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Keywords:

  • Prohormone convertase 2;
  • Proenkephalin;
  • Carboxyl

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Construction of PECT peptide chimera
  5. High-pressure gel permeation chromatography (HPGPC)
  6. RIA
  7. Production of PECT in CHO cells
  8. RESULTS
  9. PECT chimera is able to inhibit PE processing in vitro
  10. Inhibition of POMC processing is potentially due to competition of PE
  11. PECT is correctly targeted to secretory granules
  12. CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells
  13. DISCUSSION
  14. Acknowledgements
  15. References

Abstract : Prohormone convertase (PC) 2 plays an important role in the processing of neuropeptide precursors via the regulated secretory pathway in neuronal and endocrine tissues. PC2 interacts with 7B2, a neuroendocrine protein that is cleaved to a 21-kDa domain involved in proPC2 maturation and a carboxyl-terminal peptide (CT peptide) that represents a potent inhibitor of PC2 in vitro. A role for the CT peptide as an inhibitor in vivo has not yet been established. To study the involvement of the CT peptide in PC2-mediated cleavages in neuroendocrine cells, we constructed a mutant proenkephalin (PE) expression vector containing PE with its carboxyl-terminal peptide (peptide B) replaced with the 7B2 inhibitory CT peptide. This PECT chimera was stably transfected into two PC2-expressing cell lines, AtT-20/PC2 and Rin cells. Although recombinant PECT proved to be a potent (nM) inhibitor of PC2 in vitro, cellular PC2-mediated cleavages of PE were not inhibited by the PECT chimera, nor was proopiomelanocortin cleavage (as assessed by adrenocorticotropin cleavage to α-melanocyte-stimulating hormone) inhibited further than in control cells expressing only the competitive substrate PE. Tests of stimulated secretion showed that both the CT peptide and the PE portion of the chimera were stored in regulated secretory granules of transfected clones. In both AtT-20/PC2 and Rin cells expressing the chimera, the CT peptide was substantially internally hydrolyzed, potentially accounting for the observed lack of inhibition. Taken together, our data suggest that overexpressed CT peptide derived from PECT is unable to inhibit PC2 in mature secretory granules, most likely due to its inactivation by PC2 or by other enzyme(s).

Many biologically active proteins and polypeptide hormones produced via the regulated secretory pathway are synthesized as larger inactive precursor molecules. Mature active polypeptides are generated after proteolytic cleavages, sugar addition, and other posttranslational modifications such as sulfation and amidation. Proteolytic cleavage, thought to occur in the late Golgi/secretory granule compartments, usually occurs at dibasic residues, with Lys-Arg being the most common cleavage site ; however, cleavage can also occur after single basic sites (Steiner, 1998). At least some of the enzymes that are responsible for proteolytic cleavage belong to a family of mammalian subtilisin-like endopeptidases. There are seven members of this family ; these include furin and the prohormone convertases (PCs) PC1 (also known as PC3), PC2, PC4, PACE4, PC5/6, and PC7 (Steiner, 1998). Expression of the members of this family of PCs occurs either fairly ubiquitously, as in the case of furin (Hatsuzawa et al., 1990) and PACE4 (Kiefer et al., 1991), or is tissue specific, as in the case of PC1 and PC2 (Seidah et al., 1990, 1991 ; Smeekens et al., 1991 ; Day et al., 1992). Whereas furin appears to be involved in the processing of proteins that are secreted constitutively, PC1 and PC2, expressed only in neuroendocrine tissues, are thought to be involved in the processing of proteins within the regulated secretory pathway (Rouille et al., 1995).

PCs, like their polypeptide substrates, are synthesized as large inactive proproteins that require cleavage at paired dibasic sites for activation. The PC1 propeptide is cleaved in the endoplasmic reticulum at a neutral pH via an autocatalytic mechanism (Benjannet et al., 1993 ; Zhou and Lindberg, 1993 ; Lindberg, 1994 ; Milgram and Mains, 1994). In contrast, proPC2 is processed in the trans-Golgi network/secretory granules, most likely also via an autocatalytic mechanism (Guest et al., 1992 ; Benjannet et al., 1993 ; Shen et al., 1993 ; Zhou and Mains, 1994 ; Lamango et al., 1999). PC2 is also unique in that it requires association with the neuroendocrine-specific protein 7B2 for maturation and activation (Zhu and Lindberg, 1995 ; Zhu et al., 1996a, b).

7B2 is a bifunctional protein that is cleaved in the trans-Golgi network from a 27-kDa precursor protein to a 21-kDa domain and a 31-amino acid carboxyl-terminal peptide (CT peptide) (Ayoubi et al., 1990 ; Paquet et al., 1994). The 21-kDa portion of 7B2 is responsible for the facilitation and activation of proPC2 (Zhu and Lindberg, 1995). The activation requirement of proPC2 for association with 7B2 has recently been confirmed in vivo in 7B2 null mice (Westphal et al., 1999). 7B2 null mice lack PC2 activity and are unable to process peptide hormones such as adrenocorticotropin (ACTH) and proglucagon at PC2-mediated cleavage sites (Westphal et al., 1999). The 7B2 CT peptide, on the other hand, has been shown to be a potent in vitro inhibitor of PC2 (Lindberg et al., 1995). Inactivation of the CT peptide is thought to occur in secretory granules via internal cleavage at a Lys17-Lys18 site, followed by action of carboxypeptidase E (Zhu et al., 1996b). Although the CT is a nanomolar inhibitor of PC2 in vitro, the ability of the CT peptide to inhibit PC2 in a cellular environment, particularly in the secretory granules, remains to be demonstrated.

