(Received for publication, June 14, 1995; and in revised form, July 27, 1995)
From the
Dynorphin B (Dyn B-13, also known as rimorphin) is generated
from Dyn B-29 (leumorphin) by the cleavage at a single Arg residue. An
enzymatic activity capable of processing at this monobasic site has
been previously reported in neurosecretory vesicles of the bovine
pituitary and pituitary-derived cell lines. This enzyme termed
``the dynorphin-converting enzyme'' (DCE) has been purified
to apparent homogeneity from the neurointermediate lobe of the bovine
pituitary using hydrophobic chromatography on phenyl-Sepharose,
preparative isoelectrofocusing in a granulated gel between pH 4 to 6.5,
and non-denaturing electrophoresis on 5% polyacrylamide gel. DCE
exhibits a pI of about 5.1 and a molecular mass of about 54 kDa under
reducing conditions. DCE is a metallopeptidase and exhibits a neutral
pH optimum. Specific Inhibitors of soluble metallopeptidases such as
enkephalinase (EC 3.4.24.11) or enkephalin generating neutral
endopeptidase (EC 3.4.24.15) do not inhibit DCE activity indicating
that DCE is distinct from these two enzymes. Cleavage site
determination with matrix-assisted laser desorption ionization time of
flight (MALDITOF) mass spectrometry shows that DCE cleaves the Dyn B-29
N terminus to the Arg generating Dyn B-13 and Dyn
B-(14-29). Among other peptides derived from Dyn B-29, DCE
cleaves only those peptides that fit the predicted ``consensus
motif'' for monobasic processing. These data are consistent with a
broader role for the dynorphin converting enzyme in the biosynthesis of
many peptide hormones and neuropeptides by processing at monobasic
sites.
The majority of the neuropeptides are synthesized as larger precursors that undergo endoproteolysis at specific sites(1) . These sites are usually multiple basic amino acids (2, 3, 4) although some cleavage sites are single basic (``monobasic'') residues that usually fit a consensus sequence(5) .
The dynorphin precursor, Prodyn,
contains both dibasic and monobasic cleavage sites; cleavage at these
sites generates a variety of potent opioid peptides. The cleavage at
dibasic sites gives rise to - and
-neo-endorphin, dynorphin
(Dyn) A-17, Dyn B-29, and leucine
-enkephalin (Leu-Enk). The
formation of Dyn B-13 from Dyn B-29 and Dyn A-8 from Dyn A-17 requires
cleavage at monobasic sites. Several neuropeptide-processing enzymes
have been identified in mammalian
cells(2, 3, 4, 5, 6) .
Endoproteases such as furin, PC1, and PC2 are thought to preferentially
cleave peptide precursors at multibasic residue cleavage
sites(4, 7) . An endoprotease designated
``dynorphin converting enzyme'' (DCE) (
)cleaves
neuropeptides at monobasic sites(5, 8) . Following
endopeptidase activity, carboxypeptidase E (also known as
carboxypeptidase H) removes the basic amino acids from the C terminus
of the peptides(9) .
DCE is a peptide processing enzyme that
converts Dyn B-29 ()into Dyn B-13 by cleavage at single
basic residue Arg
(5, 6) . In the brain
and pituitary, the distribution of the DCE activity generally matches
that of Dyn B-13 (10, 11) . These data suggest that
DCE is physiologically involved in the processing of dynorphin peptides
at single arginine cleavage sites.
In the bovine pituitary, DCE activity is enriched in the highly purified secretory vesicles and co-sediments with Leu-Enk and carboxypeptidase E(12) . The DCE activity is also found in the regulated pathway of secretion in a number of cell lines(13, 14) . The presence of DCE activity in structurally similar (12) and functionally similar (13, 14, 15, 16) neuropeptide-containing secretory granules is also consistent with a function in opioid peptide processing since it has been shown that secretory granules and trans Golgi network are the sites of peptide processing(17) .
In
this study we used bovine neurointermediate lobe (NIL) of the pituitary
to purify DCE to apparent homogeneity. Using hydrophobic
chromatography, preparative isoelectrofocusing, and non-denaturing
electrophoresis, we have purified DCE approximately 20,000-fold. We
find that the purified DCE is a neutral metallopeptidase with a
molecular mass of 54 kDa under reducing conditions. DCE cleaves Dyn
B-29 N-terminal to the Arg directly generating Dyn B-13.
