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INTRODUCTION |
A wide variety of proteins in neuronal, endocrine, and immune
tissues undergo proteolytic processing. Many of these proteins and
peptides are intercellular messengers. Most neuroendocrine peptides are
synthesized from precursor proteins. Post-translational processing of
these precursors is a key step in the production of biologically active
peptides. In the majority of cases, this occurs by proteolysis of the
precursors at classical cleavage sites. These sites are usually
multiple basic amino acids (1). Several neuropeptide-processing enzymes
have been identified in mammalian cells (2-5). Initially the precursor
is cleaved by endoproteases of the subtilisin family of serine
proteases, such as prohormone convertases
(PCs)1 (for review, see Refs.
2 and 4). Following endopeptidase activity, carboxypeptidases such as
carboxypeptidase E (CPE, also called CPH) remove the basic amino acids
from the C termini of peptides (6). The peptides with C-terminal
extended Gly residues are processed into C-terminal amidated peptides
by peptidylglycine
-amidating monooxygenase (7). All of these
enzymes involved in the generation of regulatory peptides exhibit a
restricted neuroendocrine distribution as well as subcellular
localization to peptides containing secretory vesicles; furthermore,
they are optimally active at pH 5-6 (2-8). These properties are
consistent with the involvement of these enzymes in the generation of a
number of peptide hormones, neuropeptides, and other peptide
neurotransmitters (8, 9).
A subset of bioactive peptides is generated by processing at
nonclassical sites. These have been identified primarily by bulk purification from neuroendocrine tissues (10-12). Additionally, an
examination of cleavage sites within the precursors for endogenous peptides showed that nonclassical processing is required to release the
peptide from its precursor (13-15). Finally, mass spectrometric techniques to identify neuropeptides in the brains of mice lacking specific processing enzymes such as CPE have led to the identification of products of nonclassical cleavages (16).
Members of the metalloprotease family have been largely implicated in
the processing of bioactive peptides at nonclassical sites (17). The
majority of these proteases exhibit near neutral pH optima and a
cellular and subcellular localization that is not consistent with a
role for these enzymes in neuroendocrine peptide processing within the
intracellular milieu (18). Among them ECE-1 exhibits a neutral pH
optimum, a broad tissue distribution, and predominant cell surface
expression (19, 20). ECE-1 converts big endothelin (ET) to endothelin
through cleavage at a Trp-Val (endothelin-1 and -2) or a Trp-Ile
(endothelin-3) site (19). ECE-2 was discovered in 1995 (21) as a novel
member of the ECE-1 gene family; the two gene products share 59% amino
acid identity (18). In contrast to ECE-1, ECE-2 is optimally active at
pH 5.5 and localized to an intracellular compartment (21-24). A recent study examining the isoforms of ECE-2 has found that the ECE-2b isoform
is highly expressed in neuroendocrine tissues (brain, pituitary, and
adrenal medulla) and poorly expressed in other tissues (25). These
properties make ECE-2b an ideal candidate for intracellular processing
of neuroendocrine precursors. Despite this, relatively little has been
reported about the enzymatic properties of ECE-2b.
Here we describe studies characterizing the biochemical and kinetic
properties of ECE-2. Because this enzyme is a transmembrane protein,
purification of this protein in sufficient quantities to allow
comprehensive studies could be labor intensive and time consuming. This
problem has been overcome in the case of many Zn2+
metalloproteases, including ECE-1, through the expression of soluble
secreted enzymes (26, 27). This has enabled abundant expression and
rapid purification of these enzymes (26, 27). The resulting enzyme has
been found to exhibit virtually identical biochemical properties as
that of the endogenous enzyme (26, 27). We used a similar strategy to
produce and purify the soluble catalytic portion of ECE-2 that is
common to all isoforms (ECE-2a1, -2a2, -2b1, and -2b2). In this study,
we show that purified ECE-2 exhibits an acidic pH optimum and a unique
cleavage site selectivity. Furthermore, we show that ECE-2 is able to
generate biologically active peptides from known precursors. These
results support a role for ECE-2 in the nonclassical processing of
regulatory peptides.