The opioid peptide precursor proenkephalin (PE) contains 12 paired basic residues that represent cleavage sites for the liberation of intermediate and mature-sized enkephalins. The endoproteolytic enzymes most likely to be responsible for cleavage of PE at these sites are PC1 and PC2 (Mathis and Lindberg, 1992 ; Breslin et al., 1993 ; Johanning et al., 1996a, 1998). PE is normally processed in a semisequential order, with the initial cleavage occurring at the carboxyl-terminal end, resulting in the liberation of peptide B. PC1 performs the early cleavages of PE, although overexpression and antisense studies have shown that the later cleavages of this precursor, which liberate the smaller opioid peptides such as Met-enkephalin (Met-enk), are accomplished by PC2 (Johanning et al., 1996a).

In the present study, we have asked whether it is possible to inhibit PC2 in endocrine cells using a CT peptide construct. As 7B2 is a bifunctional protein with two opposing activities (i.e., activation and inhibition), the purpose of the present work was to uncouple the two opposing biological activities and examine the role of the CT peptide independently of the 21-kDa activating domain. We targeted the 7B2 CT peptide to secretory granules by fusing it to the N-terminal portion of PE (Fig. 1). Both PE as well as the endogenously expressed opioid peptide precursor proopiomelanocortin (POMC) contain known PC2-mediated cleavage sites (Zhou et al., 1993 ; Johanning et al., 1996a) that can be used to assess potential inhibition. Our studies show that although the CT peptide is liberated from the chimera and is correctly targeted to the secretory granules, PC2-mediated processing of PE and POMC is not blocked in chimeraexpressing AtT-20/PC2 and Rin cells. In addition, we have shown that the lack of PC2 inhibition in chimeraexpressing Rin and AtT-20 cells is potentially due to inactivation of the CT peptide in mature secretory granules.

image

Figure 1. Structure of rat PE and the PECT chimera constructed for this study.

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Construction of PECT peptide chimera

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Construction of PECT peptide chimera
  5. High-pressure gel permeation chromatography (HPGPC)
  6. RIA
  7. Production of PECT in CHO cells
  8. RESULTS
  9. PECT chimera is able to inhibit PE processing in vitro
  10. Inhibition of POMC processing is potentially due to competition of PE
  11. PECT is correctly targeted to secretory granules
  12. CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells
  13. DISCUSSION
  14. Acknowledgements
  15. References

Rat PE was amplified via PCR from the plasmid pEV/rENK. Two primers were used, one directed toward the 5′ end of PE and the other directed toward the carboxyl-terminal end of PE but lacking peptide B. The CT peptide was amplified via PCR from rat 7B2 using the following primers : 5′ CTAGTTGTGGTTTGTCCA 3′, 5′ CCCGTGGAGCCAGAAGAA 3′, 5′ GTGAAGGGCAGCTGCCTT 3′, 5′ TTAGTGAACCGTCAGATC 3′. Fusion of PE and the CT peptide was accomplished via complementary sequences attached to the 3′ end of amplified PE and the 5′ end of the amplified CT peptide fragment. A final round of PCR was used to amplify the entire PECT chimera, which was then digested with BamHI and HindIII and cloned into pCEP4 (InVitrogen). There is a Lys-Arg site between the PE and CT peptide portion of the chimera. Confirmation of the accuracy of the construct was obtained by DNA sequencing in both directions.

Cell culture, transfection, and selection

AtT-20 and AtT-20 cells stably expressing mouse PC2 (Zhou and Mains, 1994) were used in these studies. These cells were grown in Dulbecco’s modified Eagle’s (DMEM) highglucose medium (Life Technologies, Gaithersburg, MD, U.S.A.) containing 10% NuSerum (Irvine Scientific) and 2.5% fetal bovine serum (FBS ; Irvine Scientific) at 37°C in an atmosphere of 5% CO2. DG44 CHO cells were used for the expression of PECT via the dihydrofolate reductase-coupled method as previously described (Lindberg et al., 1991). CHO cells were grown in medium lacking nucleosides (α-minimal essential medium) (Life Technologies) and containing 10% dialyzed heat-inactivated FBS. Rin cells (a rat insulinoma cell line) were grown in DMEM low-glucose medium containing 10% FBS at 37°C in an atmosphere of 5% CO2.

Both parental and PC2-expressing AtT-20 and Rin cells were stably transfected with the PECT expression vector using the Lipofectin method described previously (Zhu and Lindberg, 1995). Approximately 1 × 106 cells were plated in a 10-cm dish and allowed to grow overnight. The next day, the cells were washed with phosphate-buffered saline (PBS) and incubated in 3 ml of OptiMem (Life Technologies) containing 30 μl of Lipofectin (Life Technologies), 30 μg of vector cDNA, and 15 μl of gentamycin (100 μg/ml) at 37°C. After 5 h, 7 ml of growth medium containing 100 μg/ml hygromycin (Sigma Chemicals, St. Louis, MO, U.S.A.) and 300 μg/ml active G418 was added to each plate ; plates were fed twice weekly with the same medium. After ~2 weeks, 24 hygromycin- and G418-resistant clones were selected and subcloned into a 24-well plate using the soft agar method described previously (Lindberg and Zhou, 1995). Clones were screened using Met-enk-RGL and CT RIAs, and the highest-expressing (designated B), second highest-expressing (designated MB), and low-expressing (designated L) clones were frozen and used for the experiments described here.

Metabolic labeling and immunoprecipitation

Approximately 500,000 AtT-20 or AtT-20/PC2 cells per well were seeded into six-well plates and allowed to grow at 37°C with 5% CO2 until 70-80% confluent. The cells were washed twice with warm PBS and starved for 20 min at 37°C in 1 ml of methionine-deficient RPMI medium (ICN Biomedicals, Irvine, CA, U.S.A.) containing 10 mM HEPES (pH 7.4). Cells were then incubated (pulse) for 20 min in 1 ml of methionine-free medium containing 1.0 mCi of [35S]methionine and 2% dialyzed FBS. For steady-state labeling, cells were incubated for 6 h in 1 ml of valine-free medium containing 1.0 mCi of [3H]valine (specific activity, 29 Ci/mmol ; Amersham Life Sciences, Arlington Heights, IL, U.S.A.). Following the pulse period, cells either were homogenized directly in 1 ml of acid mix [1 M acetic acid, 20 mM HCl, and 0.1% β-mercaptoethanol (BME)] or were further incubated in 1 ml of warm chase medium (DMEM high-glucose medium containing 10 mM HEPES and 2% FBS) for various time periods and then homogenized in acid mix. The frozen cells were thawed and centrifuged for 10 min at 4°C, and the supernatants were collected and lyophilized overnight. The dried cell extracts were resuspended in 200 μl of AG buffer (0.1 M sodium phosphate, pH 7.4, 1 mM EDTA, 0.1% Triton, 0.5% Nonidet P-40, and 0.9% NaCl) and centrifuged for 5 min, and 150 μl was used for immunoprecipitation.

