From the a Department of Histology and Embryology, Cell Biology Program, Institute of Biomedical Sciences, h Department of Biochemistry, e Institute of Chemistry and Heart Institute (InCor), University of São Paulo, 05508-900, São Paulo, SP, Brazil, the c Institute of Chemistry and f Department of Pharmacology, State University of Campinas, 13083-970, Campinas, SP, Brazil, and the j Interdisciplinary Center of Biochemistry Investigation (CIIB), University of Mogi das Cruzes, Mogi das Cruzes, 08780-911, Mogi das Cruzes, SP, Brazil
Received for publication, November 25, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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Endopeptidase 24.15 (EC 3.4.24.15;
ep24.15), neurolysin (EC 3.4.24.16; ep24.16), and
angiotensin-converting enzyme (EC 3.4.15.1; ACE) are metallopeptidases
involved in neuropeptide metabolism in vertebrates. Using catalytically
inactive forms of ep24.15 and ep24.16, we have identified new peptide
substrates for these enzymes. The enzymatic activity of ep24.15 and
ep24.16 was inactivated by site-directed mutagenesis of amino acid
residues within their conserved HEXXH motifs,
without disturbing their secondary structure or peptide binding
ability, as shown by circular dichroism and binding assays. Fifteen of
the peptides isolated were sequenced by electrospray ionization tandem
mass spectrometry and shared homology with fragments of intracellular
proteins such as hemoglobin. Three of these peptides (PVNFKFLSH,
VVYPWTQRY, and LVVYPWTQRY) were synthesized and shown to interact with
ep24.15, ep24.16, and ACE, with Ki values ranging
from 1.86 to 27.76 µM. The hemoglobin Endopeptidase EC 3.4.24.15 (ep24.15; also referred to as thimet
oligopeptidase) and endopeptidase EC 3.4.24.16 (ep24.16; also referred
to as neurolysin) were initially detected in and purified from rat
brain homogenates (1, 2). The cloned rat brain ep24.16 (3) showed 80%
similarity and 63% identity with the previously cloned rat testis
ep24.15 (4). Both peptidases share most of their natural substrates,
including bradykinin, neurotensin, opioids, angiotensin I, and
gonadotrophin-releasing hormone (5, 6). All of these natural substrates
are hydrolyzed at the same peptide bond and at similar rates, except
for neurotensin, which is hydrolyzed by ep24.15 and ep24.16 by cleavage
of its Arg8-Arg9 and
Pro10-Tyr11 bonds, respectively (7).
Functional studies have suggested that ep24.15 and ep24.16 inactivate
neuropeptides inside and outside the central nervous system. The
central administration of
Z-(Leu,Asp)Phe- Outside the central nervous system,
N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate,
an inhibitor of both ep24.15 and ep24.16, potentiates
bradykinin-induced hypotension, which suggests that one or both of
these peptidases participate in the metabolism of bradykinin (12). In
macrophages, CFP-AAF-pAb, a mixed inhibitor of ep24.15 and ep24.16 (7),
reduces antigen presentation through the major histocompatibility
complex class I (MHC-I)1 but
not through MHC-II (12). Conversely, liposome-mediated introduction of
ep24.15 into macrophages stimulates the antigen presentation of MHC-I,
but not that of MHC-II. The observation that ep24.15 can degrade or
bind to several MHC-I antigenic peptides (13, 14), which are 8-11
amino acid fragments generated in the cytoplasm by proteasomes (15,
16), raises the possibility that ep24.15 and ep24.16 participate in the
intracellular metabolism of peptides. The nature of such peptides is unknown.
Angiotensin I-converting enzyme (ACE; peptidyldipeptidase A) is a zinc
metallopeptidase that cleaves the COOH-terminal dipeptide from
angiotensin I to produce the potent vasopressor octapeptide angiotensin
II (17) and inactivates bradykinin by the sequential removal of two
COOH-terminal dipeptides (18). In addition to these two main
physiological substrates, which are involved in blood pressure
regulation and water and salt metabolism, ACE also cleaves
COOH-terminal dipeptides from various oligopeptides with a free COOH
terminus. ACE has also been implicated in a range of physiological
processes unrelated to blood pressure regulation, such as immunity,
reproduction, and neuropeptide regulation, based on its localization
and/or the in vitro cleavage of a range of biologically
active peptides. The role of ACE in blood pressure control and water
and salt metabolism has been defined mainly by the use of highly
specific ACE inhibitors (19). These inhibitors are effective in the
treatment of hypertension, congestive heart failure, and diabetic
nephropathy (20-22). Moreover, ACE has recently been implicated in the
hydrolysis in vivo of the tetrapeptide Ac-Ser-Asp-Lys-Pro,
which modulates hematopoietic stem cell proliferation by preventing
their recruitment into the S phase (23). The acute administration of
captopril, an ACE inhibitor, produces a 7-fold increase in the plasma
concentration of Ac-Ser-Asp-Lys-Pro in normal volunteers, thus
demonstrating the importance of ACE in the metabolism of this substrate
(24).
