From the Department of Pharmacology, University of
Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada, the ¶ Laboratory
of Neuropeptide Structure and Metabolism, Clinical Research Institute
of Montreal, Montreal, Quebec, Canada, the
Montreal Children's
Hospital Research Institute, Montreal, Quebec H3H 1P3, Canada, and the
** Department of Biochemistry and Molecular Biology, Louisiana State
University Medical Center, New Orleans, Louisiana 70112
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ABSTRACT |
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Endoproteolytic processing of the 26-kDa protein
precursor prodynorphin (proDyn) at paired and single basic residues is
most likely carried out by the proprotein convertases (PCs); however, the role of PCs at single basic residues is unclear. In previous studies we showed that limited proDyn processing by PC1/PC3 at both
paired and single basic residues resulted in the formation of 8- and
10-kDa intermediates. Because PC2 is colocalized with proDyn, we
examined the potential role of this convertase in cleaving proDyn. PC2
cleaved proDyn to produce dynorphin (Dyn) A 1-17, Dyn B 1-13, and
-neo-endorphin, without a previous requirement for PC1/PC3. PC2 also
cleaved at single basic residues, resulting in the formation of the
C-peptide and Dyn A 1-8. Only PC2, but not furin or PC1/PC3, could
cleave the Arg-Pro bond to yield Dyn 1-8. Structure-activity studies
with Dyn A 1-17 showed that a P4 Arg residue is
important for single basic cleavage by PC2 and that the P1
Pro residue impedes processing. Conversion of Dyn A 1-17 or Dyn B
1-13 into leucine-enkephalin (Leu-Enk) by PC2 was never observed;
however, Dyn AB 1-32 cleavage yielded small amounts of Leu-Enk,
suggesting that Leu-Enk can be generated from the proDyn precursor only
through a specific pathway. Finally, PC2 cleavages at single and paired
basic residues were enhanced when carried out in the presence of
carboxypeptidase (CP) E. Enhancement was blocked by GEMSA, a specific
inhibitor of CPE activity, and could be duplicated by other
carboxypeptidases, including CPD, CPB, or CPM. Our data suggest that
carboxypeptidase activity enhances PC2 processing by the elimination of
product inhibition caused by basic residue-extended peptides.
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INTRODUCTION |
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Opioid peptides are produced by the endoproteolytic processing of
three distinct polypeptide precursors: proopiomelanocortin, proenkephalin, and prodynorphin
(proDyn).1 ProDyn is the
precursor of leucine-enkephalin (Leu-Enk) C-terminally extended
sequences including dynorphin (Dyn) A 1-17, Dyn A 1-8, Dyn B 1-13,
and -neo-endorphin (
NE) (1). Flanking single and paired basic
residue cleavage sites are observed within the proDyn precursor (see
Fig. 1). Even though proDyn processing appears to be a complex model to
study processing (2), it is common for neuropeptide precursors to have
multiple cleavage sites. It is interesting to note that in
vivo processing of this precursor results in defined sets of
neuropeptide products such as Dyn A 1-17 and Dyn B 1-13, but little
if any Leu-Enk is ever observed in the same neurons (2, 3). This
suggests that not all apparent basic residue cleavage sites are used
within the secretory pathway or that the intracellular processing
enzymes responsible for proDyn processing have specific preferences for
these processing sites.
As with other neuropeptide precursors, the paired basic residues within proDyn are most likely processed by the family of enzymes known as the proprotein convertases (PCs) (4). These include furin/paired amino acid cleaving enzyme (PACE) (5, 6), PC1/PC3 (7-9), PC2 (7, 10), PC4 (11, 12), PACE4 (13), PC5/PC6 (14, 15), and LPC/PC7/PC8 (16-18). The fact that PC1/PC3 and PC2 are principally expressed in neurons and endocrine cells (19-21) suggests that they are candidate enzymes for the processing of neuropeptide precursors such as proDyn (2). In previous studies we tested the cleavage specificity of PC1/PC3 for proDyn (22). PC1/PC3 cleaved proDyn at both single and paired basic residues to yield processing intermediates of 8 and 10 kDa (Fig. 1). This study suggested that further processing by another enzyme(s) was required to achieve biologically active opioid peptides. Based on the processing profiles of other protein precursors such as proopiomelanocortin (23), proenkephalin (24), and pro-insulin (25) and also based on colocalization studies in the central nervous system (20, 26, 27), we concluded that PC2 was the best candidate to produce the expected final proDyn products.