For immunoprecipitation, 150 μl of the cell extracts was incubated with 100 μl of 20% prehydrated protein A-Sepharose (Pharmacia Biotech, Piscataway, NJ, U.S.A.) at 4°C with constant rocking. The samples were centrifuged for 5 min and the supernatants were transferred to fresh tubes and incubated overnight at 4°C with 10 μl of polyclonal rabbit antiserum JH93 directed against α-melanocyte-stimulating hormone (α-MSH) (Mains et al., 1990) or Met-enk-RGL (Lindberg and White, 1986), respectively. The samples were then centrifuged at 4°C, and 100 μl of 20% protein A-Sepharose beads was added to the supernatant, which was then incubated at 4°C with constant rocking for 1 h. The samples were centrifuged and the beads washed three times with AG buffer, then once with 0.5 M NaCl and once with PBS. Immunoprecipitated proteins were extracted from the beads by adding 40 μl of 1 M acetic acid and 80 μl of 8 M urea in 32% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA), incubated at room temperature for 15 min, and centrifuged, and the supernatants were removed. Supernatants were either frozen at -70°C or were immediately size fractionated.

High-pressure gel permeation chromatography (HPGPC)

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Construction of PECT peptide chimera
  5. High-pressure gel permeation chromatography (HPGPC)
  6. RIA
  7. Production of PECT in CHO cells
  8. RESULTS
  9. PECT chimera is able to inhibit PE processing in vitro
  10. Inhibition of POMC processing is potentially due to competition of PE
  11. PECT is correctly targeted to secretory granules
  12. CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells
  13. DISCUSSION
  14. Acknowledgements
  15. References

The HPGPC system consisted of a Bio-Sil TSK precolumn (7.5 × 7.5 mm ; Bio-Rad) and two high-pressure gel permeation columns connected in series, a Protein Pak SW 300 column (300 × 7.8 mm ; Waters, Milford, MA, U.S.A.) and a Bio-Sil TSK-125 column (300 × 7.5 mm ; Bio-Rad). A 100-μl aliquot of each sample was injected into the column array, which was eluted at a flow rate of 0.5 ml/min ; 1-min fractions were collected. The eluant used was 32% ACN and 0.1% TFA. Radioactivity in each fraction was determined by liquid scintillation spectroscopy after the addition of 5 ml of cocktail (Beckman).

To determine the amount of Met-enk-RGL and CT peptide in chimera-expressing AtT-20/PC2, AtT-20/PC2, and PE-expressing AtT-20/PC2 cells, cells were grown in 10-cm dishes until ~70% confluent and then homogenized in 1 ml of acid mix and frozen. The homogenates were then thawed and centrifuged for 10 min at 4°C, and the clear supernatant was removed and lyophilized overnight. Cell extracts were then resuspended in 250 μl of 32% ACN and 0.1% TFA and centrifuged, and 100 μl of the clear supernatant was injected onto the HPGPC system. Ten micrograms of carrier protein, bovine serum albumin (BSA), was added to each fraction collected to prevent adsorption to tube walls. Duplicate 50- to 100-μl aliquots of each fraction were dried by vacuum centrifugation in the presence of additional BSA, resuspended in 100 μl of radioimmunoassay (RIA) buffer (0.1 M sodium phosphate, pH 7.4, containing 0.1% heat-treated BSA, 50 mM NaCl, 0.1% sodium azide, and 0.1% BME), and assayed using specific enkephalin or CT peptide RIAs.

RIA

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Construction of PECT peptide chimera
  5. High-pressure gel permeation chromatography (HPGPC)
  6. RIA
  7. Production of PECT in CHO cells
  8. RESULTS
  9. PECT chimera is able to inhibit PE processing in vitro
  10. Inhibition of POMC processing is potentially due to competition of PE
  11. PECT is correctly targeted to secretory granules
  12. CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells
  13. DISCUSSION
  14. Acknowledgements
  15. References

RIAs for both Met-enk-RGL and the CT peptide were carried out by a procedure described previously (Lindberg, 1986). In brief, samples were incubated with 10,000 cpm of iodinated peptide and the appropriate dilution of antiserum in a 300-μl final volume at 4°C overnight. For the CT peptide assay, antiserum 23B6 (Zhu et al., 1996b) was used at a final dilution of 1:2,500 ; and for Met-enk-RGL, antiserum Cass (Lindberg and White, 1986) was used at 1:30,000. Standards for the CT peptide ranged from 0.05 to 5 pmol and for Met-enk-RGL from 1 to 500 fmol. To separate the antibody-bound labeled peptide from unbound labeled peptide, 1 ml of 25% polyethylene glycol and 100 μl of 7.5% carrier γ-globulin (in PBS) were added ; the samples were vortex-mixed vigorously, kept on ice for 30 min, and then centrifuged for 20 min at 5,000 g in a refrigerated tabletop centrifuge. The supernatant was aspirated and radioactivity in pellets determined using an LKB γ counter. The β-endorophin RIA was performed as previously described (Lindberg et al., 1994).