In this study, we show that ep24.15 and ep24.16, when catalytically
inactivated by site-directed mutagenesis of amino acid residues within
their HEXXH motifs, can be used to identify new endogenous
peptides present in crude peptide extracts prepared from rat tissues.
The ep24.15 or ep24.16 enzyme-bound peptides were isolated and many of
them fully sequenced by electrospray ionization tandem mass
spectrometry. Based on these sequences, synthetic peptides were
prepared and shown to interact strongly with ep24.15, ep24.16, and ACE.
One of the peptides identified here (PVNFKFLSH), derived from the
Site-directed Mutagenesis and Protein Expression--
A
QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was
used to introduce a specific point mutation in the wild type ep24.15 or
ep24.16 cDNA cloned into the expression vector pGEX4t-2 (7).
Oligonucleotide primers were synthesized with mismatches coding for
alanine, based on prokaryotic codon usage rules to obviate the use of
rare codons that could hinder subsequent protein expression. Point
mutations were specified as H473A, E474A, H477A, and E502A for ep24.15,
and H474A, E475A, H478A, and E503A for ep24.16. PCR was done in a
50-µl mixture using 50 ng of template plasmid DNA, 14 pmol of each
primer, 10 nmol of dNTPs, and 2.5 units of turbo Pfu DNA
polymerase (Stratagene) in 0.5× Pfu polymerase reaction
buffer. The thermocycler was programmed for an initial denaturation at
95 °C for 1 min followed by 16 cycles of 95 °C for 30 s,
55 °C for 1 min, and 68 °C for 15 min, with a final incubation at
72 °C for 10 min. One microliter (20 units) of DpnI (New
England Biolabs) was added to the sample (50 µl) and incubated at
37 °C for 16 h. The sample was then denatured at 65 °C for 30 min. Two microliters of the final sample were used to transform competent Escherichia coli XL1-blue cells by
electroporation. Putative positive colonies were confirmed by
double-stranded template dideoxy sequencing (25). Expression and
purification of the wild type or mutant proteins for biochemical
characterization were done as described (7), with all enzymes stored at
SDS-PAGE--
The homogeneity of the recombinant enzyme
preparations was assessed by electrophoresis under reducing conditions
in 8% polyacrylamide gels containing SDS-PAGE, as described previously
(26). Protein bands were detected by staining the gels with Coomassie
Brilliant Blue R-250 (Bio-Rad).
DNA Sequencing--
DNA was sequenced using a
multicapillary MegaBace1000 sequencer, according to the protocol
supplied with the DYEnamic ET dye terminator cycle sequencing kit
(Amersham Biosciences).
Peptide Synthesis--
Peptides were synthesized by the
Resgen-Invitrogen Corporation using Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry.
Peptide Extract from Rat Tissues--
A crude peptide extract
from rat brain or spleen was prepared as previously described (27).
Briefly, male Wistar rats were killed and the brain and spleen were
removed and rapidly frozen in liquid nitrogen prior to storage at
Enzyme-Peptide Binding Assay--
Enzyme (1-5 nmol)-peptide
complexes were produced by incubating a specific synthetic peptide or
the peptide extract with catalytically inactive ep24.15 or ep24.16 in
200 µl of buffer (25 mM Tris-HCl, pH 7.5, containing 125 mM NaCl and 0.1% of bovine serum albumin) for 30 min at
room temperature. At the end of this period, the reaction mixture was
layered onto a dried Sephadex G-25 column (previously washed and
equilibrated with Tris-buffered saline followed by
centrifugation to remove the buffer) and centrifuged at 1000 × g for 2 min. The flow-through (~200 µl) was collected and the peptide content analyzed by high performance liquid
chromatography (HPLC) using a Chromolith performance column (4.6 mm × 100 mm; Merck), with a linear gradient of 5-35%
acetonitrile in 0.1% trifluoroacetic acid, for 20 min, and at a flow
rate of 1 ml/min, as previously described (7). Control experiments were
done by: (i) adding an excess of dynorphin A1-13 (30 µM) to the reaction mixture as a specific competitive
inhibitor for ep24.15 and ep24.16, (ii) performing the assay in the
presence of wild type active ep24.15 and ep24.16, and (iii) preforming
the assay in the absence of ep24.15 and ep24.16 (reaction mixture
containing only 0.1% of bovine serum albumin).
Peptide Sequencing by ESI-MS/MS--
Peptides were sequenced by
positive nano-electrospray ionization (nano-ESI+) using
peptide-containing aliquots collected during HPLC. Typical conditions
were a capillary voltage of 1 kV, a cone voltage of 30 V, and a
desolvation gas temperature of 100 °C. The protonated peptides were
subjected to collision-induced dissociation with argon in the 15-45 eV
collision energy range. All of the mass spectrometry experiments were
done with a Q-TOF mass spectrometer (Micromass, UK) in Qq-orthogonal
time-of-flight configuration. Peptide sequences were determined
manually from the ESI-MS/MS product ion mass spectra with the help of
the PepSeq software (Micromass).
