Biomedical subjects

L D Fricker

Publications and source records attributed to L D Fricker.

At least 19 recordsLinked to original sources

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

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↗

PubMed1995

Purification and characterization of carboxypeptidase D, a novel carboxypeptidase E-like enzyme, from bovine pituitary.

Carboxypeptidase E (CPE) is involved in the biosynthesis of most neuropeptides and peptide hormones. Until recently, CPE was the only intracellular carboxypeptidase thought to be involved in neuroendocrine peptide processing. However, the finding that fat/fat mice, which have a mutation within the CPE gene that inactivates the enzyme, are capable of a reduced amount of insulin processing suggests that another carboxypeptidase is present within the secretory pathway. We have detected a CPE-like enzyme, designated CPD, which has many properties in common with those of CPE. Like CPE, CPD is a metallocarboxypeptidase that has a pH optimum of 5.5-6. The Km and Kcat values for a series of short peptide substrates show only minor differences between CPD and CPE. Several active site-directed inhibitors also show generally similar potency toward the two enzymes, although guanidinoethylmercaptosuccinic acid is approximately 10-fold more potent, and hippuryl-Arg is approximately 100-fold more potent as an inhibitor of CPD than of CPE. A major difference between the two enzymes is the molecular masses; CPE is 50,000-56,000, whereas CPD is approximately 180,000. Also, CPD does not elute from a substrate affinity column when the pH is raised to 8, which elutes CPE, although CPD can subsequently be eluted by arginine. Both CPE and CPD are present in purified bovine anterior pituitary secretory vesicles, but the tissue distribution of CPD is more uniform than that of CPE. Antisera to the N- and C-terminal regions of CPE do not recognize CPD. The partial N-terminal amino acid sequence of bovine CPD shows 30-40% homology with an N-terminal region of bovine and rat CPE and 70% homology with a duck protein known as gp180, a hepatitis B virus particle binding protein that shows 47% homology to CPE. Taken together, these results suggest that CPD is a novel secretory pathway enzyme that may be the bovine homologue of gp180.

Amino Acid Sequence↗

PubMed1995

Calcium- and pH-dependent aggregation of carboxypeptidase E.

Carboxypeptidase E (CPE) is involved with the biosynthesis of numerous peptide hormones and neurotransmitters. Several forms of CPE have been previously detected in neuroendocrine cells, including a form which is soluble at pH 5.5 (S-CPE), a form which can be extracted from membranes with 1 M NaCl at pH 5.5 (M1-CPE), and a form which requires both 1% Triton X-100 and 1 M NaCl for extraction from membranes at pH 5.5 (M2-CPE). Like other peptide processing enzymes, CPE is known to be sorted into peptide-containing secretory vesicles of the regulated pathway. One mechanism that has been proposed to be important for sorting of regulated pathway proteins is Ca2+ and pH-induced aggregation. CPE purified from bovine pituitary membranes aggregates at pH 5.5 when the concentration of CPE is 0.3 micrograms/microliters or higher, but not when the concentration is 0.01 microgram/microliters. Aggregation of CPE is pH-dependent, with very little aggregation occurring at pH 6 or above. At pH 5.0-5.5, the M2 form of CPE shows a greater tendency to aggregate than the other two forms. At pH 6, Ca2+ concentrations from 1-30 mM increase the aggregation of M1- and M2-CPE, but not S-CPE. The aggregation of M2-CPE does not explain the apparent membrane binding of this protein since the aggregate is solubilized by 1% Triton X-100 at pH 5.5 or by pH 6.0, whereas M2-CPE is not extracted from membranes under these conditions. Taken together, these results are consistent with a model in which the decreasing pH and increasing Ca2+ levels in the trans Golgi network induce the aggregation of CPE, which contributes to the sorting of this protein into regulated pathway secretory vesicles.

Amino Acid Sequence↗

PubMed1995

Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity.

Mice homozygous for the fat mutation develop obesity and hyperglycaemia that can be suppressed by treatment with exogenous insulin. The fat mutation maps to mouse chromosome 8, very close to the gene for carboxypeptidase E (Cpe), which encodes an enzyme (CPE) that processes prohormone intermediates such as proinsulin. We now demonstrate a defect in proinsulin processing associated with the virtual absence of CPE activity in extracts of fat/fat pancreatic islets and pituitaries. A single Ser202Pro mutation distinguishes the mutant Cpe allele, and abolishes enzymatic activity in vitro. Thus, the fat mutation represents the first demonstration of an obesity-diabetes syndrome elicited by a genetic defect in a prohormone processing pathway.

Amino Acid Sequence↗

PubMed1994

The C-terminal region of carboxypeptidase E is involved in membrane binding and intracellular routing in AtT-20 cells.