Enzyme assays

The fluorogenic substrate pRTRK-methylcoumarin amide was used to measure PC2 activity. The assay was performed in 100 mM sodium acetate (pH 5) containing 5 mM CaCl2, 0.1% Brij, and 0.22 μg of PC2 (final concentration 66 nM). Samples were preincubated for 30 min at room temperature in duplicate in the presence of various concentrations of 27-kDa 7B2 or the PECT chimera. The substrate was then added at a final concentration of 200 μM, and incubation was carried out at 37°C for various time points ranging from 30 min to 5 h. Liberated aminomethylcoumarin was measured with a microtiter plate fluorometer (Cambridge Technology, Cambridge, MA, U.S.A.). For determination of inhibition of PE cleavage, 50 nM PE or 100 nM PECT (final concentration) was incubated in 100 mM sodium acetate (pH 5) containing 5 mM CaCl2, 0.1% Brij, and 0.22 μg of PC2 (final concentration 66 nM) for various times in a reaction volume of 50 μl. The reactions were stopped by boiling the samples for 2 min. Aliquots of 5 μl were removed from each sample, resuspended in 100 μl of Tris-BSA buffer containing carboxypeptidase B (CPB ; 0.02 U/ml final concentration ; Worthington, Freehold, NJ, U.S.A.), and incubated at 37°C for 30 min. The reaction was again stopped by boiling for 2 min, and samples were assayed for Met-enk-RGL by RIA as described above. The enzyme/substrate ratio was ~1:1 for PE and ~1:2 for PECT.

Production of PECT in CHO cells

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Construction of PECT peptide chimera
  5. High-pressure gel permeation chromatography (HPGPC)
  6. RIA
  7. Production of PECT in CHO cells
  8. RESULTS
  9. PECT chimera is able to inhibit PE processing in vitro
  10. Inhibition of POMC processing is potentially due to competition of PE
  11. PECT is correctly targeted to secretory granules
  12. CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells
  13. DISCUSSION
  14. Acknowledgements
  15. References

The PECT chimera was overexpressed in DG44 CHO cells essentially as previously described for PE (Lindberg and Zhou, 1995). Positive clones were selected and followed by western blotting using CT peptide antiserum. Amplification was carried out to 50 μM methotrexate. For collection of PECT from conditioned medium, cells were grown in roller bottles, incubated overnight in 100 ml of OptiMem containing 100 μg/ml aprotinin, and harvested for 4 successive days. Following acidification to 0.1% with TFA and filtration, this conditioned medium was used for the single-step purification of PECT via reverse-phase chromatography on a semipreparative Vydac C4 column, essentially as previously described for PE (Lindberg and Zhou, 1995).

Stimulation of peptide release

Approximately 500,000 chimera-expressing AtT-20/PC2 and Rin cells were plated in 35-mm wells and allowed to grow until ~90% confluent. Cells were washed twice with PBS and then incubated in 0.7-1 ml of OptiMem with 100 μg/ml aprotinin and 5 μg/ml BSA at 37°C for 2 h. Cells were then washed once with PBS, OptiMem was added, and cells were incubated for 3 h at 37°C to collect basal medium. The cells were again washed with PBS, and a stimulation medium was then applied, which for AtT-20 cells consisted of 0.7-1 ml of OptiMem/BSA/aprotinin plus 100 nM phorbol 12-myristate 13-acetate (PMA), for 3 h at 37°C. For Rin cells, a solution containing 1 μM PMA, 1 mM isobutylmethylxanthine (IBMX), and 15 mM glucose was used to stimulate peptide release. The medium was collected, spun briefly to remove floating cells, and frozen. A 100-μl aliquot from both basal and stimulated medium was assayed using both Met-enk-RGL and CT peptide RIAs. CPB treatment was performed prior to Met-enk-RGL assay. Results are expressed per 35-mm well per 3-h incubation period.

Hydrolysis of 125I-labeled CT peptide

Recombinant PC1 or recombinant PC2 was incubated with 10,000 cpm of 125I-labeled 7B2 CT peptide in 100 mM sodium acetate buffer (pH 5.0) in the presence of 5 mM CaCl2 and 0.2% Brij. After incubation for various times at 37°C, samples were size-fractionated by HPGPC, and radioactivity in fractions was determined by γ spectroscopy.

PECT chimera is able to inhibit PE processing in vitro

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Construction of PECT peptide chimera
  5. High-pressure gel permeation chromatography (HPGPC)
  6. RIA
  7. Production of PECT in CHO cells
  8. RESULTS
  9. PECT chimera is able to inhibit PE processing in vitro
  10. Inhibition of POMC processing is potentially due to competition of PE
  11. PECT is correctly targeted to secretory granules
  12. CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells
  13. DISCUSSION
  14. Acknowledgements
  15. References

Given the fact that the CT peptide is a potent in vitro inhibitor of recombinant PC2, we examined the ability of recombinant PECT to inhibit PC2 directly. For these experiments, we overexpressed PECT in CHO cells using the dihydrofolate reductase-coupled methotrexate amplification method (Lindberg and Zhou, 1995). The ability of purified recombinant PECT to inhibit purified recombinant mouse PC2 was determined using the standard fluorogenic substrate, pRTKR-aminomethylcoumarin. Interestingly, we found that PECT was almost as potent as 27-kDa 7B2 in inhibiting recombinant PC2 (IC50 = 10 nM and 27 nM, respectively) (Fig. 2A). We then investigated whether PC2-mediated cleavage of the PE portion of PECT could occur under these in vitro conditions. Recombinant PECT was able to completely inhibit PC2-mediated processing of itself to free Met-enk-RGL (Fig. 2B). In the absence of PECT, 2.5 pmol of PE was processed to Met-enk-RGL immunoreactive (ir) peptides after 4 h of incubation, corresponding roughly to 100% cleavage ; in the presence of PECT, essentially no cleavage occurred. This demonstration of effective inhibition of PC2 by the PECT chimera in vitro supports the idea that the CT peptide can act as an effective inhibitor when attached to other carrier proteins.

image

Figure 2. Recombinant PECT is a potent in vitro inhibitor of PC2. A : Fluorogenic assay. Various concentrations of PECT and recombinant histidine-tagged 27-kDa 7B2 were assayed for their inhibitory potency against PC2. Circles, rat 27-kDa 7B2 ; squares, PECT. B : Inhibition of PC2-mediated hydrolysis of PE. Recombinant PECT and PE were incubated for various times with recombinant PC2. PE processing was determined by assessing liberation of the internal peptide Met-enk-RGL by RIA.