Determination of the Peptide Bonds Cleaved--
The peptide
bonds cleaved were identified by isolating the fragments by HPLC
followed by ESI-MS/MS mass spectrometry sequencing, as described above.
Peptide Sequence Homologies--
To identify the putative
protein precursors of the peptides sequenced by ESI-MS/MS, a protein
data base (www.ncbi.nlm.nih.gov/blast) was searched for short, nearly
exact matches (rodentia origin), as previously described (28). When the
perfect match for a given peptide was not found in a large protein
sequence, more than one putative protein precursor containing part of
the identified peptide was listed.
Enzyme Activity Assay and Determination of Kinetic
Parameters--
The enzymatic activity of wild type and mutant ep24.15
and ep24.16 was determined in duplicate in a continuous assay using the
quenched fluorescent substrate (QFS)
(7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Pro-dLys-(2,4-dinitrophenyl), as previously described (29, 30). ACE (Sigma) enzymatic activity was
measured similarly using the internally quenched fluorescent peptide
Abz-FRK(2,4-dinitrophenol)P-OH (31). The relative inhibition constants (Ki) of the new synthesized peptides were
determined in parallel with well known substrates or competitive
inhibitors as a reference. The following equations were used to
calculate the Ki values: Ki = Ki,app/(1 + [S]/Km), where [S] = molar concentration of the
substrate, Km = Michaelis-Menten constant, and
Ki,app = apparent inhibition constant,
assuming [S] = Km (10 µM) (26). The
Ki,app was calculated using the
equation, Vo/Vi = 1 + [I]/Ki,app, where
Vo = velocity of hydrolysis without the inhibitor,
Vi = velocity of hydrolysis in the presence of the
inhibitor, and [I] = molar inhibitor concentration. In a plot of
(Vo/Vi) Circular Dichroism (CD)--
The secondary structure of selected
mutants displaying a substantial decrease in catalytic and inhibitor
binding capacity was examined by CD spectroscopy using a Jasco 720 spectropolarimeter. The instrument calibration was verified using an
aqueous solution of d10-camphorsulfonic acid,
and the CD spectra were collected in the wavelength range of 190 to 260 nm at 0.5-nm intervals, with a resolution of 0.5 nm, a response time of
0.5 s, a scan speed of 10 or 20 nm/min for 4 or 5 scans, a cell
path length of 0.01 or 0.02 cm, and a temperature of 20-22 °C.
Samples were prepared in 10 mM Tris-HCl (pH 7.4). Secondary
structure estimation of the proteins was done using the SELCON3
algorithm (27).
Protein Concentration--
For the CD experiments, protein
concentrations were determined as described by Gill and von Hippel
(33). For all other purposes, protein concentrations were determined by
the Bradford assay (34) using bovine serum albumin as standard.
Action of Selected Peptides on Blood Pressure in Anesthetized
Rats--
To examine the action of peptides PVNFKFLSH, LVVYPWTQRY, and
FDLTADWPL on blood pressure, male Wistar rats (~200 g) were
anesthetized with sodium pentobarbital (>60 mg/kg, intraperitoneal;
HypnolTM, Cristália, Itapira, SP, Brazil) and placed
under a heating lamp to maintain body temperature. The trachea was
cannulated to facilitate breathing, and the left carotid artery and
left femoral vein were cannulated with polyethylene tubing for the measurement of arterial blood pressure and drug/peptide administration, respectively. The cannulas were kept patent with heparinized 0.9% (w/v) saline. The arterial pressure was recorded continuously via a
pressure transducer (Abbott, Chicago, IL) coupled to a
computer-controlled data acquisition system (Transonics Systems, Inc.,
Ithaca, NY). The experiments were initiated after at least 15 min for
stabilization. Bradykinin (BK), angiotensin II, and peptides
(PVNFKFLSH, LVVYPWTQRY, and FDLTADWPL) were dissolved and administered
in 0.9% saline. For PVNFKFLSH, the doses tested ranged from 0.001 to
10 µg/kg, whereas for LVVYPWTQRY and FDLTADWPL, only two doses (10 and 100 µg/kg) were examined. The order of dose administration was
randomized in all experiments. The responsiveness of the preparations
was assessed by administering a single dose of BK (3 µg/kg) and
angiotensin II (3 µg/kg) before peptides PVNFKFLSH, LVVYPWTQRY, and
FDLTADWPL, and then at the end of the experiment to assess whether
there was any alteration in the response to these two agonists. In
separate experiments, enalapril (2 mg/kg, intravenously) was given
10-15 min before administration of the lowest doses (0.001, 0.01, and 0.1 µg/kg) of PVNFKFLSH to assess the influence of ACE inhibition on
the action of this peptide. In all cases, bolus intravenous injections
(100 µl) of peptides were washed in a further 100 µl of
saline. The animal protocols and procedures described here were
done in accordance with the NIH Guide for the Care and Use of
Laboratory Animals and the general principles for the care and use of
animals established by the Brazilian College for Animal Experimentation (COBEA).