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EXPERIMENTAL PROCEDURES |
Materials--
Big endothelin-1 (human, 1-38), substance P,
neurotensin, [Arg8]vasopressin, joining peptide
(bovine),
-endorphin (rat), dynorphin B (Dyn B), Dyn A, Dyn A-8,
bovine adrenal medulla (BAM) peptide E, BAM 22, and luteinizing
hormone-releasing hormone were purchased from Peninsula Laboratories,
Inc. (San Carlos, CA). Adrenocorticotropic hormone (ACTH, rat),
bradykinin, and BAM 18 were obtained from Phoenix Pharmaceuticals, Inc.
(Belmont, CA), and angiotensin I, II, and III were acquired from Sigma.
Little PEN-LEN was synthesized by Invitrogen, internally quenched
fluorescent substrate McaBk2 ((7-methoxycoumarin-4-yl)acetyl-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys-(2,4-dinitrophenyl)) was custom synthesized by Sigma-Genosys, and all other peptides were
synthesized at the peptide synthesis facility at Albert Einstein College of Medicine. DEAE-Sepharose fast flow was obtained from Amersham Biosciences AB (Uppsala, Sweden), TalonTM
metal affinity resin was purchased from Clontech
(Palo Alto, CA), and the C18 column reverse-phase column
was acquired from Vydac (Hesperia, CA).
Expression and Purification of Soluble ECE-2--
To generate a
secreted soluble form of ECE-2, the N-terminal transmembrane domain was
removed and replaced with the signal peptide from rat CPE corresponding
to amino acids 1-42. Following the CPE cDNA, we inserted an
oligonucleotide encoding six His residues followed by human ECE-2
cDNA (corresponding to amino acids 81-766). The DNA fragment was
cloned into the baculovirus expression vector pVL1392, and recombinant
virus expression was carried out as described previously (28).
Sf9 insect cells growing in Sf900-II serum-free medium
(Invitrogen) were co-transfected with recombinant ECE-2 cDNA in
pVL1392 and BaculoGold viral DNA (Pharmingen) using the calcium
phosphate procedure. After culturing for 5 days, 100 µl of
medium were used to infect 106 Sf9 cells in a
25-cm2 flask. Then 1 ml of the medium from this second
infection was used to infect 108 Sf9 cells in 50 ml
of medium growing in a shaker flask. After 3 days of infection,
the medium was analyzed for the expression of enzyme activity. The
medium was collected by centrifugation at 20,000 × g
for 60 min, and the resulting supernatant was frozen at
80 °C
until use.
Approximately 300 ml of medium from 6 × 108
Sf9 cells infected with ECE-2-expressing baculovirus was thawed
and centrifuged at 5000 × g for 30 min, and the
supernatant was filtered through a 0.2-µm filter. All subsequent
operations were at 0-4 °C unless otherwise noted. The enzyme was
concentrated by passing the medium through Amicon ultrafiltration
membrane YM100 (Millipore Corp., Bedford, MA). For purification, the
concentrated material was loaded onto a DEAE-Sepharose fast flow anion
exchange column in 20 mM Tris-Cl, pH 7.1 (Buffer A). The
column was washed with 10 column volumes of Buffer A and eluted with a
linear gradient of 0-0.5 M NaCl in Buffer A. The fractions
were assayed for ECE-2 activity as described below and for protein
levels using BCA reagent (Pierce). The peak of ECE-2 activity eluted
around 0.25 M NaCl. Fractions containing the highest
activity were pooled and loaded onto a TalonTM-Sepharose
Co2+ affinity resin column equilibrated with Tris-Cl
buffer, pH 7.1, containing 300 mM NaCl. Previously we have
found the Ni2+ affinity resin to be unsuitable for
purification of ECE-2. The enzyme bound to Co2+ resin was
washed with the sodium acetate buffer, pH 6.0, containing 300 mM NaCl and was eluted with the same buffer adjusted to pH 5.0. Fractions containing ECE-2 activity were subjected to SDS-PAGE and
visualized by silver staining.