These data support the premise that DCE is a monobasic processing
endoprotease involved in the generation of Dyn B-13.
Antiserum ``17S'' was used for the detection of Dyn B-13. It is a highly selective and sensitive antiserum(8) . Radioimmunoassay with this antiserum allows the detection of picomolar concentrations of Dyn B-13 in the presence of nanomolar concentrations of Dyn B-29.
For preparative IEF, a gel slurry was prepared by mixing 4 g of
Sephadex G-75 IEF-grade (Sigma) with 95 ml of 10% glycerol (v/v), 2.5
ml of ampholyte (Pharmacia) at pH 4.0-6.5 and 1 mM dithiothreitol. The slurry was poured onto a glass plate (24
11 cm) and dried with a light stream of cool air until the
weight decreased by 35%. The plate was placed on the cooling unit of an
LKB 2117 Multiphor platform. Electrode strips were soaked in either 40
mM glutamic acid (anode) or 200 mM histidine
(cathode) and placed at the end of the IEF plate. The plate was
prefocused for 2 h at 8 watts/plate at 4 °C. The
concentrate-containing peak of DCE activity from the phenyl-Sepharose
column was applied in the pH 6.0 region of the gel and focused for 8 h
at 8 watts/plate at 2-4 °C. A stable pH gradient was
established during this time. The resin was then sectioned into 40
fractions (0.5 cm wide) and each fraction eluted with 10 volumes of 0.1 M sodium phosphate buffer, pH 7.5, containing 10% glycerol.
DCE activity was assayed in fractions as described above. Protein
estimation was using BCA reagent (Pierce). In addition, we estimated
protein concentration using the silver-stained gel following
electrophoresis of the fractions containing the peak of activity. The
pH gradient was determined by extracting a small segment of each
fraction with deionized water and then directly measuring the pH.
The preparative IEF chromatography procedure alone gives another
80-100-fold purification and DCE runs with a pI of about 5.1. The
DCE is extracted from the gel, concentrated on a Centriprep 100
(Amicon) concentrator, and the protein subjected to non-denaturing
electrophoresis(18) . For this, 5% polyacrylamide gels were
used and the samples were not denatured by boiling and SDS or
-mercaptoethanol were excluded during sample preparation. 0.5
mM thioglycollate was prerun to remove ammonium persulfate and
other free radical reaction products generated in the gel
system(19) . The electrophoresis was carried out at 4 °C.
Following electrophoresis, the activity in 0.5-mm sections was
extracted with 50 mM sodium phosphate buffer, pH 7.5,
containing 0.1 mM dithiothreitol and 0.1% Triton X-100. The
DCE activity in each fraction was determined as described above. The
peak of DCE activity corresponded to a protein band around 180 kDa as
visualized by silver staining of the gel (data not shown). This is
interesting because DCE activity by gel-exclusion chromatography also
exhibited a molecular mass of about 180 kDa(12) .
The peak of activity was pooled, concentrated on Centricon-100 (Amicon), and subjected to electrophoresis under denaturing conditions and visualized by silver-staining.
To determine the cleavage site specificity of DCE in Dyn B-29 and in Dyn B-29-derived peptides (shown in Table 6), 1 nmol of peptide was incubated with 100 ng of purified enzyme in 50 mM sodium phosphate buffer, pH 7.5, containing 0.1% Triton X-100 for 20 min, 1 or 18 h at 37 °C. The reaction was terminated by incubation at 100 °C for 10 min. The reaction mixture was subjected to MALDITOF-mass spectrometry (Dr. Ronald Beavis, Skirball Institute, NYU Medical Center, New York).
The intramolecularly quenched fluorescent peptides were synthesized as described previously(20) ; these peptides have ortho-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine as a donor-acceptor pair at the N and C termini of the peptides, respectively. N-(2,4-Dinitrophenyl)ethylenediamine was attached to glutamine in all peptides, a necessary result of the solid-phase peptide synthesis strategy employed. An automated bench-top simultaneous multiple solid-phase peptide synthesizer (PSSM 8 system from Shimadzu) was used for the solid-phase synthesis of all the peptides by the Fmoc procedure.