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PE is efficiently processed in both chimera-expressing AtT-20 and Rin cells

AtT-20 cells and AtT-20/PC2 cells were stably transfected with an expression vector encoding PECT, and the clones were screened by RIA for the CT peptide. Three to four chimera-expressing clones were selected, grown, and used for the experiments described here. The PE portion of the PECT chimera contains a PC2-mediated internal cleavage site for Met-enk-RGL production (Breslin et al., 1993 ; Johanning et al., 1996a). We have previously shown that the cleavage of PE to Met-enk-RGL is enhanced in PC2-expressing AtT-20/PE cells compared with non-PC2-expressing AtT-20/PE cells (Johanning et al., 1996a). We therefore investigated the ability of cellular PC2 to liberate the internal Met-enk-RGL sequence by performing RIA for this peptide in size-fractionated cell extracts prepared from chimera-expressing AtT-20 and Rin cells. (It should be noted that the 5.3-kDa and the 18-kDa amino-terminally extended forms of Met-enk-RGL cross-react in the Met-enk-RGL assay ; CPB was used to remove C-terminal basic residues in all cases to reveal the immunoreactive C terminus of this molecule.) Figure 3B shows that in PECT-expressing AtT-20/PC2 cells, 84% of the total Met-enk-RGL immunoreactivity recovered from the column was present in the form of the free octapeptide Met-enk-RGL ; in contrast, only 44% of the total Met-enk-RGL immunoreactivity recovered from the column was processed in AtT-20 cells expressing the chimera but lacking PC2 (Fig. 3A), indicating that PC2 was most likely the enzyme responsible for this cleavage event. Similarly efficient processing of PECT to free Met-enk-RGL was observed in Rin cells, which naturally contain PC2 (Fig. 4B). In opposition to the idea that the CT peptide might represent a PC2 inhibitor, no difference was observed in the content of mature Met-enk-RGL in cells expressing PE (Fig. 4A) and PECT (Fig. 4B).

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Figure 3. PECT is processed to Met-enk-RGL in AtT-20 and in AtT-20/PC2 cells. RIA of Met-enk-RGL immunoreactivity in sizefractionated extracts of AtT-20 chimera-expressing cells without (A) and with (B) PC2 shows that this PC2-mediated cleavage is not inhibited. Results represent total immunoreactivity per fraction. Similar results were obtained for four different clones of each cell line.

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image

Figure 4. PECT is processed to Met-enk-RGL in Rin cells. RIA of Met-enk-RGL immunoreactivity in PE-expressing (A) and PECT-expressing (B) Rin cells shows that PC2 is not inhibited. Results represent immunoreactivity per fraction. Similar results were obtained for two different clones.

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To investigate the kinetics of PC2-mediated processing of PE in PECT cells, pulse chase/immunoprecipitation experiments were performed. We found that after 3.5 h, 10% of newly synthesized PE was processed to Met-enk-RGL in PECT-expressing AtT-20 cells and 5% was processed in PE-expressing control cells (data not shown). The higher degree of processing in the chimera-expressing cells as opposed to natural PE may be an effect of the introduced mutation, as all PE mutants constructed to date exhibit more complete precursor processing for reasons that are not yet clear (Johanning et al., 1996b). These results are consistent with the above RIA data and confirm the inability of the CT peptide to inhibit PE processing in AtT-20/PC2 cells. Taken together, the results from both the RIA and the pulse chase studies suggest that when presented in the context of the PECT chimera, the CT peptide is not able to inhibit intracellular PC2-mediated cleavage of PE.

Inhibition of POMC processing is potentially due to competition of PE

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Construction of PECT peptide chimera
  5. High-pressure gel permeation chromatography (HPGPC)
  6. RIA
  7. Production of PECT in CHO cells
  8. RESULTS
  9. PECT chimera is able to inhibit PE processing in vitro
  10. Inhibition of POMC processing is potentially due to competition of PE
  11. PECT is correctly targeted to secretory granules
  12. CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells
  13. DISCUSSION
  14. Acknowledgements
  15. References

The processing of endogenous POMC was used to further assess the potential inhibition of PC2 in PECT cells. POMC, like PE, is cleaved at dibasic amino acid residues to liberate bioactive peptides (Mains et al., 1990). The POMC product β-lipotropic hormone (β-LPH) is the precursor to β-endorphin, and the processing of β-LPH to β-endorphin is mediated partially by PC2 (Zhou et al., 1993). In both PECT- and PE-expressing AtT-20/PC2 cells, we found that there was substantially less β-endorphin than in AtT-20/PC2 cells (data not shown). These results suggest that the decrease in β-endorphin in PECT-expressing and PE-expressing cells is due to the PE portion of the chimera and not to the CT peptide, potentially by competition of PE and POMC for PC2.