Assay for ECE-2 Activity--
McaBk2 was dissolved in 100%
Me2SO, and the concentration of the peptide was determined
spectrophotometrically with an extinction coefficient of 14,000 M
1 cm
1. ECE-2 activity
routinely was assayed with 10 µM McaBk2 in 0.2 M sodium acetate buffer, pH 5.5, containing 0.01%
detergent C12E8 (octaethylene glycol dodecyl
ether, Calbiochem) unless indicated otherwise. For pH dependence
studies, sodium citrate, Tris acetate, or sodium acetate buffers were
used. Substrate hydrolysis was monitored on a Fluoromax plate reader
with excitation at 320 nm and emission at 405 nm, and initial velocity
was determined.
Western Blotting--
Polyclonal antisera were custom generated
against the C-terminal 16 amino acids of ECE-2; this sequence differs
from the sequence of the C terminus of ECE-1. The fractions containing
ECE-2 activity were analyzed by Western blotting as described (29)
using a 1:1000 dilution of antiserum. This antiserum is able to
recognize the ~100-kDa truncated ECE-2 secreted from baculovirus
cells (Fig. 1B) as well as the 120-kDa form of
endogenous ECE-2 (data not shown). The signal was blocked
completely by the immunogenic peptide, suggesting that the antiserum is
specific to ECE-2.
Identification of Peptide Hydrolysis Products by Mass
Spectrometry--
Approximately 1 nmol of each peptide listed in Table
II was incubated with purified ECE-2 in 0.2 M sodium
acetate, pH 5.5, at 37 °C. The reaction was carried out for various
time periods and terminated by quick freezing. Peptides from samples
were isolated using Zip-Tip hydrophobic chromatography columns
and analyzed by matrix-assisted laser desorption ionization
time-of-flight (MALDI-TOF) mass spectrometry on a Perceptive Biosystems
Voyager-DE STR mass spectrometer as described (16, 30).
Approximately 100 laser shots were summed per spectrum.
-Cyano-4-hydroxycinnamic acid saturated in 30% acetonitrile and
0.1% trifluoroacetic acid in water was used as a matrix. External
calibration was performed with [des-Arg1]bradykinin ((M + H)+ = 904.4681) and neurotensin ((M + H)+ = 1672.9170).
Determination of Kinetic Constant for ECE-2 Hydrolysis of
Different Peptides--
Approximately 0.2-100 µM
peptides were incubated with 0.5-3 nM purified ECE-2 in
0.2 M sodium acetate buffer, pH 5.5, containing 0.01%
C12E8 (v/v) at 37 °C. Reactions were carried
out for various times and quenched by the addition of trifluoroacetic
acid of 0.1%. Prior to injection, the trifluoroacetic acid and
acetonitrile concentrations of the sample were adjusted to 0.05 and
15%, respectively. The samples were loaded onto a C18
column (0.46 × 15 cm) and eluted using a linear gradient of
15-65% acetonitrile in 0.05% trifluoroacetic acid. The peptides were
detected by measuring absorbance at 215 nm. The initial rate of
substrate hydrolysis (V0) was determined by
measuring the appearance of product under initial rate conditions (less
then 10% substrate hydrolysis). V0 values were
plotted as a function of substrate concentration ([S]) and fit to the
Michaelis-Menten equation using Prism (version 2.0) software.
kcat values were calculated using the equation
kcat = Vmax/[E] with a subunit molar mass
of 100 kDa. Identity of peptide peaks was confirmed by MALDI-TOF mass
spectrometry as described (16, 30).
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RESULTS |
Eukaryotic Expression and Purification of ECE-2--
To facilitate
the biochemical characterization, we expressed ECE-2 as a soluble
secreted protein in a eukaryotic expression system. For this, the
transmembrane domain (including the N-terminal region) was removed from
ECE-2b to enable expression as a soluble enzyme and replaced with the
signal sequence and proregion of a secretory vesicle protein, CPE, to
enable targeting of the recombinant enzyme to the secretory pathway.
Thus the cDNA fragment encoding the entire luminal domain (that
contains the catalytic region) of ECE-2b was fused in frame with the
cDNA encoding the signal sequence and proregion of CPE; the
resulting sequence consisted of residues 1-42 of CPE, six His
residues, and residues 81-766 of ECE-2b. The secreted medium
from cells infected with recombinant virus was subjected to
purification using anion exchange and metal ion affinity
chromatography. For the determination of ECE-2 activity, the
intramolecularly quenched fluorescent peptide substrate McaBk2 was
used (31). This reagent originally was developed as a substrate for
ECE-1 and has 7-methoxycoumarin-4-yl and dinitrophenyl as the donor and
acceptor at the N and C termini, respectively. The substrate is readily
hydrolyzable by ECE-2; this simple and fast assay proved to be ideal
for the rapid purification and characterization of ECE-2.