Carboxypeptidase E (CPE), a neuropeptide processing enzyme, is present in neuroendocrine tissues in soluble and membrane-associated forms. The membrane-associated forms do not contain a conventional transmembrane-spanning domain; instead, the C-terminal region of CPE has been proposed to form an amphiphilic helix which binds to the membrane. To test this, and to investigate the possible contribution of this C-terminal sequence to the intracellular sorting of CPE into the regulated pathway, the C-terminal region of CPE was attached to albumin and the recombinant proteins expressed in AtT-20 cells. Albumin itself showed little association with membranes under the conditions examined. A construct containing albumin with only 9 residues of CPE, corresponding to a highly charged region immediately preceding the potential amphiphilic helix region, showed generally similar membrane binding and secretion rates as albumin alone. When the C-terminal 51 amino acids of CPE were attached to the C terminus of albumin and the recombinant protein detected with an antisera raised against the C terminus of CPE, virtually all of the protein was membrane-associated. This finding suggests that the C-terminal region of CPE functions as a membrane anchor. The secretion of albumin with the C-terminal region of CPE was stimulated by a phorbol ester and by forskolin, although the magnitude of the stimulation was smaller than the effect of these compounds on the secretion of CPE. These results imply that the C-terminal region of CPE contains the membrane anchor and contributes to the sorting of this protein into the regulated pathway.

Amino Acid Sequence↗

PubMed1994

Regulation of carboxypeptidase E. Effect of Ca2+ on enzyme activity and stability.

Carboxypeptidase E (CPE), an enzyme that functions in the post-translational processing of bioactive peptides, is a member of the metallocarboxypeptidase gene family. A 12-residue region of CPE has 70% amino acid identity with the bacterial enzyme carboxypeptidase T (CPT); in CPT, this region has been identified previously as the Ca(2+)-binding region (Teplyakov, A., Polyakov, K., Obmolova, G., Strokopytov, B., Kuranova, I., Osterman, A., Grishin, N., Smulevitch, S., Zagnitko, O., Galperina, O., Matz, M., and Stepanov, V. (1992) Eur. J. Biochem. 208, 281-288). Using 45Ca2+ binding, we determined that CPE binds Ca2+. To investigate the potential function for the interaction of CPE with Ca2+, we investigated the effect of Ca2+ on aggregation, thermostability, and enzyme activity of CPE. CPE does not aggregate under a variety of Ca2+ concentrations at either pH 5.5 or 7.5, and with protein concentrations ranging from 10 to 100 micrograms/ml. Whereas Ca2+ generally stabilizes proteins to thermal denaturation, CPE was destabilized by Ca2+ and stabilized by low concentrations of EGTA. The Ca(2+)-induced destabilization of CPE was more pronounced at pH 8 than at lower pH values. At pH 8, CPE was unstable even at 37 degrees C, with approximately 40% loss of activity upon incubation for 30 min in the absence of added Ca2+ and 70% loss of activity upon incubation in the presence of 10 mM CaCl2. Enzyme activity was not influenced by added Ca2+, but was stimulated by micromolar concentrations of EGTA; kinetic analysis showed this stimulation to be due to a change in Vmax, and not Km. Taken together, these data suggest that Ca2+ plays a role in the regulation of CPE activity.

Amino Acid Sequence↗

PubMed1994

Expression of the carboxypeptidase E gene: characterization of the initiator-binding proteins.

Several of the genes for enzymes involved in peptide hormone processing, such as carboxypeptidase E (CPE), do not contain a TATA box. The region surrounding the major transcription initiation site of the CPE gene has sequence homology with the 'initiator' (Inr) elements of the TATA-less terminal deoxynucleotidyltransferase (TdT) gene, and the adenovirus major late (AdML) and other promoters. To investigate the promoter region of the CPE gene, GH4C1 cells were transiently transfected with constructs containing the luciferase reporter gene attached to various portions of the rat CPE gene (-395 to +45). Positive regulator elements were detected in positions -84 to -12 and +30 to +47. However, the Inr-like element of the CPE gene (-12 to +20) produced detectable luciferase activity in the absence of upstream and downstream sequences. This region of the CPE gene was much more active when expressed in the normal (sense) orientation than when expressed in the antisense orientation. A mutation within the consensus sequence between CPE and other Inr elements was much less active than the wild-type sequence. Interestingly, deletion of the Inr and surrounding sequences produced a large increase in the transcription from upstream sites, suggesting that proteins which bind at, or near, the Inr sequence suppress transcription from other sites. To characterize GH4C1 nuclear proteins which bind to the CPE gene, Southwestern blotting, UV cross-linking, and gel shift analyses were performed. The Southwestern analysis showed that the CPE and AdML Inr sequences labeled several proteins of similar sizes which are distinct from the transcription factor USF; this factor has been previously reported to bind to the AdML Inr sequence. A CPE Inr-binding protein co-purifies with an AdML Inr-binding protein on a CPE Inr affinity column. Gel shift assays showed that with some binding conditions, the proteins that bind to the CPE sequence also bind to the TdT and AdML Inr elements. Taken together, these results indicate that the -12 to +20 region of the CPE gene has the properties of an Inr element which binds some, but not all, of the factors which bind to other Inr elements.

Animals↗