median eminence, and choroid plexus. Taken together, these data suggest a widespread function for CPX-2, possibly as a binding protein rather than an active carboxypeptidase.

Amino Acid Sequence↗

PubMed1998

Immunohistochemical localization of carboxypeptidases E and D in the human placenta and umbilical cord.

Carboxypeptidase E (CPE) is highly concentrated in neuroendocrine tissues and is the only carboxypeptidase detected in mature secretory vesicles. Carboxypeptidase D (CPD), a carboxypeptidase with CPE-like activity, is widely distributed in tissues and is present in the trans-Golgi network. Previous work had shown that both CPE and CPD are expressed in the human placenta and that CPD is expressed at much higher levels than CPE. The present work provides evidence for the co-localization of CPE and CPD to basal plate extravillous trophoblasts and maternal uteroplacental vascular endothelial cells, chorionic villous endothelial cells, amnionic epithelial cells, and umbilical venous and arterial smooth muscle cells. Whereas the intensity of CPD immunostaining is similar in the placenta and umbilical cord, CPE staining in the placenta is much weaker than in the umbilical cord, suggesting that CPD plays a more important role in the processing of placental peptides. Immunoelectron microscopy of umbilical venous smooth muscle cells shows subcellular localization of both enzymes to the rough endoplasmic reticulum. In addition, CPE is present just subjacent to the cell membrane. The difference in cellular and subcellular localization between the two enzymes indicates that they perform distinct functions in the processing of placental peptides and proteins.

Carboxypeptidase H↗

PubMed1998

Intracellular trafficking of metallocarboxypeptidase D in AtT-20 cells: localization to the trans-Golgi network and recycling from the cell surface.

Carboxypeptidase D (CPD) is a recently discovered membrane-bound metallocarboxypeptidase that has been proposed to be involved in the post-translational processing of peptides and proteins that transit the secretory pathway. In the present study, the intracellular distribution of CPD was examined in AtT-20 cells, a mouse anterior pituitary-derived corticotroph. Antisera to CPD stain the same intracellular structures as those labeled with furin and wheat germ agglutinin. This distribution is distinct from carboxypeptidase E, which is localized to the secretory vesicles in the cell processes. The perinuclear distribution of CPD is detected even when the AtT-20 cells are treated with brefeldin A for 1-30 minutes, suggesting that CPD is present in the trans-Golgi network (TGN). Although CPD is predominantly found in the TGN, an antiserum to the full length protein is internalized within 15-30 minutes of incubation at 37 degrees C. In contrast, an antiserum raised against the C-terminal region of CPD does not become internalized, suggesting that this domain is cytosolic. The antiserum to the full length CPD is internalized to a structure that co-stains with furin and wheat germ agglutinin, but is distinct from transferrin recycling endosomes. The internalization of CPD is not substantially affected by treatment of the AtT-20 cells with brefeldin A. These data are consistent with the cycling of CPD to the cell surface and back to the TGN. The TGN localization of CPD raises the possibility of a role for this enzyme in the processing of proteins that transit the secretory pathway.

Animals↗

PubMed1997

Characterization of Aplysia carboxypeptidase E.

Carboxypeptidase E (CPE) is involved in the biosynthesis of peptide hormones and neurotransmitters. To determine whether a recently reported Aplysia californica cDNA encodes a CPE-like enzyme, this cDNA was expressed in the baculovirus system. The Aplysia CPE is optimal at pH 5.5-6.5 and is inhibited by chelating agents and by the sulfhydryl reagent p-chloromercuriphenyl sulfonate. The effect of divalent cations and active site-directed inhibitors on enzyme activity are generally similar for Aplysia and rat CPE. Western blot analysis using antisera to the N- and C-terminal regions of the Aplysia CPE show that the Aplysia CPE is present in atrial glands and ovotestis. This Aplysia CPE is purified on a p-aminobenzoyl-Arg Sepharose affinity column under conditions that selectively purify rat CPE. Taken together, these results suggest that the previously cloned cDNA represents a CPE-like enzyme that is expressed in Aplysia tissue.

Amino Acid Sequence↗

PubMed1997

Cloning and expression of human carboxypeptidase Z, a novel metallocarboxypeptidase.