ECE-2 was purified ~100-fold from the secreted medium (Table
I). The level of purification was judged
by SDS-PAGE and visualized by silver staining (Fig.
1A). Western blotting analysis
with a polyclonal antiserum generated to the C-terminal 16-residue
peptide shows enrichment of the ~100-kDa band representing soluble
recombinant ECE-2 (Fig. 1B). The purified ECE-2 exhibits an
apparent Km of about 9 µM for the
McaBk2 substrate.

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Fig. 1.
Purification of ECE-2 activity from the media
of Sf9 cells overexpressing ECE-2. A, fractions
containing ECE-2 activity were subjected to purification by ion
exchange and metal ion chromatography as described under
"Experimental Procedures." The resulting fractions were subjected
to electrophoresis under denaturing conditions on an 8% polyacrylamide
gel. B, Western blot analysis of ECE-2 using a polyclonal
antiserum directed against the C-terminal region of ECE-2. The
positions of protein standards (in kDa) are indicated.
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Characterization of ECE-2 Activity: pH Optimum and Divalent Cation
Sensitivity--
We characterized the purified ECE-2 activity by
examining its pH optimum and sensitivity to divalent cations. Previous
studies with crude ECE-2 (membranes from cells expressing recombinant ECE-2) showed that the enzyme was optimally active at pH 5.5 (21). Our
results with purified ECE-2 agree with this earlier observation in that
the purified enzyme exhibits a sharp pH dependence (Fig. 2A). The pH optimum is
5.0-5.5, and the enzyme has <20% of the maximal
activity at pH 4.5 or 6.0 and is inactive at pH 6.5 (Fig. 2A). This acidic pH optimum of ECE-2 contrasts with the near
neutral pH optimum of ECE-1 (19, 20, 31). These results are consistent with a role in peptide processing for ECE-1 in the extracellular milieu
and for ECE-2 in the intracellular milieu.

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Fig. 2.
Characterization of purified ECE-2.
A, pH optimum of purified ECE-2 was determined with 10 µM McaBk2 in sodium acetate (circles), Tris
acetate (squares), and sodium citrate (triangles)
at the indicated pH values. The data represent means of quadruplicate
determinations. Error bars show the standard error of the
mean. B, the effect of metal ions on purified ECE-2 was
determined with 10 µM McaBk2 in 0.2 M sodium
acetate buffer, pH 5.5. The reaction was carried out in the presence of
the indicated concentration of metal ion. "Relative units"
represent fluorescence units generated per min, and the data represent
means of triplicate determinations.
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The sensitivity of ECE-2 to divalent ions was tested using
ZnCl2, CoCl2, NiSO4,
CuSO4, MnCl2, MgCl2, and
CaCl2. ECE-2 is activated to a small but significant extent
by Co2+ ions at all concentrations examined (Fig.
2B). In response to Zn2+ and Cu2+,
the enzyme exhibits a biphasic response in that low concentrations (up
to 10 µM) activate and higher concentrations inhibit
ECE-2 activity. Ni2+ ions inhibit the activity at all
concentrations tested. Mn2+, Mg2+, or
Ca2+ ions had no significant effect on ECE-2 activity.
Taken together, these results support the involvement of metal ions at
the active site.
ECE-2 Hydrolyzes a Variety of Bioactive Peptides--
Next
we examined 30 distinct peptides and a pool of 12 peptides that differ
only in the penultimate position for their ability to serve as
substrates of ECE-2 (Table II), and we
identified the products by MALDI-TOF mass spectrometry (Fig.