To confirm the idea of potential competition between PE and POMC, pulse chase/immunoprecipitation experiments were performed using an antibody directed toward the NH2-terminal end of ACTH. This antibody reacts with POMC, ACTH, and α-MSH. In AtT-20 cells, POMC is cleaved to ACTH by PC1, and ACTH is then further cleaved by PC2 to a smaller peptide, α-MSH (Zhou et al., 1993). The processing of ACTH to α-MSH is mediated fairly specifically by PC2 (Zhou et al., 1993). In cells lacking PC2, ACTH is not internally processed (Fig. 5A). On the other hand, in AtT-20 cells expressing PC2, ACTH was processed to α-MSH (Zhou et al., 1993, Fig. 5B). Interestingly, in both PECT-expressing and PE-expressing cells containing PC2, ACTH was not processed to α-MSH (Fig. 5C and D). Collectively, our data suggest that the CT peptide is not responsible for inhibition of PC2-mediated cleavage of POMC to α-MSH ; rather, it is the PE portion of the chimera that is likely to be responsible for blocking PC2-mediated cleavages of POMC, by directly competing with POMC for PC2. Although nonquantitative due to cross-reactivity issues, our RIAs of stored peptide products suggest that the expression level of PE and PECT in AtT-20/PC2 cells is roughly equivalent to that of the endogenous precursor POMC (15-27 pmol/plate of Met-enk-RGL immunoreactivity vs. 17-20 pmol/plate of β-endorphin immunoreactivity).

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Figure 5. Inhibition of POMC processing by PECT is potentially due to competition between PE and POMC for PC2. AtT-20 (A), AtT-20/PC2 (B), chimera-expressing AtT-20/PC2 (C), and PE-expressing AtT-20/PC2 (D) cells were labeled in medium containing [35S]methionine for 20 min and chased in methionine-deficient medium for 0 and 2 h. Extracts were immunoprecipitated with ACTH/α-MSH antiserum JH93 and fractionated by HPGPC.

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PECT is correctly targeted to secretory granules

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Construction of PECT peptide chimera
  5. High-pressure gel permeation chromatography (HPGPC)
  6. RIA
  7. Production of PECT in CHO cells
  8. RESULTS
  9. PECT chimera is able to inhibit PE processing in vitro
  10. Inhibition of POMC processing is potentially due to competition of PE
  11. PECT is correctly targeted to secretory granules
  12. CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells
  13. DISCUSSION
  14. Acknowledgements
  15. References

Given the effectiveness of the PECT chimera in inhibition of PC2 in vitro, we wanted to assess whether the CT portion of the PECT chimera was properly targeted to and stored within the regulated secretory pathway ; for this purpose, stimulus-secretion studies were performed. To detect storage of PE and the CT peptide in secretory granules, aliquots of both basal and stimulated media were assayed by RIAs directed against either the CT peptide or Met-enk-RGL.

Tables 1 and 2 show that the CT peptide was secreted from PECT-expressing cells similarly to Met-enk-RGL-ir peptides, both in AtT-20 cells expressing and in those lacking PC2. For CT peptide and Met-enk-RGL-ir peptides, the stimulation induced by exposure to the secretagogue PMA was two- to threefold in PC2-expressing cells and in cells lacking PC2. The release of both peptides could be similarly stimulated from Rin cells with a secretagogue cocktail (Table 3). The stimulated peptide release experiment was repeated in its entirety three times with two to five different clones, with similar results. We found no statistical difference between the secretion of Met-enk-RGL and the CT peptide (p > 0.05) or between cells with or without PC2 (p > 0.05). Taken together, these results suggest that both the CT peptide- and the PE-related products derived from the chimera are stored similarly within regulated secretory granules.

Table 1. Stimulation of peptide release in AtT-20 PE-CT cellsCells were stimulated in the presence of 100 nM PMA for 3 h.
Basal release Stimulated release
Clone no.CT peptide (pmol)Met-enk-RGL (pmol)CT peptide [pmol (-fold increase)]Met-enk-RGL [pmol (-fold increase)]
1B5.15.48.0 (1.6)8.9 (1.6)
3B5.52.517 (3.0)6.0 (2.4)
13M5.82.512 (2.2)3.9 (1.6)
15L1.50.42.4 (2.2)1.1 (2.8)
Average -fold increase 2.22.1
Table 2. Stimulation of peptide release in AtT-20/PC2 PECT cellsCells were stimulated in the presence of 100 nM PMA for 3 h.
Basal releaseStimulated release 
Clone no.CT peptide (pmol)Met-enk-RGL (pmol)CT peptide [pmol (-fold increase)]Met-enk-RGL [pmol (-fold increase)]
21B7.41022 (3.0)16 (1.6)
24M8.22319 (2.3)49 (2.1)
13L2.84.413 (4.6)15 (3.4)
Average -fold increase 3.32.4
Table 3. Stimulation of peptide release in Rin PECT cellsCells were stimulated in the presence of 1 μM PMA, 1 mM IBMX, and 15 mM glucose for 3 h.
 Basal releaseStimulated release
Clone no.CT peptide (pmol)Met-enk-RGL (pmol)CT peptide [pmol (-fold increase)]Met-enk-RGL [pmol (-fold increase)]
7B (PECT)241556 (2.3)18 (1.2)
6MB (PECT)7.5629 (3.9)10 (1.7)
Average-fold increase  3.01.6

CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Construction of PECT peptide chimera
  5. High-pressure gel permeation chromatography (HPGPC)
  6. RIA
  7. Production of PECT in CHO cells
  8. RESULTS
  9. PECT chimera is able to inhibit PE processing in vitro
  10. Inhibition of POMC processing is potentially due to competition of PE
  11. PECT is correctly targeted to secretory granules
  12. CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells
  13. DISCUSSION
  14. Acknowledgements
  15. References

We have previously proposed that PC2 is the enzyme responsible for inactivation of the CT peptide by cleavage at an internal dibasic (Lys-Lys) site, an action followed by removal of C-terminal basic amino acids by carboxypeptidase E (Zhu et al., 1996b). We therefore speculated that the CT peptide might already be hydrolyzed/inactivated by PC2 in chimera-expressing cells. To investigate this possibility as well as to verify actual removal of the CT peptide from PECT by cleavage at the paired basic fusion site, we examined the molecular mass of CT-ir peptides in chimera-expressing cells. Figure 6A shows that chimera-expressing AtT-20 cells exhibit only one peak of CT immunoreactivity, which corresponds to the position of the cleaved CT peptide. Although AtT-20 cells lacking PC2 cannot cleave the CT peptide internally when presented as 27-kDa 7B2 (Zhu et al., 1996b ; unpublished observations), surprisingly, the CT peptide was also efficiently internally cleaved in chimera-expressing AtT-20 cells lacking PC2 (Fig. 6B). As previously observed in AtT-20 cells, in Rin PE/27-kDa 7B2 cells, the CT peptide was incompletely cleaved, with the intact peptide eluting at fraction 38 (Fig. 6D) ; in contrast, the CT peptide is completely cleaved in Rin PECT cells (Fig. 6C). Thus, the cellular handling of the CT peptide appears to differ when the molecule is synthesized in the form of 27-kDa 7B2 or in the form of PECT. This experiment was repeated three times with similar results.