Statistical Analysis--
The blood pressure changes were
expressed as the mean ± S.E. of the peak changes in mean arterial
blood pressure (in mm Hg) relative to the values recorded immediately
prior to peptide administration. Differences between doses and
treatments were compared using Student's t test or analysis
of variance followed by the Tukey test, as appropriate. A value of
p < 0.05 indicated significance.
Site-directed mutagenesis of the cDNA encoding rat testis
ep24.15 and pig liver ep24.16 was used to prepare mutants in which the
histidine and glutamic acid residues of the HEXXH motif
conserved within an active site -chain fragment
PVNFKFLSH, which we have named hemopressin, produced
dose-dependent hypotension in anesthetized rats, starting
at 0.001 µg/kg. The hypotensive effect of the peptide was potentiated
by enalapril only at the lowest peptide dose. These results suggest a
role for hemopressin as a vasoactive substance in vivo. The
identification of these putative intracellular substrates for ep24.15
and ep24.16 is an important step toward the elucidation of the role of
these enzymes within cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(PO2CH2)(Leu,Asp)Ala-Lys-Met, a fully specific endopeptidase ep24.15 inhibitor (8), prolongs the
forepaw licking latency of mice in the hot plate test and following the
injection of submaximally active doses of neurotensin (9). Likewise,
the intracerebroventricular administration of Pro-Phe-
(PO2CH2)-Leu-Pro-NH2, a
selective ep24.16 inhibitor (10), significantly increases the
neurotensin-induced antinociception of mice in the hot plate test
(11).
1 chain of hemoglobin, was among the best natural
substrates identified so far for these enzymes, and caused
dose-dependent hypotension in rats. This peptide, which we
have named hemopressin, may have a role in blood
pressure regulation and in cardiovascular disease.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C for subsequent analysis.
80 °C. Tissues from five rats were added to boiling 0.1 M acetic acid and homogenized (Polytron, Brinkmann). The
tissue homogenates were boiled for 10 min, centrifuged at 50,000 × g for 30 min at 4 °C, and the supernatant was filtered through a Millipore centrifugal filter unit with a NMCO of 5,000. The
filtrate was adjusted to pH 7.4 with 1 M Tris-HCl (pH 7.4) and then used in the experiments described below.
1 versus [I], the slope is
1/Ki,app. To determine the
Ki values, five solutions with synthetic peptide
concentrations ranging from 0.1 to 100 µM were used to
construct the graph (Vo/Vi)
1 versus [I]. The relative hydrolysis ratio was
determined using peptides at a concentration of 100 µM,
under zero-order kinetics, with less than 10% of the substrate
consumed by the end of the incubation period, which varied from 30 min
to 2 h. The enzyme concentration varied from 5 to 50 ng/assay.
All assays were done in triplicate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix were genetically substituted.
Two additional glutamate residues carboxyl to the HEXXH
motif, Glu502 in ep24.15 and Glu503 in ep24.16,
were also mutated. The wild-type ep24.15 and ep24.16 have previously
been expressed in DH5
E. coli in a catalytically active
form that resembles the proteins isolated from mammalian tissue (7).
Isopropyl-1-thio-
-D-galactopyranoside induction of
transformed DH5
E. coli triggers a
time-dependent overexpression of specific proteins, the
apparent molecular weight of which corresponds to the calculated mass
of ep24.15 or ep24.16 fused with glutathione S-transferase;
the maximal production of the fusion proteins similarly reached a
plateau by 4 h (data not shown). Proteolytic removal of
glutathione S-transferase and subsequent purification of the recombinant proteins allowed the recovery of apparently homogenous peptidases based on SDS-PAGE analysis (Fig.
1A). The production yield
(~0.5 mg/liter of culture) was similar for all expressed proteins,
suggesting that mutation of the above mentioned amino acid residues did
not affect the relative levels of ep24.15 or ep24.16 expression in
DH5
E. coli.
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Fig. 1.
SDS-PAGE and circular dichroism spectra of
wild type and mutated ep24.15 and ep24.16. A, SDS-PAGE
electrophoresis followed by Coomassie Blue staining showing the
homogeneity of the wild type and mutated ep24.15 and ep24.16 enzymes
(15 µg each) obtained after a single-step affinity chromatography on
a Sepharose-glutathione S-transferase column. B,
CD spectra were obtained using a Jasco model 720 spectrometer at 0.5-nm
intervals in the wavelength range of 190 to 260 nm. The settings used
were a resolution of 0.5 nm, a response time of 0.5 s, a scan
speed of 10 or 20 nm/min (4 or 5 scans), a cell path length of 0.01 or
0.02 cm, and a temperature of 20-22 °C. The samples were prepared
in 10 mM Tris-HCl (pH 7.4).