3). Among the biologically active
peptides that were previously found to be cleaved by other related
metalloproteases, big ET-1 and bradykinin are cleaved, respectively, at
sites Trp21-Val22 and
Pro7-Phe8, which also are used by ECE-1 (31,
32). Interestingly, [des-Arg1]bradykinin is not cleaved
even though the Pro7-Phe8 site is present in
this peptide. This suggests that there is a minimum length requirement
for cleavage by ECE-2 (discussed further below). Neurotensin,
angiotensin I, and substance P are cleaved at sites
(Pro10-Tyr11,
Pro7-Phe8, and
Gly9-Leu10, respectively) that also are cleaved
by neprilysin. ECE-2 does not cleave luteinizing hormone-releasing
hormone, which has been shown to be cleaved by neprilysin and ECE-1,
albeit less efficiently (32). Furthermore, a number of peptides, such
as angiotensin II and III, [Arg]vasopressin, ACTH, and
-neoendorphin, that are hydrolyzed by other metalloproteases are not
cleaved by ECE-2 (Table II), suggesting that this enzyme exhibits
unique substrate specificity.
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Table II
Hydrolysis of bioactive peptides by ECE-2: determination of cleavage
sites by MALDI-TOF mass spectometry
The arrows represent primary cleavages and arrowheads represent
secondary cleavages.
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Fig. 3.
Cleavage site determination using MALDI-TOF
mass spectrometry. One nanomole of peptide was incubated in the
presence or absence of purified ECE-2 (5 ng) in 0.2 M
sodium acetate buffer, pH 5.5, for 100 min at 37 °C. The reaction
was terminated by quick freezing, and the samples were subjected to
MALDI-TOF mass spectrometry as described (16, 28).
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Among the peptides derived from opioid peptide precursors,
proopiomelanocortin-derived peptides (
-endorphin, ACTH, and
J-peptide) are not cleaved by ECE-2 (Table II). In contrast,
proenkephalin-derived peptides, namely peptide E, BAM 22, and BAM 18, were found to be good substrates for ECE-2 (Fig. 3 and Table II). The
primary site of cleavage of peptide E is between Gly23 and
Phe24 leading to the generation of BAM 23 (Fig. 3). Peptide
E also is cleaved between Asp16 and Tyr17,
albeit less efficiently, to generate BAM 16. Another proenkephalin A-derived peptide, BAM 22, also is cleaved by ECE-2 at a single site
leading to the generation of BAM 12 (Table II). Finally, BAM 18 is
cleaved by ECE-2 at Asp16-Tyr17 and at
Glu12-Trp13 generating BAM 16 and BAM 12, respectively. Although the three substrates share substantial homology
in their N-terminal region, the sites of processing of these peptides
appear to have only a partial overlap. Furthermore, the fact that
peptide E and BAM 22, which differ by only three amino acids at their C
termini, are processed at entirely different sites suggests a role for peptide length and/or secondary structure in the recognition of the
substrate by ECE-2.
Among the peptides derived from prodynorphin, Dyn B is cleaved by
ECE-2, whereas Dyn A or Dyn A-8 is not (Table II). Among the
pro-SAAS-derived peptides, only PEN-LEN is processed by ECE-2 (Fig. 3). The major cleavage is at Arg21-Val22
leading to the generation of PEN-21, and minor cleavages are at
Ala18-Leu19 and
Leu20-Arg21 (Fig. 3 and Table II). To address
the amino acid requirement at and around the cleavage site, a number of
synthetic peptides were tested as substrates. These 9-14-residue
peptides represent various portions of carboxypeptidases A-5, D, and
E. One set of peptides was a mixture containing 12 amino acid
substitutions at the position penultimate to the C terminus; the
individual peptides present in the mixture were detectable by MALDI-TOF
mass spectrometry. None of these peptides were cleaved by ECE-2.
From the analysis of sites within the 10 peptides that were cleaved (as
well as those that were not), it appears that ECE-2 prefers cleaving at
sites containing an aromatic residue (Trp, Tyr, or Phe) or an aliphatic
residue with a large branched side chain (Ile, Val, or Leu) at the P1'
site. A wide range of amino acids is tolerated in the P1-position,
although a Pro, Gly, or charged residue (Glu, Asp, Lys, or Arg) is
often present at the P1 site of the most efficiently cleaved peptides.