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Processing of protein precursors also requires the action of carboxypeptidases to yield a fully mature peptide (28). Once endoproteolysis has occurred C-terminal to a single or paired basic residue site, a carboxypeptidase is required for C-terminal trimming of the basic residue(s). Although CPE was long thought to be the only carboxypeptidase to carry out this function within secretory pathways, novel carboxypeptidases have now been discovered that carry out this function, including CPD (29). This study focuses on proDyn processing by PC2 and examines the importance of carboxypeptidase activity on the efficiency of PC2 activity.
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MATERIALS AND METHODS |
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cRNA Probes and in Situ Hybridization-- The rat proDyn cRNA probe was transcribed from a BamHI-HincII fragment of 733 nucleotides subcloned into pGEM4. The cRNA for rat PC2, achieved by reverse transcription-polymerase chain reaction from a rat striatum library (Stratagene, La Jolla, CA) represents a 425-base pair fragment covering the rat sequence from bases 1812 to 2236 (7). 35S-UTP and 35S-CTP (1250 Ci/mmol, Amersham) were used for radioactive label.
The in situ hybridization colocalization studies were carried out as described previously (30). Male Sprague-Dawley rats (250-300 g) were used for these studies. Sections were simultaneously hybridized with an 35S-UTP/35S-CTP-labeled PC2 cRNA probe (specific activity, 2.6 × 105 Ci/mmol) and a digoxigenin-UTP-labeled proDyn cRNA probe. ProDyn mRNA signal appeared as dark blue (or purple) staining, whereas PC2 mRNA appeared as silver autoradiographic grains, which could be revealed as white grains when viewed in dark field light microscope observations. Controls for specificity of hybridization were carried out by pretreatment of brain sections with RNase A or by the use of sense strand probes of the same size and specific activity. No specific labeling was observed in these experiments.Recombinant PC2 and proDyn--
Recombinant mouse proPC2 was
purified from the conditioned medium of Chinese hamster ovary PC2/7B2
cells as described previously (31). Three different preparations were
used during the course of the work with similar results. Rat proDyn was
overexpressed in dhfr Chinese hamster ovary cells and
purified as described previously (22). No significant loss of PC2
activity is observed when the enzyme is incubated at temperatures below
42.5 °C (31). The specific activity of PC2 was 0.9 nmol/h/µg using
the Cbz-Arg-Ser-Lys-Arg-aminomethylcoumarin substrate. Recombinant
mouse PC1/PC3 and furin were purified from baculovirus (32). Each of
these proteins was purified to apparent homogeneity. Analysis was
carried out using SDS-polyacrylamide gel electrophoresis with Coomassie
staining or Western blotting using specific antibodies.
Peptides and in Vitro Studies-- The peptides Dyn A 1-17, Dyn A 1-8, Dyn A 1-9, porcine leumorphin, Dyn AB 1-32, and Dyn A 2-17 were purchased from Peninsula Laboratories (Belmont, CA). Dyn A 1-17 peptide analogs (see Fig. 5) were synthesized on an automated peptide synthesizer (Applied Biosystems) using Fast-Moc chemistry (33). The following side chain protecting groups were used: tert-butyloxy-carbonyl for Lys, tert-butyl for Tyr and Asp, trityl (triphenylmethyl) for Trp, and 2,2,5,7,8-pentamethylchroman-6-sulfonyl for Arg side chains, respectively. The crude peptides obtained upon cleavage from the resin and complete deprotection were purified by reverse phase HPLC performed on a semi-prep CSC-Excil C18 column (25 × 1.0 cm, Chromatography Sciences Co., St. Laurent, QC, Canada) using a linear gradient of 20-60% acetonitrile in 0.1% trifluoroacetic acid. The peptides were fully characterized by electron spray mass spectroscopy and amino acid analysis.