Figure 1: Purification of DCE activity from bovine pituitary soluble fraction. A, hydrophobic chromatography on phenyl-Sepharose. DCE activity (hatched bars) is expressed as Rel. Units are in nanomoles of immunoreactive-Dyn B-13 formed/min/200 µl. Protein (solid line) is in mg/200 µl; protein estimation was using Bradford reagent (Bio-Rad). The ammonium sulfate gradient is in dashed lines. B, preparative isoelectrofocusing. DCE activity (hatched bars) expressed as Rel. Activity is in nanomoles of irradiated-Dyn B-13 formed/min/fraction. Protein (solid line) relative units (mg/ml); protein estimation was using BCA reagent. pH gradient (dotted line) was determined by extracting a small segment of each fraction with deionized water and then directly measuring the pH.
The preparative IEF on a granulated gel between pH
4-6.5 gives another 70-fold purification and about a 10-20%
yield (Table 1). DCE runs with a pI of about 5.1 (Fig. 1B). At this point only three major protein bands
can be visualized in the fraction containing peak of DCE activity (Fig. 2). The non-denaturing electrophoresis in 5% gel results
in approximately 4-6-fold purification and results in the
homogeneous purification of DCE. DCE runs as a 180-kDa protein under
these non-denaturing electrophoresis conditions (data not shown), and
when the peak of DCE activity is subjected to denaturing
electrophoresis followed by silver-staining, a single band of 54 kDa is
detected (Fig. 2). Increasing the reducing conditions by boiling
in 2% SDS, 5% -mecaptoethanol, treatment with iodoacetamide, or
treatment with guanidine hydrochloride (22) do not result in
further changes in the size of this protein (data not shown).
Figure 2: Silver-stained gel following denaturing electrophoresis using SDS. Fractions containing DCE activity were subjected to SDS-polyacrylamide gel electrophoresis under denaturing conditions in 7.5% gel. The numbers on top correspond to the various steps in purification: M, molecular weight markers (Sigma); 1, homogenate; 2, ammonium sulfate supernatant; 3, phenyl-Sepharose eluate; 4, isoelectrofocusing eluate; 5, non-denaturing gel eluate. Arrows point to DCE seen as a protein band at about 54 kDa. The numbers on the left and right margins represent molecular masses in kDa.
Figure 3: pH profile for DCE activity. The reaction was carried out as described except buffers were 50 mM sodium citrate (open squares), 50 mM sodium phosphate (closed circles), and 50 mM Tris-Cl (open circles) at indicated pH values. Data represents means of triplicate values. Relative activity is in nanomoles of immunoreactive-Dyn B-13 formed/h/µg. The experiment was carried out twice with less than 10% variation between experiments.
Among the chelating agents the
enzyme is more sensitive to 1,10-phenanthroline (Table 3). 1
mM 1,10-phenanthroline completely inhibits the activity
whereas 5 mM EDTA or EGTA cause only a 73-78% inhibition
of activity. 5 mM CDTA causes only a 30% inhibition of
activity. In order to see if the DCE activity is modulatable by
cations, we examined the effect of divalent cations on DCE activity (Table 4). CaCl, MgCl
, or MnCl
do not show appreciable activation of DCE; ZnCl
and
CoCl
, in contrast, cause substantial inhibition of activity
at 1 or 5 mM, respectively. Inhibition by these heavy metal
ions could be due to the ability of these metal ions to bind free SH
groups in the cysteine residue. These data suggest that DCE is a
thiol-sensitive metalloprotease.
Other well characterized thiol-sensitive metallopeptidases include soluble neutral endopeptidases EC 3.4.24.15(23) , EC 3.4.24.16(24) , EC 3.4.24.11(25) , N-arginine dibasic convertase(26) , and amidorphin Gly-generating enzyme(27) . We used specific peptide inhibitors of EC 3.4.24.15, EC 3.4.24.16, or EC 3.4.24.11 and potent inhibitors of N-arginine dibasic convertase or amidorphin Gly-generating enzyme in order to examine their effects on DCE activity. DCE activity is not inhibited by these inhibitors at 100 µM (Table 5). In addition the size, pH profile, and the protease inhibitor profiles for these enzymes are distinct from that of DCE (Table 5). These data suggest that DCE is distinct from these other neutral metallopeptidases.