However, a notable exception is big ET-1, where the cleavage site
presumably is exposed because of the cysteine bridge. Because ECE-2
does not cleave most of the potential sites that contain a P1' aromatic
or aliphatic residue, there must be additional constraints that limit
the activity of this enzyme. Inspection of the hundreds of potential
sites in the 42 peptides tested revealed several additional features that distinguish between cleaved and uncleaved sequences: cysteines are
not present surrounding the cleavage site (from P3 to P3'), acidic
residues are not present in the P3 to P3' sites except at the
P1-position, and there are no prolines in the P1'- to P3'-positions. In
addition, the cleavage sites are located between 7 and 23 residues from
the N terminus and between 2 and 17 residues from the C terminus. Finally, if two sites are present that both fit these preferences, the
enzyme appears to prefer the site closer to the C terminus. The
peptidyldipeptidase-like activity seen with ECE-2 also has been
reported for other metalloendoproteases, including neprilysin and ECE-1
(31, 32).
Kinetic Analyses of ECE-2 Hydrolysis of Bioactive
Peptides--
Initial velocity dependence on substrate concentration
was determined for five representative peptides that serve as
substrates for ECE-2 (Fig. 4 and Table
III). These results show that the
processing of substrates by ECE-2 follows typical Michaelis-Menten
kinetics; this is shown for peptide E and bradykinin in Fig. 4.
Comparisons of the kinetic parameters show that McaBk2 is the best
substrate for ECE-1 and ECE-2. Although big ET-1 serves as a substrate
for both ECE-1 and ECE-2, there is a substantial difference in
catalytic rates between these two enzymes. Among the peptides processed by ECE-2, Dyn B exhibits the highest catalytic rate and lowest affinity, whereas big ET-1 exhibits the lowest catalytic rate and
highest affinity. Furthermore, the fact that various peptides are
processed at differing efficiencies suggests a role for ECE-2 in
modulating the levels of biologically active peptides.

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Fig. 4.
Kinetic analysis of ECE-2 hydrolysis of two
representative bioactive peptides. Purified ECE-2 (5 ng) was
assayed using the indicated concentrations of the various peptides.
The reaction mixtures were subjected to high pressure liquid
chromatography analysis, and peptides were detected by absorbance at
215 nm. The initial rate of substrate hydrolysis was determined by
measuring the appearance of product under initial rate conditions (less
than 10% substrate hydrolysis). Representative figures from the
analysis of peptide E and bradykinin hydrolysis are shown.
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Table III
Steady-state kinetic constants for hydrolysis of bioactive peptides by
ECE-2
In the case of Dyn B and peptide E where sequential cleavages of the
substrate were observed upon prolonged incubation, the initial velocity
measurements were performed under conditions where only the first
cleavage occurred.
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DISCUSSION |
In this study, we have characterized the enzymatic properties of
ECE-2. Using recombinant purified ECE-2, we show that the enzyme has an
acidic pH optimum and is completely inactive at pH 7; neither the
McaBk2 peptide nor larger peptide substrates including big ET-1 are
cleaved by ECE-2 at neutral
pH.2 This is in
contrast to ECE-1, which exhibits maximal activity at pH 7.0 for the
cleavage of larger peptides, including big ET-1, and a pH optimum of
~6.0 for shorter peptide substrates (20, 33). Taken together, these
results are consistent with the processing of peptides at the cell
surface by ECE-1 and in an intracellular compartment by ECE-2.
ECE-2b exhibits a neuroendocrine distribution (20, 21, 34, 35). A study
examining the distribution of ECE-2b by in situ
hybridization analysis has found that the localization is restricted to
neurons and to areas of the central nervous system enriched
in neuropeptides (34). Another study examining the distribution
of ECE-2b within the endothelial cells by immunocytochemistry localized
the enzyme to an intracellular compartment (23, 24). Consistent with
this, the processing compartment of ECE-2b activity was found to be
intracellular because co-expression of the enzyme with endothelin
precursor was required for the generation of ET-1, whereas
co-incubation of cells individually expressing the two was not
sufficient to generate ET-1 (21). Taken together, the neuroendocrine
distribution, acidic pH optimum, and subcellular localization to a
peptide-containing compartment make ECE-2b an ideal candidate for an
intracellular processing enzyme of neuroendocrine precursors.
Analysis of processing of biologically active peptides revealed that
ECE-2 cleaves some but not all peptides that are processed by other
Zn2+ metalloendopeptidases (such as ECE-1 and neprilysin).