To ensure that the mutated ep24.15 and ep24.16 had not lost enzymatic
activity as a result of gross structural alterations during mutagenesis
and subsequent protein expression, the secondary structures of these
enzymes were compared with those of the catalytically active wild-type
proteins. The CD spectra suggested that both ep24.15 and ep24.16 had a
typical -helix secondary structure (Fig. 1B) that was not
significantly modified by any of the mutations that inactivated the
catalytic activity (Table I). The effects of individual mutations on the catalytic activity of ep24.15 and ep24.16 were assessed using a QFS. As expected, complete ablation of
enzymatic activity was observed when point mutations were made for
H473A, E474A, H477A, and E502A in ep24.15, or H474A, E475A, H478A, and
E503A in ep24.16.
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Following the structural characterization of the wild type and mutant ep24.15 and ep24.16, we examined whether the inactive enzymes would bind to bioactive peptides such as dynorphin A1-13 and bradykinin. Initial binding assays done with 125I-dynorphin A1-13 suggested a similar ability of all inactive enzymes to bind this peptide (data not shown). Therefore, the E474A and E475A mutants were selected for further experiments, as this specific glutamic acid is believed to be involved directly in substrate catalysis, whereas the other residues are involved in zinc ion coordination (19). To further characterize the ability of the E474A and E475A mutants to bind bioactive peptides, such as bradykinin and dynorphin A1-13, enzyme-peptide complexes were allowed to form in solution and the excess of unbound peptide was removed by gel filtration. The resulting complexes were analyzed by HPLC. The results from these assays (Table II) supported the idea that catalytically inactive ep24.15 (E474A) and ep24.16 (E475A) retained the ability to bind bioactive peptides such as bradykinin and dynorphin A1-13.
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To assess the usefulness of these mutants for isolating new substrates
for ep24.15 and ep24.16, the E474A and E475A mutants were incubated
with a rat brain peptide extract and the enzyme-peptide complexes were
separated from the excess of free peptides by gel filtration and then
subjected to HPLC. The presence of either E474A or E475A in the
incubation with peptide extract was critical for obtaining increasing
amounts of specific peptide peaks, as shown in the HPLC chromatograms
(Fig. 2). In control experiments without
the inactive enzymes to complex and arrest the peptides, only small
peaks were seen (Fig. 2). For the moment, it is unclear whether these
smaller peaks represent lower amounts of peptides bound to the inactive
enzymes. Equivalent experiments done with active ep24.15 or ep24.16
produced chromatograms without a significant increase in the peptide
peaks, when compared with those obtained using the inactive enzymes
(data not shown). Hence, catalytic inactivation before incubation with
the crude peptide extracts was important to recover larger amounts of
the putative natural substrates of these enzymes. To determine whether
binding of the peptides by the inactive enzymes involved a specific
interaction, dynorphin A1-13 (30 µM), a
potent competitive inhibitor of both ep24.15 and ep24.16, was added to
the peptide extracts. Dynorphin A1-13 clearly reduced the
number of peptide peaks observed in the chromatograms (Fig. 2,
A and B). Thus, it is reasonable to conclude that
the above procedures were appropriate for identifying peptides that
interact specifically with ep24.15 and ep24.16.
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To identify the peptides that bound to inactive ep24.15 and ep24.16,
the peptide peaks were collected manually during HPLC (Fig.
2C) and were further analyzed by nano-ESI-MS/MS. A
representative deconvoluted ESI-MS/MS product-ion mass spectrum, which
allowed the complete sequencing of peptide PVNFKFLSH, is shown in Fig. 3. The isotopic cluster separation of 0.5 mass/charge ratio (m/z) units of the precursor
ion and its m/z reveals that doubly charged, doubly protonated peptide molecules of mass 1088.92 (M + H) were formed
by ESI ionization and mass selected for MS/MS sequencing. The sequence
of this peptide, and that of several other peptides sequenced from
similar ESI-MS/MS spectra, was found to match specific sequences in
various proteins (Table III).
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To confirm whether the sequenced peptides were in fact ep24.15 and/or ep24.16 substrates, and to further validate this new method, four of the 15 peptides identified were chemically synthesized. One of these four peptides (FDLTADWPL) was selected for synthesis because it did not appear to be arrested by ep24.15 or ep24.16, as shown in the HPLC chromatograms (Fig. 2C). The other three peptides (LVVYPWTQRY, VVYPWTQRY, and PVNFKFLSH) were selected for synthesis because they were efficiently arrested by ep24.15 and ep24.16 (Fig. 2C). The constant of inhibition (Ki values) and relative hydrolyzes ratio for these peptides were determined in parallel with known bioactive peptides such as bradykinin, angiotensin I and II, and dynorphin A1-13 (Table IV).