Between 5 and 20 µg of purified recombinant proDyn or various proDyn-derived synthetic peptides were incubated with PC2 (200 ng/reaction). The incubations were carried out in 50 mM sodium acetate, pH 5.0, containing 5 mM CaCl2 and 0.1% Brij-30. Incubation times varied from 30 min to 24 h depending on the experiment. In some experiments different carboxypeptidases were coincubated in the reaction mixture. These included CPE, CPD, CPM (provided by Dr. Lloyd Fricker, Albert Einstein College of Medicine), and CPB (Sigma). The specific activity of CPE was determined using the substrate dansyl-Phe-Ala-Arg. 10 ng of CPE completely cleave 50 nmol of Ala-Arg bonds in 1 h. In the described experiments, where CPE was used, excess CPE conditions (>200 ng/24 h incubation) were always added to ensure that all C-terminal basic residues would be cleaved. Similar conditions (>200 ng/reaction) were also applied with the other carboxypeptidases, CPD, CPM, and CPB. To terminate the reaction, the samples were diluted 1:1 (vol:vol) with acidified methanol (0.1 N HCl). The samples were then submitted to gel filtration chromatography or reverse phase HPLC analysis.Chromatography and RIAs--
For HPLC, the samples were injected
using an automated injector system (Hitachi L-7200) onto a Beckman
5-µm C18 column (25 × 0.46 cm). The samples were
separated using a 15-35% acetonitrile gradient in 0.1%
trifluoroacetic acid increasing at a rate of 0.5%/min. Peaks were
detected by UV light at 210 nm and evaluated by peak height. All
integrations were performed automatically with the Hitachi D-7000 HPLC
System Manager program. HPLC fractions were dried down and submitted to
amino acid analysis. Gel chromatography was performed using Sephadex
G-50 using a running buffer of 0.1% formic acid with 0.1% bovine
serum albumin (34). G-50 fractions were collected, dried, resuspended,
and submitted to RIA. For both HPLC and G-50 chromatography peptide
standards were run under the same conditions were used to determine the
elution times of the peptide of interest. For the G-50 chromatography
dextran blue and cobalt chloride were also used as markers of void and
total volumes, respectively. The recovery yields for gel chromatography (85%) and HPLC analysis (95%) have been determined using
radioactively labeled peptides as described previously (34).
Fluorogenic Enzyme Assay-- The effect of CPE on the hydrolysis of Cbz-Arg-Ser-Lys-Arg-aminomethylcoumarin (MCA) by PC2 was studied in a 50-µl reaction volume containing 6 ng of activated PC2, 200 mM substrate, in 0.1 M sodium acetate buffer, pH 5.0, containing 5 mM calcium chloride and 0.1% Brij-30. Reactions were carried out in duplicate, and the liberated aminomethylcoumarin was estimated by fluorometry at the time intervals stated. The experiment was repeated once with similar results.
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RESULTS |
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In Situ Hybridization Colocalization Studies of PC2 and proDyn-- In Fig. 2, we provide direct evidence of the colocalization of proDyn and PC2 in the central nervous system. Using a combined in situ hybridization technique, we observed PC2 mRNA within proDyn expressing neurons in the striatum. Other brain regions were also examined, including the paraventricular and supraoptic nuclei, the hippocampus, and the cortex. In each of these regions, proDyn and PC2 mRNA colocalization was observed. However, anterior lobe gonadotrophs, which are known to express proDyn (38), co-expressed PC1/PC3 but not PC2.2
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Processing of Recombinant proDyn by PC2 in Vitro--
To determine
whether PC2 was important in the formation of biologically active
opioid peptides, we tested the effect of PC2 on proDyn precursor
processing in vitro. Purified recombinant proDyn and PC2
were incubated overnight and fractionated using size exclusion
chromatography (Fig. 3, A and
B) and HPLC chromatography (Fig. 3, C,
D, E, and F). The same experiment was
also carried out in the presence of CPE (Fig. 3B). The
fractions were analyzed with RIAs using well characterized antibodies
(34) to different regions of proDyn. The data demonstrated that PC2
could generate each of the biologically active opioid peptides,
including Dyn A 1-17 (Fig. 3D), Dyn B (Fig. 3E),
and NE (Fig. 3F), from the proDyn precursor protein.
Processing intermediates (10 kDa) were observed (Fig. 3, A
and B), and smaller intermediates such as Dyn AB 1-32 were
also identified (data not shown). Because processing of the proDyn
precursor was not complete, the values of each of the obtained final
opioid products examined vary as seen in Fig. 3 (C,
D, E, and F). For example, in the case
of Dyn A 1-17, the immunoreactive content varies from 1 to 3 nmol,
whereas C-peptide immunoreactivity is in the 20-40-nmol range. It
should be remembered that the Dyn A 1-17 peptide was further processed
to Dyn A 1-8 and that significant levels of Dyn AB 1-32 were also
observed, both peptide species contributing to the lower Dyn A 1-17
immunoreactivity observed in the HPLC fractions collected.