A novel cDNA, designated carboxypeptidase Z (CPZ), was identified based on its homology to known metallocarboxypeptidases. Northern blot analysis shows bands of 2.1 and/or 2.6 kilobases in all tissues examined. The major form of CPZ mRNA in human salivary gland encodes a protein with an open reading frame of 641 amino acids. In addition, three variants were found that presumably arise due to alternative intron splicing. The 641-amino acid protein contains an 18-residue signal peptide-like sequence, a 120-residue region that shows 23-29% amino acid identity with a Cys-rich domain found in frizzled proteins and in type XVIII collagen, and then a 390-residue carboxypeptidase domain with 49% amino acid identity to carboxypeptidases E and N. The 641-amino acid form of CPZ expressed in the baculovirus system cleaves 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Phe-Ala-Arg, although the level of enzyme activity was approximately 10-fold lower than either carboxypeptidase E or D expressed using the same viral system. The CPZ activity is more active at neutral pH than at pH 5.5 and is inhibited by active site-directed inhibitors of metallocarboxypeptidases. In summary, CPZ is a novel metallocarboxypeptidase that is active toward substrates with C-terminal basic amino acids.

Amino Acid Sequence↗

PubMed1997

The pro region is not required for the expression or intracellular routeing of carboxypeptidase E.

Carboxypeptidase E (CPE) is initially synthesized as a larger precursor containing an additional 14-residue propeptide that is highly conserved between human and rat. Previous studies have established that the proenzyme is enzymically active and that deletion of the pro region does not affect the expression of the active enzyme. In the present study the function of the pro region was examined both by deleting this region from CPE and by attaching this region to the N-terminus of albumin. CPE lacking the pro region is sorted into the regulated secretory pathway in AtT-20 cells, based on confocal microscopy and examination of the stimulated secretion of the protein. Stimulation of AtT-20 cells with either forskolin or phorbol 12-myristate 13-acetate induces the secretion of wild-type CPE and of CPE lacking the pro region to similar extents, indicating a similar efficiency of sorting of the mutant. When the pro region of proalbumin is replaced with the pro region of CPE followed by expression in AtT-20 cells, the protein is not sorted into the regulated pathway, based on the lack of stimulated secretion. Confocal microscopy suggests that the proCPE/albumin protein is retained in the endoplasmic reticulum to a greater extent than is proalbumin. Pulse-chase analysis indicates that the pro region of CPE is not efficiently removed from the N-terminus of albumin, and the small amount of propeptide cleavage that does occur takes place soon before secretion of the protein. In contrast, confocal microscopy indicates that the majority of the propeptide is removed from CPE, and that this cleavage occurs in the trans-Golgi network or soon after sorting into the secretory vesicles. Taken together, these results suggest that the pro region of CPE is not required for the expression or intracellular routeing of this protein.

Amino Acid Sequence↗

PubMed1997

Cloning and sequence analysis of cDNA encoding rat carboxypeptidase D.

Carboxypeptidase D (CPD) is a recently described 180-kD enzyme with carboxypeptidase E-like enzymatic properties. CPD has been proposed to be present in the secretory pathway and to contribute to peptide hormone processing in the Cpe(fat)/Cpe(fat) mouse, which lacks functional CPE. Sequence analysis of cDNA clones encoding rat CPD show the protein to contain an amino-terminal signal peptide, three carboxypeptidase-like domains, a putative transmembrane domain, and a 60-amino-acid cytoplasmic tail. Whereas active site, substrate-binding, and metal-binding residues of other metallocarboxypeptidases are conserved in the first two domains of CPD, several of the critical residues are not conserved in the third domain; this third domain is not predicted to form an active carboxypeptidase. The overall homology between rat CPD and the duck homolog gp180 is high, with 75% amino acid identity. The three carboxypeptidase domains show 66%, 83%, and 82% amino acid identity between rat CPD and duck gp180. Homology is also high in the transmembrane domain (86%) and in the cytoplasmic tail (97%). The mouse Cpd gene maps to the medial portion of chromosome 11, approximately 45.5 cM distal to the centromere. Northern blot analysis of CPD mRNA shows major bands of approximately 8 and 4 kb in many rat tissues, and additional species ranging from 1.4 to 5 kb that are expressed in some tissues or cell lines. CPD mRNA is detectable in most tissues examined, and is most abundant in hippocampus, spinal cord, atrium of the heart, colon, testis, and ovaries. In situ hybridization of CPD mRNA shows a distribution in many cells in rat brain and other tissues, with high levels in hippocampus, olfactory bulb, and the intermediate pituitary. The broad distribution is consistent with a role for CPD in the processing of many peptides and proteins that transit the secretory pathway.