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The three peptides selected on the basis of a specific interaction with the inactive enzymes prevented the hydrolysis of QFS by ep24.15 or ep24.16 in a competitive enzyme assay, in contrast to the fourth peptide (FDLATDWPL), which did not inhibit the enzymes (Table IV). To estimate the affinity of these peptides for ep24.15 and ep24.16, the relative constant of inhibition (Ki) was determined. Peptide FDLTADWPL had a Ki above 100 µM, and was not efficiently degraded by these enzymes, even after prolonged incubations. On the other hand, peptides that specifically interacted with the inactive ep24.15 and ep24.16 enzymes had Ki values in the micromolar range (Table IV). Peptide PVNFKFLSH had the highest affinity for both enzymes, with a Ki eight times lower for ep24.16 (3.43 µM) than for ep24.15 (27.8 µM). Despite these differences, this peptide was a better substrate for ep24.15 and ep24.16 than bradykinin. Peptides LVVYPWTQRY and VVYPWTQRY, which differed by a single NH2-terminal amino acid, had similar Ki values for both ep24.15 and ep24.16. However, the relative hydrolysis ratio of VVYPWTQRY was five times higher for ep24.15 compared with ep24.16. In contrast, the peptide with the NH2-terminal leucine, LVVYPWTQRY, was degraded at least five times faster by ep24.16 than by ep24.15 (Table IV), suggesting that a large nonpolar amino acid residue at the NH2-terminal position could be of importance for defining specific substrates or inhibitors for ep24.15 or ep24.16.
The cleavage products of the peptides LVVYPWTQRY, VVYPWTQRY, and PVNFKFLSH, digested by either ep24.15 or ep24.16, were identified by ESI-MS/MS sequencing. Contrary to previously described natural substrates for ep24.15 and ep24.16, hydrolysis of the peptides LVVYPWTQRY, VVYPWTQRY, and PVNFKFLSH involved at least three peptide bonds (Table V). These results agreed with data obtained using several synthetic peptides (36, 37), suggesting that these enzymes could also simultaneously hydrolyze more than one peptide bond in natural substrates.
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Peptidases are not peptide-specific (38), and ep24.15 and ep24.16 share a series of substrates with ACE (7). Because the physiological function for ACE in the cardiovascular system is well known (20, 21), we examined the kinetic parameters of the peptides identified in this study. The ep24.15 and ep24.16 substrates LVVYPWTQRY, VVYPWTQRY, and PVNFKFLSH also interacted with ACE, with Ki values ranging from 1.7 µM up to 26 µM (Table IV). ACE hydrolyzed the peptide PVNFKFLSH more efficiently than it did angiotensin I or bradykinin (Table IV).
The effects of three of the peptides identified here (FDLTADWPL,
LVVYPWTQRY, and PVNFKFLSH) were examined on the blood pressure of
anesthetized rats. The intravenous injection of PVNFKFLSH produced immediate hypotension, the extent of which varied according to the dose
(Fig. 4A). The rapid fall in
blood pressure elicited by PVNFKFLSH was similar to that seen with
bradykinin, but required a lower dose (0.01 versus 3 µg/kg
for bradykinin). LVVYPWTQRY also produced hypotension at 10 µg/kg,
whereas FDLTADWPL produced a slight effect only at 100 µg/kg. Based
on this effect on blood pressure, PVNFKFLSH was named
hemopressin. Whereas enalapril significantly enhanced the
hypotensive response to bradykinin (decrease in mean arterial blood
pressure:
14.9 + 4.2 mm Hg versus
28.2 + 2.4 mm Hg
before and after enalapril, respectively; n = 5 each,
p < 0.05), this ACE inhibitor had a significant effect
only on the lowest dose of PVNFKFLSH. The responses to bradykinin were
potentiated after the administration of PVNFKFLSH while those to
angiotensin II were unaffected; LVVYPWTQRY had no such effect on the
responses to these two agonists (Fig. 4B).
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DISCUSSION |
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A major finding of the present study was the identification of a
new peptide substrate for ep24.15, ep24.16, and ACE that causes
hypotension. This peptide (named hemopressin) is a fragment of the -chain of hemoglobin. This is the first report to identify intracellular protein fragments as natural substrates for endopeptidase 24.15 (ep24.15) and neurolysin (ep24.16). These findings suggest a role
for ep24.15 and ep24.16 in intracellular peptide metabolism.
The substitution of amino acids His474, Glu475, His477, or Glu502 abolishes the enzymatic activity of ep24.15 (39). The importance of the corresponding residues in ep24.16 for catalysis has not yet been experimentally demonstrated, but may be predicted from the recently determined tertiary structure (35). Indeed, substitution by alanine of the corresponding residues on ep24.16 (H474A, E475A, H478A, and E503A) completely abolished the enzymatic activity. Because all of the proteins examined were expressed to an equivalent level, it is unlikely that the outcome observed with any of the substitutions resulted from improper protein folding. Analysis of the secondary structures by circular dichroism confirmed the similarity of the mutated proteins to the wild type, and indicated that inappropriate protein folding did not cause the loss of enzymatic activity. In addition, deconvolution of the circular dichroism data supported the previous assumption of secondary structure homology between these two oligopeptidases. In an attempt to identify new natural substrates for ep24.15 and ep24.16, catalytically inactive forms of these enzymes were used to identify peptides present in rat brain and spleen extracts.