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In Vitro Processing of Dyn A 1-17 by PC2--
We first studied
the formation of Dyn A 1-8 by PC2 using Dyn A 1-17 as the peptide
substrate (Fig. 4). Dyn A 1-17 peptide was incubated with determined amounts of PC2 active enzyme in an
overnight incubation. The amount of PC2 activity was measured using a
fluorogenic assay with the substrate pGlu-Arg-Thr-Lys-Arg-MCA. Each
sample was analyzed by reverse phase HPLC enabling the separation of
each of the processed peptides. These experiments were carried out in
the absence of CPE, and therefore if cleavage occurred in between the
ArgPro site of Dyn A 1-17, we would expect the formation of Dyn A
1-9 and Dyn A 10-17. The data demonstrated that Dyn A 1-17 was
cleaved into two distinct peptides, which co-eluted with Dyn A 1-9 and
Dyn A 10-17 peptide standards (Fig. 4B). To obtain
unequivocal identification of these peptides, the fractions were
collected and submitted to amino acid analysis. The two peaks obtained
were identified as Dyn A 1-9 and Dyn A 10-17. There was no evidence
for the production of Leu-Enk-Arg-Arg (Dyn A 1-7), demonstrating that
PC2 had a cleavage preference for the single basic residue site rather
than a paired basic residue site in the Dyn A 1-17 substrate. In Fig.
4C, we tested the specificity of the cleavage reaction using
the C-terminal (CT) peptide of 7B2. The CT peptide was shown to be a
highly potent (i.e. at nM concentrations) and
specific inhibitor of PC2 activity (41). The CT peptide completely
abolished the cleavage of Dyn A 1-17 by PC2.
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Processing of Dyn A 1-17 Analogs by PC2--
To investigate the
involvement of amino acids surrounding the single Arg cleavage site, we
prepared a series of Dyn A 1-17 synthetic peptide analogs (Fig.
5). Cleavage of each peptide was compared
with the processing of Dyn A 1-17. The conditions were set such that
the measured amount of PC2 activity (based on fluorogenic assay)
cleaved 75% of the Dyn A 1-17 peptide. Substitution of Arg9 by Ala9 resulted in the complete lack of
cleavage, demonstrating the importance of this basic residue and
further confirming the site of PC2 cleavage. It is worth noting that
PC2 was still unable to cleave the paired basic amino acid residues to
produce Leu-Enk-Arg-Arg from [Ala9]-Dyn A 1-17. The
[Lys9]-Dyn A 1-17 analog was inefficiently processed
(1.3%), suggesting that a single Lys residues at the P1
cleavage position produces a poor substrate. The
[Ala6]-Dyn A 1-17 analog was not cleaved by PC2, whereas
efficient cleavage of the [Ala7]-Dyn A 1-17 was
observed, demonstrating that an Arg in P4 is important for
cleavage of Dyn A 1-17, whereas an Arg in P3 is not. These
data indicate that the motif RXXR is critical for the processing of Dyn A 1-17. Because a Pro residue at P1 is
predicted to impede processing, we tested an [Ala10]-Dyn
A 1-17 analog. This peptide was more efficiently cleaved by PC2 than
Dyn A 1-17. Other substitutions, with related Pro analogs, where
Pro10 was replaced with DPro10 or
hydroxy-Pro10 showed reduced cleavage. We also tested the
influence of other basic residues located C-terminal to the cleavage
site by synthesizing an [Ala11]-Dyn A 1-17 analog. This
peptide analog was cleaved more efficiently by PC2, suggesting that the
basic residue (i.e. Lys) at P2
interferes with
PC2 cleavage. Finally, we examined the peptide
des[Tyr1]-Dyn A 1-17 (Dyn A 2-17). The Tyr residue is
known to be conformationally important for interactions of Dyn A 1-17
with opioid receptors; however, the effect of conformation on
processing is unexplored. The data show that
des[Tyr1]-Dyn A 1-17 was well cleaved as compared with
Dyn A 1-17 and that removal of the Tyr residue had only a minor effect
on the ability of PC2 to cleave this peptide.