Amino Acid Sequence↗

PubMed1997

Beta-cell lines derived from transgenic Cpe(fat)/Cpe(fat) mice are defective in carboxypeptidase E and proinsulin processing.

A spontaneous point mutation in the coding region of the carboxypeptidase E (CPE) gene in Cpe(fat)/Cpe(fat) mice affects proinsulin processing. Cell lines derived from the pancreatic beta-cells of Cpe(fat)/Cpe(fat) mice were generated by crossing C57BLKS/J-Cpe(fat)/+ mice with NOD mice expressing the simian virus 40 large T oncogene under the control of the rat insulin II promoter. Two cell lines, designated NIT-2 and NIT-3, were cultured from adenomatous islets obtained from F2 littermates and were compared with the NIT-1 cell line previously developed from mice with wild-type CPE. Electron microscopy of the cultured NIT-2 and -3 cells showed increased numbers of enlarged and electron-lucent granules compared with NIT-1 cells. Pro-CPE, but not the mature form of CPE, is present in NIT-2 and -3 cells, and neither pro-CPE nor CPE are secreted into the medium. Immunocytochemistry shows the pro-CPE to be localized to an endoplasmic reticulum-like structure in NIT-3 cells. Proinsulin is less extensively processed in NIT-2 and -3 cells than in NIT-1 cells, indicating that the Cpe(fat) mutation affects both the endopeptidase and carboxypeptidase reactions. The secretion of insulin/proinsulin from NIT-2 and -3 cells is significantly elevated by secretagogues, indicating that CPE is not required for sorting proinsulin into the regulated pathway.

Animals↗

PubMed1997

Effect of carboxypeptidase E deficiency on progastrin processing and gastrin messenger ribonucleic acid expression in mice with the fat mutation.

Proforms of gastrointestinal peptides are cleaved at paired basic residues into intermediate forms. Paired basic residues at the C-terminal then are excised by carboxypeptidases before the peptide is amidated. An obese mouse, called Cpe(fat)/Cpe(fat), has a missense mutation in carboxypeptidase E (CPE) with no pancreatic CPE activity and a reduced processing of pancreatic proinsulin to insulin. The purpose of this study was 1) to look for the presence of CPE in the antrum of the stomach, duodenum, and colon in the Cpe(fat)/Cpe(fat) mouse; 2) to determine whether CPE is involved in the processing of progastrin (Pro-G) to its carboxyl-terminal amidated form; and 3) to determine whether a decrease in amidated gastrin results in an up-regulation of stomach gastrin messenger RNA (mRNA) levels. In Cpe(fat)/Cpe(fat) mice, CPE activity was absent in the antrum and colon. In Cpe(fat)/Cpe(fat) mice, amidated gastrin levels were reduced significantly. Levels of the precursor for amidated gastrin (gastrin-Gly-Arg-Arg) were markedly elevated. Gastrin mRNA levels were increased approximately 2-fold over the levels in Cpe(fat)/Cpe(fat) mice. These results indicate that CPE is needed for processing progastrin to gastrin in the stomach and that amidated gastrin exerts an inhibitory feedback effect on gastrin mRNA levels.

Animals↗

PubMed1996

Carboxypeptidase E activity is deficient in mice with the fat mutation. Effect on peptide processing.