All of the peptides isolated and sequenced here were within the size range previously reported for natural and synthetic substrates of ep24.15 and ep24.16 (36, 37, 53-55). Using a series of peptides structurally related to bradykinin, Oliveira et al. (53) showed that 5 amino acids was the minimum substrate size for ep24.15. Similar results were obtained for ep24.15 and ep24.16 using synthetic fluorescent substrates (37). The smallest peptide isolated here also contained 5 amino acids, which corroborated previous findings (37, 53). On the other hand, orphanin, a neuropeptide containing 17 amino acids, is the largest natural substrate described so far for ep24.15 (54). Using fluorescent substrates in a detailed, systematic analysis of the influence of substrate size on ep24.15 and ep24.16 catalysis, Oliveira et al. (37) confirmed that 17 amino acids was indeed the largest substrate size for both enzymes. The largest peptides identified here contained 16 amino acids, which also agrees with these earlier studies (37, 53-55).
There is increasing evidence that ep24.15 and ep24.16 may play a major role in the intracellular metabolism of peptides, probably at a stage beyond the proteasome (13-15, 40). As shown here, we have identified several putative intracellular substrates for ep24.15 and ep24.16. Because the substrates for ep24.15 and ep24.16 must be peptides containing 5-17 amino acids, there is a need for a proteolytic system able to generate such small peptides from larger proteins. The 20 S proteasome, a multicatalytic proteinase complex, is the main intracellular extralysosomal proteolytic system involved in ubiquitin-dependent and -independent intracellular proteolysis (41). In degrading cytosolic, mitochondrial, and nuclear proteins (42, 43), the proteasome generates peptides from 3 to 22 residues in size (16, 44, 45). The peptides generated by the proteasome are therefore within the optimum size range for substrates of ep24.15 and ep24.16 (14, 37). Of the new ep24.15 and ep24.16 substrates identified here, at least seven are hemoglobin fragments. Interestingly, ep24.15 is present in large amounts in human erythrocytes, where hemoglobin also occurs in large quantities (46). Short hemoglobin fragments have been shown to be generated directly by the proteolytic action of the proteasome (47, 48). Whereas additional studies will be necessary to clarify the putative enzymes involved in the generation of hemopressin in vivo, it seems reasonable to suggest that ep24.15 and ep24.16 may function in the later steps of intracellular protein degradation. The mechanisms whereby the peptides isolated here escaped degradation is unknown.
In addition to their well known receptor-mediated functions, some
peptides also play a role in intracellular processes. For example,
calmodulin-dependent protein kinase II is a multifunctional protein kinase with an important role in controlling a variety of
cellular functions in the central nervous system (49). A 13-amino acid
peptide (KKALRRQEAVDAL), known as autocamtide-2-related inhibitory
peptide, is a highly specific inhibitor of
calmodulin-dependent protein kinase II (50). In
Bacillus subtilis, the RapA and RapB proteins are
aspartylphosphate phosphatases that specifically dephosphorylate the
Spo0F~P intermediate response regulator of the phosphorelay signal
transduction system for sporulation (51). The RapA phosphatase activity
on Spo0F~P is inhibited in vivo by a pentapeptide
generated from the phrA gene, which displaces Spo0F~P from a preformed complex with RapA (51). The c-Jun
NH2-terminal kinase, a member of the stress-activated group
of mitogen-activated protein kinases, is inhibited by a cell-permeable
peptide that decreases intracellular c-Jun NH2-terminal
kinase signaling and confers long-term protection to pancreatic
-cells against interleukin-1
-induced apoptosis (52). Thus, by
acting on the intracellular metabolism of peptides, ep24.15 and ep24.16
could contribute to the maintenance of cellular homeostasis.
Of the endogenous globin fragments identified in the present study,
three (LVVYPWTQRY, VVYPWTQRY, and the fragment VYPWT) are apparently
related to a family of peptides known a hemorphins, which are derived
from the degradation of the -chain of human hemoglobin and show
morphine-like activity based on their ability to inhibit the
contractions of electrically stimulated guinea pig ileum (Refs. 56 and
57 and references therein). LVVYPWTQRY and VVYPWTQRY are identical to
human LVV-hemorphin-7 (57, 58) and VV-hemorphin-7 (59), respectively,
except for their terminal amino acid residue, which is tyrosine instead
of arginine and may reflect the rodent origin of our peptides.
Peptide PVNFKFLSH produced potent hypotension in anesthetized rats.
This peptide is derived from the 1-chain of rodent
hemoglobin and shares no sequence identity with the hemorphins of the
-chain of human hemoglobin. Other peptides derived from the
-chain identified here included HHPGDFTPAMHASLDK and two truncated
fragments of this peptide, but their effect on blood pressure was not
examined. The mechanism by which PVNFKFLSH produces hypotension is
still unclear but could involve a variety of pathways, including ion channel activation or blockade, the stimulation of nitric oxide (NO)
formation through as yet unidentified receptors, the release of
vasodilator peptides such as atrial natriuretic factor, or the
inhibition of endogenous peptidase activity which could lead to an
increase in circulating levels of hypotensive peptides.