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Single Basic Cleavage of PC2 in Leumorphin-- As a comparative measure of PC2 processing at single basic residues, we investigated the single basic residue cleavage in leumorphin (Dyn B 1-29). In this peptide there is no Arg residue in the P4 position; however, an Arg is found at P8 (Fig. 6). The proDyn C-peptide was cleaved from recombinant proDyn by PC2, and thus we would also expect cleavage of leumorphin by PC2. Dyn B 1-29 was completely cleaved to yield Dyn B 1-14 (i.e. Arg-extended form of Dyn B 1-13) and Dyn B 15-29 (i.e. the proDyn C-peptide).
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Enhanced Processing in the Presence of Carboxypeptidase-- Addition of CPE appears to result in more efficient processing of proDyn by PC2 (Fig. 3, A and B). Further evidence of the enhancement of PC2 processing produced by CPE is provided using the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-MCA. This substrate was incubated with PC2 with or without the addition of CPE (Fig. 7). The addition of CPE resulted in a 25% increased PC2 processing efficiency.
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CPE Enhancement of PC2 Processing on Dyn AB 1-32-- We further investigated the enhancement effect of CPE on PC2 processing using a more complex peptide that has multiple cleavage sites. Dyn AB 1-32 is an intermediate in the formation of Dyn A 1-17 and Dyn B 1-13. Within this peptide, four potential cleavage sites are found (i.e. two Arg-Arg sites, one Lys-Arg site, and a single Arg) (Fig. 10). The incubation of PC2 and Dyn AB 1-32 resulted in the formation of only Dyn A 1-19 (i.e. Lys-Arg extended) and Dyn B 1-13 (Fig. 10B). We could not detect the formation of either Dyn A 1-9 or Leu-Enk-Arg-Arg. However, when the same experiment was carried out in the presence of CPE (Fig. 10C), enhanced PC2 processing was revealed by the detection of Dyn A 10-17, Leu-Enk, Dyn B 8-13, and Dyn A 1-8.
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Comparative Kinetic Measurements of PC2 Processing-- We compared the Km values of Dyn AB 1-32, Dyn A 1-17, and leumorphin (in the absence of CPE). These values were approximately the same for the three peptides (Dyn AB 1-32, 100 ± 17 µM; Dyn A 1-17, 79 ± 20 µM; and leumorphin 77 ± 19 µM). This suggests that the three peptides bind with equal affinity to PC2. However, we observed a major difference in turnover rates. Using the same cleavage conditions (i.e. enzyme and substrate concentrations) for the three peptides, we measured the time required for PC2 to cleave 50% of each substrate (i.e. t1/2). The values obtained were as follows: leumorphin, t1/2 = 1.25 h; Dyn A 1-17, t1/2 = 13 h; and Dyn AB 1-32, t1/2 = 19 h. Thus, leumorphin was cleaved at least 10 times faster than Dyn A 1-17 and 15 times faster than Dyn AB 1-32.
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DISCUSSION |
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A major finding of the present study is that PC2 can process proDyn both at single and paired basic residues to produce biologically active opioid peptides. Taken together with data demonstrating the neuronal co-expression of PC2 and proDyn, a strong case can be made that PC2 is an important proDyn processing enzyme in the brain.
The distribution of proDyn mRNA, proDyn-derived peptides, and PC2
mRNA has been well studied (1, 3, 20, 34-37). ProDyn has been
shown to be differentially processed in the brain and pituitary (34).
Processing is more complete in the brain, resulting in final opioid
peptide products, whereas only processing intermediates are observed in
the anterior pituitary. A major difference between these two tissues
are the low levels of PC2 in the anterior pituitary (21). We have
previously proposed that PC2 would be critical for the processing of
final proDyn opioid peptide products based on correlations between the
in vivo cellular expression of PC2 and completeness of
proDyn processing (26), where lack of PC2 expression correlated with
lack of proDyn processing. The data of the present study (Fig. 3)
provide direct evidence that PC2 is responsible for the formation of
final opioid active peptide products (Dyn A 1-17, Dyn A 1-8, Dyn B
1-13, and NE), which contrasts with our previous data showing that
PC1/PC3 processed proDyn primarily into 8- and 10-kDa intermediates
(22). To produce these final opioid active peptides, PC2 must cleave
proDyn not only at paired basic residues but also at single basic
residues. Two such sites are found in Dyn A 1-17 and leumorphin,
resulting in the formation of Dyn A 1-8 and the C-peptide,
respectively. The data of previous studies (22) with those presented
here showed that PC1/PC3 could generate the C-peptide (22) but not Dyn
A 1-8. Therefore, PC1/PC3 has a redundant function with PC2 to produce
the C-peptide, but the formation of Dyn A 1-8 is exclusive to PC2.