DCE is fairly stable at
23 and 37 °C and retains essentially 100% activity during 30 min of
incubation at these temperatures; however, a 30-min incubation at 45
°C causes 60% loss of activity. DCE is also differentially
sensitive to organic solvents. 1% of ethanol or isopropanol causes up
to 30% of inactivation, whereas, 5% MeSO causes no
inactivation. Approximately a 20% loss of activity is detected only
when Me
SO is increased to 10% (data not shown).
The
cleavage site specificity of DCE was determined using MALDITOF mass
spectrometry. Dyn B-29 alone was detected as a single peak with a mass
ion of about 3528 (Fig. 4). Upon incubation with the purified
DCE for 1 h at 37 °C, two additional peaks of mass ions 1570 and
1975 are detected; this mass is identical to the predicted mass of Dyn
B-13 and Dyn B-29-(14-29), respectively (Fig. 4). Longer
incubation (18 h) of enzyme with the substrate results in substantial
conversion of the substrate to only two products with masses identical
to Dyn B-13 and Dyn B-29-(14-29) (data not shown). These data
suggest that DCE cleaves Dyn B-29 N-terminal to Arg. DCE
displayed typical Michaelis-Menten kinetics with an apparent K
for the substrate of 0.2 µM and an
apparent V
of about 2-8 µmol/min/µg (Fig. 5).
Figure 4:
Cleavage site determination using MALDITOF
mass spectrometry. Purified DCE (1 ng) was incubated with 1 nmol of Dyn
B-29 in 50 mM Tris-Cl buffer, pH 7.5, containing 0.1% Triton
X-100, for 1 h at 37 °C. The reaction was terminated by boiling for
10 min. Reaction mixture with boiled enzyme was used as control. The
samples were subjected to MALDITOF mass spectrometry. Top
panel, Dyn B-29 with buffer; bottom panel, Dyn B-29 with
DCE. MALDI-MS, m/z (M+H) for Dyn B-29 is
3528 (formula weight is 3527), for Dyn B-13 is 1571 (formula weight is
1569), and for Dyn B-29-(14-29) is 1975 (formula weight is
1974).
Figure 5:
Kinetic determination using Dyn B-29 as
substrate. Purified DCE (1 ng) was assyed with Dyn B-29 as substrate as
described under ``Materials and Methods'' except that Dyn
B-29-(9-22) was excluded from the reaction mixture. Kinetic
constants were determined from linear regression analysis of plots of
the inverse of the reaction veocity versus the inverse of the
substrate concentration (Lineweaver-Burke plot). The experiment was
performed twice in triplicate, with less than 10% variation in the K and V
values.
In order to test the hypothesis that DCE recognizes
peptides that fit the consensus for monobasic processing, we
synthesized a number of peptides with substitutions at sites predicted
to be involved in the recognition by the enzyme. The peptide
representing Dyn B-29-(6-16) was efficiently cleaved by DCE. By
MALDITOF mass spectrometry the site of cleavage was determined to be
N-terminal to the Arg (Table 6). The substitutions of
the Arg
to a Gly or Nle resulted in the absence of
cleavage of the peptide as determined by MALDITOF spectrometry (Table 6). Similarly, substitution of the Ser
to Val or Phe and Thr
to Tyr resulted in
absence of processing. These results suggest that the residues around
the cleavage site play an important role in the recognition of Dyn B-29
by DCE.
In the present study we have purified the monobasic processing endopeptidase designated DCE from NIL to apparent homogeneity. Using only a few steps including preparative isoelectrofocusing, the enzyme was purified 20,000-fold. The fairly high yield and the quick purification protocol has made possible studies characterizing the enzyme. The limited amount of the enzyme in the NIL is consistent with a selective role for the enzyme in neuropeptide biosynthesis. The evidence for such a role derives from our earlier finding that within NIL the enzyme appears to be associated with neurosecretory vesicles where it is associated with Dyn B-13(12) . Also, in the brain and in peripheral tissue, the distribution of DCE shows about 10-fold variation, which has similarities to the distribution of neuropeptides (11) . DCE is distinct from any of the previously described neutral metallopeptidases(23, 24, 25, 26, 27, 28) . Unlike the other metallopeptidases, DCE is extremely sensitive to PCMBS being inhibited completely 5 µM. Additionally, selective inhibitors that inhibit other neutral metallopeptidases do not have any effect on the DCE activity (Table 5).