In experiments not described here, we have observed that PVNFKFLSH does
not contract or relax vascular (aorta) or nonvascular (guinea pig
ileum) smooth muscle preparations. This finding is similar to the
inability of hemorphins to contract isolated endothelium-denuded aortic
strips from rats (60), and suggests that PVNFKFLSH probably does not
have a direct action on vascular smooth muscle. The involvement of NO
in the observed hypotension merits investigation, although Moisan
et al. (57) observed that the blockade of NO production by
L-N-nitro-L-arginine
methyl ester did not influence the hypertensive response to hemorphins.
Exogenous and endogenous peptides may be metabolized by a variety of peptidases, including the three enzymes studied here. To examine the influence of ACE on the hypotensive responses to PVNFKFLSH, rats were treated with enalapril to block this enzyme. Although the treatment was effective in potentiating BK-induced hypotension, it had little effect on the responses to PVNFKFLSH, except at the lowest dose of the peptide. This finding will have to be explored further in light of the role of ACE, and other peptidases such as the ep24.15 and ep24.16, in the metabolism of PVNFKFLSH. Experiments to address this aspect using specific inhibitors are in progress in our laboratory.
The ability of PVNFKFLSH to potentiate the hypotension to BK without
affecting the hypertension to angiotensin II is interesting, although
it is still unclear whether this response is selective for BK or
applies to vasodilatory peptides in general. This action of PVNFKFLSH
could involve the sensitization of intracellular pathways to subsequent
stimulation by BK or could involve the specific inhibition of a
peptidase(s), possibly ACE, that degrades BK. The inhibition of ACE by
PVNFKFLSH could influence the metabolism of other peptide
substrates by this peptidase. The hypotensive action of PVNFKFLSH
may involve therefore peptidase- and nonpetidase-mediated pathways. Finally, the observation that FDLTADWPL, derived from a nonhemoglobin molecule(s), caused little hypotension compared with
the varied effects observed for fragments from the (PVNFKFLSH) and
(hemorphins and LVVYPWTQRY) chains of hemoglobin confirms data in
the literature indicating that the degradation of hemoglobin is an
important source of bioactive peptides, and could provide a lead for
investigating the biological activities of the other peptides
identified in this study.
In summary, we have demonstrated the feasibility of using catalytically
inactive forms of ep24.15 and ep24.16 to identify new bioactive peptide
substrates for these enzymes. However, the methodology should be
applicable to other enzyme systems. One of the new substrates
identified (hemopressin) is a fragment of the hemoglobin
-chain and reduces blood pressure in anesthetized rats. Further
functional analyses will be necessary to evaluate the pharmacological
and physiological relevance of hemopressin and of the other peptides
identified in this study.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Marc J. Glucksman for help, Dr. James L. Roberts for providing the wild type ep24.15 cDNA, Drs. Akira Kato and Shigerisa Hirose for providing the wild type ep24.16 cDNA, Dr. I. A. Smith for providing the QFS, Drs. Vitor Oliveira and Maurício C. Araujo for help with the kinetic experiments, Dr. Heitor Moreno, Jr. (Department of Pharmacology, UNICAMP) for allowing the use of his blood pressure monitoring system, and Dr. Shirley Schreier for allowing us to use her Jasco 720 spectropolarimeter. Dr. Lloyd D. Fricker provided suggestions and criticisms on the manuscript and Dr. Antonio C. M. Camargo gave important advice on various aspects of this study.
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FOOTNOTES |
---|
* This work was supported in part by the São Paulo State Research Foundation Grants 96/1451-9, 99/01983-9, 00/04297-8, 00/11176-2, and 01/07544-9.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.
b Supported by a São Paulo State Research Foundation Ph.D, postdoctoral fellowship Grant 99/07738-6, and is a Ph.D. student in the Molecular Biology Program at the Paulista Medical School/UNIFESP, São Paulo.
d Supported by São Paulo State Research Foundation Ph.D and postdoctoral fellowship Grant 00/11524-0.
g Supported by São Paulo State Research Foundation Ph.D and postdoctoral fellowship Grant 00/01419-5.
i Supported by São Paulo State Research Foundation Ph.D and postdoctoral fellowship Grant 99/06541-4.
k Supported by research fellowships from the Brazilian National Research Council (CNPq).
l To whom correspondence should be addressed: Laboratório de Comunicação Celular, Av. Prof. Lineu Prestes 1524, Salas 431/435, São Paulo, SP, Brazil, 05508-900. Tel./Fax: 55-11-3091-7310; E-mail: eferro@usp.br.
Published, JBC Papers in Press, December 24, 2002, DOI 10.1074/jbc.M212030200
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ABBREVIATIONS |
---|
The abbreviations used are:
MHC, major
histocompatibility complex;
ACE, angiotensin I-converting enzyme;
HPLC, high performance liquid chromatography;
QFS, quenched fluorescent
substrate;
BK, branykinin;
Dyn, dynorphin A1-13;
Aib, -aminoisobutyric acid;
NO, nitric oxide.
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