Since PC1/PC3 can cleave at single basic residues, its inability to
produce Dyn A 1-8 suggested that neighboring amino acids might be
restricting catalysis. It is possible that the Pro residue at the
P1 position was causing an important steric hindrance. Conformational constraints can restrict proteolysis by trypsin and
tryptic-like enzymes, especially when the Pro residue is found following the Arg cleavage site, as is the case with Dyn A 1-17. Indeed, trypsin cleaving C-terminal to single basic residues cannot cleave an Arg
Pro bond. However, when this Arg-Pro motif is preceded by Gln (i.e. Gln-Arg
Pro), the arginyl bond becomes
susceptible again to trypsin action (47, 48). Similarly, based on the results presented using Dyn A 1-17 analogs, we believe that the presence of an Arg in the P4 position permits PC2 to carry
out the required catalysis. Thus, we suggest that the motif
RXXR
in Dyn A 1-17 is recognized by PC2 for cleavage, a
recognition sequence that is largely preferred to the other potential
cleavage site present in the same substrate, namely the
Arg6-Arg7 pair. On the other hand, the presence
of an Arg in the P4 position does not appear to be an
essential requirement for PC2 cleavage at single basic sites because we
also observed the cleavage of leumorphin by PC2 (Fig. 6). In the case
of leumorphin we propose that the Arg in position P8 could
be important. In this case PC2 cleavage would conform with the rules
proposed by Devi (42) for single basic cleavage, which predicts that
basic residues at position P4, P6, or
P8 are important (43).
Although Dyn A 1-17 is cleaved by PC2, the data obtained using peptide
analogs (Fig. 5) provide evidence that both the Pro9 and
Lys11 residues (at the P1 and P2
positions, respectively) contribute restrictions to PC2 cleavage. Thus,
structural constraints and charged residues at the P
positions within
the Dyn A 1-17 substrate are responsible for the incomplete processing
of Dyn A 1-17 into Dyn A 1-8. These data may have some bearing on
what is known about proDyn peptides in vivo. The varying
ratios of Dyn A 1-17:Dyn A 1-8 that are found in different brain
regions (49) could be a function of PC2 enzyme activity. There is
growing evidence that both PC2 levels (21, 50) and activity (39) are
regulated, and thus the Dyn A 1-17:Dyn A 1-8 ratio could be altered
by changes in PC2. Although both Dyn A 1-17 and Dyn A 1-8 are
selective for the
opioid receptor, Dyn A 1-8 is more selective for
the
opioid receptor than Dyn A 1-17. Regarding behavior,
and
receptor are associated with aversive and rewarding behaviors. It
is conceivable that modulation of PC2 activity within proDyn expressing
neurons could have physiological consequences through Dyn A 1-17
processing.
Our data provide evidence for PC2 as the Dyn A 1-8 generating enzyme;
however, an alternative possibility would be that a metalloendopeptidase cleaves Dyn A 1-17 N-terminal to the single Arg site, directly producing Dyn A 1-8 without the need for a subsequent carboxypeptidase (44-46). Although the present data demonstrate the specificity of PC2 to cleave Dyn A 1-17 at the Arg
Pro site (i.e. PC1/PC3 and furin had no effect), it
does not exclude the above hypothesis. However, we have recently
examined brain extracts of mice lacking active PC2 (51) and could not measure any Dyn A 1-8 immunoreactivity, whereas control mice showed normal levels of Dyn A 1-8. These recent observations suggest that no
redundant function exists in the formation of Dyn A 1-8.