Carboxypeptidase E (CPE) is involved in the biosynthesis of many peptide hormones and neurotransmitters. Mice with the fat mutation have previously been found to have a point mutation in the cpe gene, and to have greatly reduced levels of CPE-like enzyme activity in the pituitary and pancreatic islets (Naggert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135-142). In the present report, we examined CPE-like activity and peptide processing in several tissues of C57BLKS/LtJ-Cpefat/Cpefat mutant (Cpefat/Cpefat) mice. Whereas CPE-like activity is detected in homogenates of Cpefat/Cpefat mouse tissues, the majority of this activity is not due to CPE based on the sensitivity to p-chloromercuriphenyl sulfonate. In addition, the Cpefat/Cpefat activity does not bind to a substrate affinity column under conditions that bind CPE. Furthermore, the enzyme activity and immunoreactive properties of the activity purified from Cpefat/Cpefat brain are distinct from those of CPE. Taken together, these data suggest that CPE is completely inactive in the Cpefat/Cpefat mice, and that all of the CPE-like activity is due to other carboxypeptidases such as carboxypeptidase D. Levels of Leu-enkephalin in Cpefat/Cpefat mouse brain are approximately 5-fold lower than those in control brain. Treatment of the Cpefat/Cpefat brain extract with carboxypeptidase B restores the level of Leu-enkephalin to the level in control brain. Interestingly, the large molecular weight enkephalin-containing peptides are elevated 2-3-fold in Cpefat/Cpefat mouse brain. These data indicate that CPE plays an important role in the processing of peptide hormones in various tissues, but that other carboxypeptidases also contribute to peptide processing. Furthermore, the increase in levels of high molecular weight enkephalin peptides in the Cpefat/Cpefat mouse suggests that CPE is required for efficient peptide processing by the endopeptidases.

Animals↗

PubMed1996

Tissue distribution and characterization of soluble and membrane-bound forms of metallocarboxypeptidase D.

Metallocarboxypeptidase D (CPD) is a recently discovered 180-kDa membrane-bound carboxypeptidase E-like enzyme (Song, L. and Fricker, L. D. (1995) J. Biol. Chem. 270, 25007-25013). In the present study, a soluble CPD-like activity has been purified to homogeneity and characterized. On denaturing polyacrylamide gels, the soluble enzyme from bovine pituitary glands appears as two bands of 170 and 135 kDa which are converted to 155 and 115 kDa by endoglycosidase F. Both of the soluble forms of CPD are recognized by an antisera raised against CPD purified from rat brain membranes. The partial N-terminal amino acid sequences of the two soluble forms are identical to each other and to the predicted N terminus of duck gp180. The soluble and membrane forms of CPD have similar pH optima, inhibitor specificities, and kinetic parameters for substrate hydrolysis. CPD-like enzymatic activity is detected in all rat tissues examined, with highest levels in pituitary, brain, and adrenal. Western blot analysis indicates that both soluble and membrane forms of CPD are present in rat brain, heart, liver, and kidney. At least four distinct 100-180-kDa forms of CPD are detected on Western blots, although an antiserum raised against the C-terminal region of rat CPD recognizes only the 180-kDa membrane-bound form. The finding that CPD is widely distributed suggests a broad role for this enzyme in the processing of proteins that transit the secretory pathway.

Animals↗

PubMed1996

The C-terminal region of carboxypeptidase E involved in membrane binding is distinct from the region involved with intracellular routing.

Carboxypeptidase E (CPE) is involved in the biosynthesis of numerous peptide hormones and neurotransmitters. Previously, the C-terminal region of CPE has been shown to participate in the binding of the protein to membranes and to also contribute to the sorting of CPE into the regulated pathway. In this study, the role of the C-terminal region of CPE was further examined using several approaches. A series of CPE mutants with C-terminal deletions was expressed in the baculovirus system; constructs with a deletion of 14 or 23 residues were expressed at levels comparable to wild-type CPE. In contrast, deletion of 33 or more residues eliminated CPE activity, and the resulting protein was not secreted from the cells. Even though CPE mutants with a deletion of 14 or 23 residues were expressed normally, the resulting protein was mainly soluble, whereas approximately 55% of wild-type CPE was membrane associated. When expressed in AtT-20 cells, CPE with a deletion of 43 C-terminal amino acids was not secreted, whereas CPE with a deletion of 23 residues was secreted via the regulated pathway. Pulse-chase analysis revealed the protein with a deletion of 43 residues to be degraded in a non-acidic intracellular compartment. To investigate whether the C-terminal region of CPE can confer membrane binding and regulated pathway sorting to another protein, portions of the CPE C-terminal region were attached to the C terminus of albumin and the fusion proteins expressed in AtT-20 cells. Of the constructs examined, only the protein containing 51 amino acids of CPE was sorted to the regulated pathway, although with reduced efficiency compared to endogenous CPE. Although the C-terminal 14 amino acids of CPE are sufficient to target albumin to membranes, this fusion protein is not sorted into the regulated pathway. Taken together, these results indicate that the C-terminal 14 amino acids of CPE are important for membrane binding and that membrane binding and sorting require distinct signals.

Albumins↗