image

Figure 6. The CT peptide is internally cleaved in PECT-expressing cells. RIA of CT peptide immunoreactivity in chimera-expressing AtT-20 cells expressing (A) or lacking (B) PC2 shows that cellular CT peptide is internally cleaved in both cell lines. RIA of CT peptide immunoreactivity in chimera-expressing (C) and 27-kDa 7B2-expressing Rin (D) cells shows that CT peptide derived from PECT is also internally cleaved in Rin cells, which naturally express PC2. Standards used included 125l-human CT peptide (1-31) and Tyro-CT (19-31).

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Due to the possibility that our RIA might preferentially detect processed CT peptide, thus skewing the immunoreactive profile, we performed valine steady-state labeling studies with CT peptide antiserum to confirm the RIA results presented above. Note that valine is represented only once in CT (19-31), but three times in intact CT peptide, thus underestimating the relative amount of the cleaved product by a factor of 3. We showed that intact CT peptide is present in the cells (Fig. 7) and thus must indeed be initially cleaved from the chimera as such. Previous studies have shown that the cleavage of the CT peptide from the PE portion of the chimera is at the joining multibasic sequence (Zhu et al., 1996b). Further, the CT peptide was found to be substantially internally hydrolyzed in AtT-20, AtT-20/PC2, and Rin PECT-expressing cells (Fig. 7). A slightly greater proportion of the CT peptide was internally cleaved at the Lys17-Lys18 site in cells containing PC2 as compared with cells lacking PC2 (Fig. 7), thus supporting the involvement of PC2 in this event (Zhu et al., 1996b). However, in agreement with the CT peptide RIA data, the majority of the CT peptide was also internally cleaved in cells lacking PC2 (Fig. 7B). These data imply that PC1 or another convertase may be able to internally cleave the CT peptide when synthesized in the context of the chimera, although not when synthesized as 27-kDa 7B2.

image

Figure 7. The CT peptide is liberated from PECT in PECT-expressing Rin and AtT-20 cells. AtT-20/PC2 (A), AtT-20 (B), and Rin (C) cells expressing PECT were labeled in medium containing [3H]valine for 6 h. Extracts were immunoprecipitated with CT peptide antiserum and size fractionated by HPGPC. Results are not corrected for the number of valines per molecule.

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To reproduce this phenomenon in vitro, we incubated recombinant PCs with recombinant 27-kDa 7B2 and PECT. However, whereas PC1 was able to rapidly remove intact CT peptide from both of these proteins, PC1 was unable to cleave the CT peptide internally when presented in the context of either 27-kDa 7B2 (Fig. 8A) or PECT (Fig. 8B). PC1 was similarly unable to internally cleave 125I-labeled CT peptide (Fig. 9B). However, in agreement with previous studies (Zhu et al., 1996b), PC2 was able to cleave this iodinated peptide (Fig. 9A). Taken together, these results demonstrate that in all PECT-expressing cells, stored CT peptide is substantially internally cleaved, either by PC2 or by other enzymes such as PACE4. The lack of ability of PC1 to mediate this cleavage in vitro suggests that it is probably not responsible for this internal cleavage event.

image

Figure 8. PC1 is unable to cleave CT peptide in vitro in the context of either 27-kDa 7B2 or PECT. Recombinant PC1 was incubated with either recombinant 27-kDa 7B2 (A) or recombinant PECT (B) for various times. Reaction mixtures were size fractionated by HPGPC and CT peptide immunoreactivity determined by RIA.

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image

Figure 9. PC2, but not PC1, can internally cleave 125I-labeled CT peptide. Radiolabeled human CT peptide (~10,000 cpm) was incubated with recombinant PC1 or PC2 for various lengths of time. The reactions were size fractionated by HPGPC, and radioactivity in fractions was determined by γ spectroscopy. Note that the iodinated residue appears in the amino-terminal fragment.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Construction of PECT peptide chimera
  5. High-pressure gel permeation chromatography (HPGPC)
  6. RIA
  7. Production of PECT in CHO cells
  8. RESULTS
  9. PECT chimera is able to inhibit PE processing in vitro
  10. Inhibition of POMC processing is potentially due to competition of PE
  11. PECT is correctly targeted to secretory granules
  12. CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells
  13. DISCUSSION
  14. Acknowledgements
  15. References

The CT portion of the 27-kDa neuroendocrine protein 7B2 has been shown to represent a potent tight binding inhibitor of the convertase PC2 (Lindberg et al., 1995 ; Apletalina et al., 1998) ; to date, this peptide represents the only known natural inhibitor of this family of enzymes. Although initial studies led us to believe that the role of this inhibitory protein might be related to the prevention of premature activation of proPC2 (Martens et al., 1994), recent studies have demonstrated that this is not the case. For example, both 27-kDa 7B2 as well as the CT peptide are completely ineffective in preventing proPC2 from activating in vitro, implying lack of access to the active site (Lamango et al., 1996, 1999). Potential occupation of the active site by propeptide in PCs is suggested by a study showing that this occurs for the related enzyme furin (Anderson et al., 1997). In the case of PC2, preliminary studies have directly demonstrated potent inhibition of recombinant PC2 by PC2 propeptide (C. Lazure, personal communication). Thus, a direct role for the CT peptide in preventing premature zymogen conversion by occupation of the active site is unlikely.