The data demonstrating enhanced PC2 processing by carboxypeptidase activity suggest an interesting relationship between the PC family of enzymes and the now growing family of carboxypeptidases (29). The data indicate that the enhanced processing effects are not due to protein-protein interactions (between PC2 and CPE) but that carboxypeptidase activity is important. Evidence supporting this notion includes the fact that GEMSA, a specific inhibitor of carboxypeptidase activity, reversed the enhancement effect. Furthermore distinct proteins with carboxypeptidase activity (i.e. CPE, CPD, CPM, and CPB) produced identical enhancement effects. The enhancement of PC2 processing by carboxypeptidase was not substrate-dependent because it was observed using recombinant proDyn, Dyn A 1-17, Dyn AB 1-32, and a fluorogenic substrate. In regards to the mechanism of PC2 processing enhancement, we propose that C-terminal trimming of single or paired basic residues removes product inhibition. In support of this, our data also showed that the exogenous addition of the product could serve to inhibit the effectiveness of PC2 processing.
We believe our findings showing enhanced processing have a direct relevance to fat/fat mouse model, which has been shown to be deficient in CPE activity (52). While examining this animal model for basic residue extended peptides, several investigators noted that processing of protein precursors (i.e. proDyn and pro-insulin) was diminished in the fat/fat mouse (53, 54), an effect that implicates reduced processing by endoproteases such as the PCs. Based on the present data, it is likely that accumulation of basic residue extended peptides within the fat/fat mouse may cause a product inhibition of enzymes such as PC2. The data presented (Fig. 10B) show that products such as Dyn A 1-8, Dyn A 10-17, or even Leu-Enk cannot be generated from Dyn AB 1-32, even after long incubation periods, unless CPE is present (Fig. 10C). The presence of Dyn A 1-19 (i.e. Lys-Arg extended) is most likely restricting further processing of Dyn AB 1-32. Within the concentrated environment of the immature secretory granule, product inhibition could represent a significant factor in the overall processing of protein precursors.
Finally, the occurrence of the differential processing of protein precursors in distinct tissues is well established. Various mechanisms are possible, including the selective cellular expression of different enzymes responsible for the cleavage of the protein precursor substrate. The present data suggest that the activity levels of a single enzyme could also be important. In the case of proDyn, all the final opioid peptide products can be obtained with PC2. However, because there are structural constraints (for example in the Arg-Pro bond of Dyn A 1-17), changes in PC2 activity could be a mechanism used to select alternate processing pathways and ultimately leading to differential cellular biological output. Further studies examining precursor-product relationships are necessary to elucidate the mechanisms of processing by PC2, as well as further studies into the influence of carboxypeptidases on PC processing.
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ACKNOWLEDGEMENTS |
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We thank Xue Wen Yuan for excellent technical
assistance and Dany Gauthier for carrying out the amino acid
compositions. We also thank Dr. Lloyd Fricker (Albert Einstein College
of Medicine) for the purified carboxypeptidase enzymes (CPE, CPD, and
CPM) and for helpful discussions. We thank Dr. Huda Akil (University of
Michigan) for the proDyn peptide antisera against Dyn A 1-17, Dyn B
1-13, NE, and C-peptide and Dr. Donald F. Steiner (Howard Hughes
Medical Institute, Chicago, IL) for providing the brain tissues of
PC2-null mice. The rat proDyn cDNA was obtained from Dr. Jim
Douglass (Amgen, Thousand Oaks, CA).
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FOOTNOTES |
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* This work was supported by Medical Research Council Grants MT-13196 and ME-13678 (to R. D.) and PG-11474 (to C. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Scholar of the Fonds de la Recherche en Santé du Quebec. To whom correspondence should be addressed: Dept. of Pharmacology, Faculty of Medicine, University of Sherbrooke, 3001 12ième Ave. Nord, Sherbrooke, Quebec J1H 5N4, Canada. Tel.: 819-564-5428; Fax: 819-564-5400; E-mail: rday{at}courrier.usherb.ca.
1
The abbreviations used are: proDyn,
prodynorphin; Leu-Enk, leucine-enkephalin; Dyn, dynorphin; NE,
-neo-endorphin; PC, proprotein convertase; HPLC, high pressure
liquid chromatography; CP, carboxypeptidase; RIA, radioimmunoassay;
dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; GEMSA,
guanidinoethylmercaptosuccinic acid; PACE, paired amino acid cleaving
enzyme; CT, C-terminal; MCA, aminomethylcoumarin.
2 R. Day and W. Dong, unpublished observation.
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REFERENCES |
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