The molecular mass of the purified enzyme under denaturing conditions is determined to be 54 kDa. Under non-denaturing conditions and upon gel-filtration chromatography, the activity exhibits a molecular mass of 180 kDa (this report and 12). It is possible that the enzyme activity exists in vivo as an oligomer resulting in a larger apparent size; this larger size also could be due to anomolous migration of the enzyme during gel-filtration and in non-denaturing gels.
DCE is a monobasic
processing enzyme that processes Dyn B-29 at a single Arg residue.
Other dynorphin processing enzymes have been shown to predominantly
process dynorphins at dibasic processing
sites(29, 30) . DCE is unique in that the enzyme
processes Dyn B-29 N-terminal to the Arg. We had previously found that
the 2800-fold purified DCE from bovine anterior pituitary cleaved Dyn
B-29 N-terminal and to the C-terminal of the
Arg(12) ; we have not observed such a cleavage in
the present study. This could be either due to the absence of another
enzyme that was copurified with DCE from the anterior pituitary or due
to regional variations in the properties of DCE. The latter possibility
is supported by the finding that some of the properties of anterior
pituitary DCE are similar but not identical to the NIL DCE; for
example, NIL DCE is inhibited by EDTA whereas the anterior pituitary or
DCE was not(12) .
It is interesting to note that metalloproteases have been predominantly found to cleave peptide hormone precursors N-terminal to basic sites (26, 27, 28) . We find that DCE cleaves Dyn B-29 at a monobasic cleavage site, Thr-Arg (this report). The adrenorphin Gly-generating enzyme cleaves bovine adrenal peptide-12P at a monobasic cleavage site, Gly-Arg(27) . The NRD convertase cleaves prosomatostatin at a dibasic cleavage site, Glu-Arg-Lys(26) . The cleavage site selectivity (N-terminal to an Arg) of these neutral metalloproteases suggest that these enzymes are related and belong to a distinct class of peptide-processing enzymes.
Monobasic enzymes are widely varied including,
cholecystokinin-8-processing serine protease(31) ,
prosomatostatin-processing serine protease(32) ,
somatostatin-28-converting aspartyl protease(33) , atrial
natriuretic factor-processing serine protease(34) , and
proenkephalin-processing thiol protease(35) . DCE is distinct
from these enzymes and from the prohormone-processing serine proteases
(furin and PC1) that have the ability to cleave precursors at monobasic
sites(36, 37, 38, 39) . This is
supported by inhibitor studies and the studies with cell lines that
have DCE activity. AtT-20 cells have high levels of PC1
mRNA(40, 41) , and BRL-3A cells have no detectable PC1
or PC2 mRNA(15) . The relative levels of furin mRNA and DCE
activity are different in these cells. For example, the level of furin
mRNA is much higher in BRL-3A cells than in AtT-20 cells, whereas DCE
activity shows the opposite distribution. ()Furin mRNA is
expressed at similar levels in brain, intestine, lung, and
kidney(42) , whereas DCE activity is 10-20-fold lower in
lung and kidney as compared with brain and intestine(11) . The
subcellular distribution of these two enzymes is also different.
Whereas furin has been localized to the Golgi
apparatus(43, 44) , DCE activity is enriched in the
secretory vesicle fraction of bovine pituitary glands (12) and
in neuroendocrine cell lines(13, 14) .
The unusual specificity of DCE had previously led us to propose that this enzyme recognizes secondary structure around the cleavage site rather than the primary structure at the cleavage site(8) . By comparing sequences that are processed at monobasic cleavage sites, we proposed that a compositional motif (or consensus) could govern the secondary structure around the cleavage site, and such a motif is recognized by DCE and DCE-like enzymes(45) . The consensus for recognition by the monobasic processing enzyme is hypothesized to require basic residues at -3, -5, or -7 position, not tolerate an aliphatic residue at +1 position, or an aromatic residue at -1 position. It is exciting to see that DCE recognizes peptides that fit only the consensus motif and does not recognize peptides with amino acid substitutions at sites that are predicted to be necessary for recognition. This makes DCE an ideal candidate for the enzyme involved not only in the generation of Dyn B-13 but also in the processing of other precursors that require cleavage at monobasic sites such as growth factors, neuropeptides, and other peptide hormones.