For example, some regulatory peptides (neurotensin, bradykinin,
angiotensin I, and substance P) that are cleaved by ECE-2 also are
hydrolyzed by ECE-1 and neprilysin. In contrast, a number of peptides
that are cleaved by neprilysin are not processed by either ECE-1 (32) or ECE-2 (Table II). Thus it appears that like ECE-1, ECE-2 is involved
in the selective processing of specific peptides as opposed to the
nonselective degradation of peptides. In the case of ECE-1, studies
using big ET-1 and peptides derived from endothelins have suggested
that hydrolysis of substrates by ECE-1 is highly dependent on substrate
conformation (35, 36). In support of this, it was found that linear big
ET-1, in which the formation of disulfide bonds was prevented by
alkylation of the four cysteines, was cleaved at multiple sites; this
is in contrast to the native big ET-1 that is cleaved at a single site
by this enzyme (26). It is likely that the recognition of substrates by
ECE-2 is also dependent on the substrate length and conformation
because we find that not all structurally related peptides are
recognized by ECE-2. Furthermore, the analysis of cleavage site
selectivity of ECE-2 suggests that the peptide length as well as
residues surrounding the cleavage site that would confer secondary
structure play an important role in the recognition of the substrate by
ECE-2.
A notable feature of ECE-2 is that the enzyme is able to process
endoproteolytically peptide intermediates to generate biologically active peptides. Big ET-1 is processed at the Trp-Val site to generate
ET-1; this site of cleavage is identical to the site used by ECE-1.
However, the fact that ECE-2 processes big ET-1 at acidic pH (as
described above) suggests that this enzyme is able to generate ET-1
intracellularly. Another potential biologically active peptide
generated by ECE-2 is BAM 12 from the endoproteolytic processing of BAM
22 (Table II). Both BAM 22 and BAM 12 were originally isolated as
enkephalin-containing opioid peptides from bovine adrenal medulla (37)
and later reported to be distributed in the substantia nigra and
pallidum of rat and human brains (38, 39). BAM 12 exhibits
opioid receptor selectivity that contrasts with the µ opioid receptor
selectivity of BAM 22 (40, 41). These results imply that differential
processing of these peptides would modulate selective activation of
opioid receptor types. In addition, differential processing could also
generate peptides that activate other G-protein-coupled receptors. A
recent study has shown BAM 22 to be the most potent ligand for sensory
neuron-specific G-protein-coupled receptors (SNSR-3 and SNSR-4) in the
dorsal root ganglion; these receptors are thought to be involved in
pain transmission (42). We find that ECE-2 efficiently processes
peptide E to BAM 23, which contains a C-terminal Gly. Because peptides
with C-terminal Gly are substrates for peptidylglycine
-amidating
monooxygenase, BAM 23 would be converted rapidly into BAM 22 amide by
this enzyme. Thus it appears that ECE-2 is capable of processing a
number of peptide intermediates leading to the generation of a variety
of endogenous ligands.
One of the peptide intermediates processed by ECE-2 is a
pro-SAAS-derived peptide, PEN-LEN. We and others have shown previously that PEN-LEN peptides with intact C termini are inhibitors of the
classical processing enzyme, prohormone convertase 1 (29, 43). In this
study, we find that ECE-2 cleaves PEN-LEN leading to the generation of
shorter PEN peptides such as PEN-21. It should be pointed out that
within the milieu of secretory vesicles, PEN-21 would be converted
rapidly to PEN-20 by CPE. PEN-20 is an endogenous peptide found in both
rat brain as well as pituitary (44). We also have shown that these
shorter PEN peptides do not inhibit PC1 activity (29, 43). The
endoproteolytic processing of PEN-LEN has functional implications
because processing at internal sites, as seen with ECE-2, that results
in the generation of shorter peptides (such as PEN-19 and PEN-20) would
lead to a loss of PC1 inhibition (29, 43). Because PC1 is involved in
the generation of a large number of neuroendocrine peptides, modulation
of PC1 activity would have a significant impact on the levels of
neuroendocrine peptides. Thus, the nonclassical processing of PEN-LEN
by ECE-2 would affect the level of PC1 inhibitory peptides and is
likely to play an important role in the regulation of neuroendocrine peptide levels in vivo.