What, then, might be the physiological role of the CT peptide ? We have postulated that the CT peptide might play a role in the intragranular regulation of PC2 activity, as our data show that it is stored within regulated secretory granules and is inactivated in these same granules by internal hydrolysis (Zhu et al., 1996b). If this is the case, then overexpression of the CT peptide in the same compartment as active PC2 should result in lowered activity of this enzyme. We have previously attempted to manipulate peptide levels by overexpressing 27-kDa 7B2 (which is also inhibitory to PC2 in vitro) in AtT-20 cells ; however, no alterations in POMC processing were seen (R. E. Mains, personal communication). We found a similar lack of inhibition of PE processing upon overexpression of 27-kDa 7B2 in Rin PE cells (data not shown). To uncouple potential facilitative effects of the 21-kDa domain from inhibitory effects of the CT peptide, we engineered a PECT chimera designed to target this potent inhibitor to the granules.

Our data show that the PECT chimera is indeed correctly targeted to a stimulatable compartment, that is, regulated secretory granules, but that PC2-mediated processing of PE and POMC is not thereby inhibited. Potential explanations that were investigated included the possibility that the CT peptide is not initially cleaved from the PECT chimera or is inactivated. With regard to the first possibility, valine-labeling studies showed that a small amount of intact CT peptide is indeed present within chimera-transfected neuroendocrine cell lines. In fact, however, cleavage of the CT peptide from the chimera was not found to be necessary for inhibition, because the recombinant PECT protein represented a potent inhibitor of recombinant PC2 in vitro. We also investigated the possibility that in PECT-expressing cells, the CT peptide was totally inactivated by internal hydrolysis at the Lys17-Lys18 pair ; when followed by action of carboxypeptidase E, this proteolysis is known to destroy the inhibitory potency of the CT peptide (Zhu et al., 1996b). Indeed, we found that stored cellular CT peptide derived from PECT was almost completely internally cleaved in all cell lines examined. This internal cleavage is most likely responsible for the lack of inhibitory potency of transfected PECT. However, stored CT peptide derived from 27-kDa 7B2 was only partially internally cleaved in both AtT-20/PC2 and Rin cells and was not at all cleaved in AtT-20 cells lacking PC2 (Zhu et al., 1996b ; this article). These data imply that there are significant differences in the interaction of the CT peptide with PC2 and with other convertases, when it is presented in the context of PECT rather than as 27-kDa 7B2, which result in its ability to be internally hydrolyzed in the former case but not in the latter. We speculate that the presence of the 21-kDa portion of 27-kDa 7B2 may somehow protect the CT peptide from internal hydrolysis during a vulnerable stage of trafficking.

Given the contradiction between the apparent lack of inhibition of transfected PECT in vivo and its potent inhibition in vitro, we analyzed the relative cellular expression levels of PC2 and PECT in Rin PECT cells by two methods, a radioactive method (steady-state [35S]methionine labeling/immunoprecipitation) and a nonradioactive method (quantitative western blotting for PC2 and RIA for CT peptide). Both methods yielded similar molar ratios of ~1.3 for total PECT/CT to proPC2/PC2 (results not shown). Whereas in vitro the high affinity of PC2 for the CT peptide results in stoichiometric inactivation of PC2 (Lindberg et al., 1995), in cells this stoichiometric ratio is apparently inadequate for inhibition, most likely due to substantial internal hydrolysis of the CT peptide. Targeting a greater quantity of CT peptide to the granules—potentially through the use of a multiple CT peptide-containing cassette—might be required for effective inhibition of granular PC2. However, as the peptide can apparently be hydrolyzed by other enzymes (unless protected by the presence of the 21-kDa portion of 7B2 ?), it may be that this strategy will not result in increased granular storage of intact CT peptide.

In vivo inhibition of subtilisin-like convertases should theoretically result in decreased activity against propeptide substrates and lowered production of bioactive peptides. This has been achieved in the case of furin using vectors encoding the modified serpin α1-antitrypsin Portland (Anderson et al., 1993 ; Benjannet et al., 1997). No such comparable inhibitors exist for PC1 and PC2, and the usefulness of antitrypsin Portland in the regulated secretory pathway is compromised by its inability to effciently traffic to the secretory granules (Benjannet et al., 1997). We have succeeded in targeting the secretory compartment with a potent PC2 inhibitor ; however, our inability to actually effect inhibition suggests that we need to better understand the regulation and subcellular site of CT peptide inactivation by various enzymes. Our challenge is to design inhibitor-bearing vectors that will effectively access PC2 at a vunerable point in the secretory pathway.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Construction of PECT peptide chimera
  5. High-pressure gel permeation chromatography (HPGPC)
  6. RIA
  7. Production of PECT in CHO cells
  8. RESULTS
  9. PECT chimera is able to inhibit PE processing in vitro
  10. Inhibition of POMC processing is potentially due to competition of PE
  11. PECT is correctly targeted to secretory granules
  12. CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells
  13. DISCUSSION
  14. Acknowledgements
  15. References

We thank Joelle Finley for assistance with cell culture, Lisa Vaughn for help with RIAs, and Dick Mains for α-MSH antiserum as well as AtT-20/PC2 cells. This work was supported by National Institutes of Health grants DK49703 and DA05084 and RCDA DA00204 to I.L. and by predoctoral fellowship DK49703-02S1 to Y.F.

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  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Construction of PECT peptide chimera
  5. High-pressure gel permeation chromatography (HPGPC)
  6. RIA
  7. Production of PECT in CHO cells
  8. RESULTS
  9. PECT chimera is able to inhibit PE processing in vitro
  10. Inhibition of POMC processing is potentially due to competition of PE
  11. PECT is correctly targeted to secretory granules
  12. CT peptide is inactivated in AtT-20 and Rin PECT-expressing cells
  13. DISCUSSION
  14. Acknowledgements
  15. References
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