From the Department of Medicine, University of California, and
Department of Veterans Affairs Medical Center, San Diego, California
92161 and the Department of Vascular Biology, The Scripps
Research Institute, La Jolla, California 92037
Received for publication, February 19, 2001, and in revised form, May 1, 2001
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ABSTRACT |
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Chromogranin A (CgA), the major
soluble protein in catecholamine storage vesicles, serves as a
prohormone that is cleaved into bioactive peptides that inhibit
catecholamine release, providing an autocrine, negative feedback
mechanism for regulating catecholamine responses during stress.
However, the proteases responsible for the processing of CgA and
release of bioactive peptides have not been established. Recently, we
found that chromaffin cells express components of the plasmin(ogen)
system, including tissue plasminogen activator, which is targeted to
catecholamine storage vesicles and released with CgA and catecholamines
in response to sympathoadrenal stimulation, and high affinity cell
surface receptors for plasminogen, to promote plasminogen activation at
the cell surface. In the present study, we investigated processing of
CgA by plasmin and sought to identify specific bioactive CgA peptides
produced by plasmin proteolysis. Highly purified human CgA (hCgA) was
produced by expression in Escherichia coli and purification
using metal affinity chromatography. hCgA was digested with plasmin.
Matrix-assisted laser desorption/ionization mass spectrometry
identified a major peptide produced with a mass/charge ratio
(m/z) of 1546, corresponding uniquely to
hCgA-(360-373), the identity of which was confirmed by reverse
phase high pressure liquid chromatography and amino-terminal microsequencing. hCgA-(360-373) was selectively liberated by plasmin from hCgA at early time points and was stable even after prolonged exposure to plasmin. The corresponding synthetic peptide markedly inhibited nicotine-induced catecholamine release from pheochromocytoma cells. These results identify plasmin as a protease, present in the
local environment of the chromaffin cell, that selectively cleaves CgA
to generate a bioactive fragment, hCgA-(360-373), that inhibits
nicotinic-mediated catecholamine release. These results suggest that
the plasminogen/plasmin system through its interaction with CgA may
play a major role in catecholaminergic function and suggest a specific
mechanism as well as a discrete CgA peptide through which this effect
is mediated.
Chromogranin A (CgA)1 is
the major soluble protein in the core of catecholamine storage vesicles
and is released with catecholamines by exocytosis from chromaffin cells
of the adrenal medulla and from sympathetic neurons (1). CgA is found
ubiquitously in secretory vesicles of neuroendocrine cells (2) and may
be an example of a prototypical prohormone precursor (3). Evidence has
accumulated to suggest that CgA may function as a precursor of several
small, biologically active, secretion-inhibitory peptides that play an
important autocrine regulatory role in neuroendocrine secretion from a
variety of cell types (4-11). In the case of catecholaminergic cells,
CgA serves as a precursor from which peptides are released that
modulate catecholamine secretion, contributing to an autocrine,
homeostatic (negative feedback) mechanism for regulating catecholamine
responses during stress (9-11).
Proteolytic processing of CgA may take place intracellularly as well as
extracellularly (i.e. after secretion into the extracellular space (9, 11, 12)), and indeed, extracellular processing appears to be
particularly important for generation of catecholamine release-inhibitory activity from the CgA molecule in the vicinity of
the chromaffin cell (9, 11). Recent studies suggest a major role for
the plasminogen/plasmin protease system in CgA processing. Plasminogen,
the zymogen for the serine protease and major fibrinolytic enzyme
plasmin, circulates at high concentration (2 µM (13, 14))
in plasma and extracellular fluid and is present in the local
environment of the chromaffin cell. We recently demonstrated that
catecholaminergic cells express components of the fibrinolytic system,
including tissue plasminogen activator (t-PA) (15), the major activator
of this system, as well as high affinity, high capacity binding sites
for plasminogen and t-PA, consistent with the presence of a local,
cellular fibrinolytic system on chromaffin cells (16). We also
demonstrated that perturbation of the local cellular fibrinolytic
system results in profound alterations in secretagogue-mediated
exocytotic release of catecholamines, suggesting a major role for
fibrinolytic molecules in the regulation of catecholamine secretion
(16). Plasmin readily cleaves CgA, and binding of plasminogen to its
cellular binding sites promotes activation of plasminogen to plasmin
and plasmin-mediated CgA processing (16). Furthermore, the peptide
mixture resulting from the interaction between plasmin and CgA markedly
inhibits nicotinic cholinergic stimulation of catecholamine release
from catecholaminergic cells (16). However, the specific peptide(s) released from CgA by plasmin, in particular those that modulate catecholamine secretion, have not been identified.
In the present study, we have focused on the identification of
regulatory peptide(s) produced by the interaction of CgA with plasmin.
We utilized a combined approach that included development of an
expression and purification system for obtaining highly purified human
CgA (hCgA) substrate and controlled plasmin-specific proteolysis,
coupled with matrix-assisted laser desorption/ionization (MALDI) mass
spectrometry, reverse phase HPLC, and amino-terminal amino acid
sequencing, to identify specific cleavage sites and specific CgA
peptide fragments resulting from the interaction of CgA and plasmin.
Our results demonstrate that processing of CgA by plasmin is sufficient
to selectively liberate a discrete CgA fragment with a pronounced
inhibitory effect on catecholamine secretion. These results suggest
that the plasminogen/t-PA system through its interaction with CgA may
play a major role in catecholaminergic function and suggest a specific
mechanism as well as a discrete CgA peptide fragment through which this
effect is mediated. These interactions between catecholaminergic and
fibrinolytic pathways thus have important implications for
cardiovascular regulation.
Construction of the Plasmid pET-(hCgA-6his), Encoding
Histidine-tagged Mature Human CgA--
Human CgA cDNA encoding the
mature protein (minus the 18-residue signal peptide) was generated by
the polymerase chain reaction using the cDNA clone, pGEM-hCgA (from
Dr. Lee Helman, NCI, National Institutes of Health) (17) as
template. The 5' primer,
5'-CATGCCATGGCTCCCTGTGAACAGCCTAT-3', encoded the N-terminal
portion of the mature protein (amino acid residues 1-6) preceded by a
NcoI site (underlined). The 3' primer, 5'-CCGCTCGAGGCCCCGCCGTAGTGCCTGCAGC-3', was complementary to
the 3'-end of the coding region (encoding amino acid residues 433-439) and contained an XhoI site (underlined). For amplification,
10 ng of cDNA were added to 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-Cl, pH 8.0, 25 mM MgCl2,
0.1% Triton X-100, with the two oligonucleotide primers (each at 50 µM), dNTPs (Life Technologies, Inc.) (each at 25 mM) and 2.5 units of Taq DNA polymerase (Applied
Biosystems, PerkinElmer Life Sciences) in a total volume of 100 µl.
Twenty-five polymerase chain reaction cycles were carried out in three
steps consisting of a 1-min denaturation step at 94 °C, 2-min
annealing at 60 °C, and 2-min extension at 72 °C using a PTC-100
Thermal Cycler (MJ Research, Inc., Watertown, MA). The polymerase chain reaction product was digested with NcoI and XhoI
(Life Technologies) and gel-purified. The resulting fragment of 1326 base pairs was directionally cloned into the expression vector, pET-28b
(Novagen, Madison, WI). The final construct was designated
pET-(hCgA-6his) and encoded mature human CgA (439 residues) with the
addition of the residues MPW at the amino terminus and the residues
LEHHHHHH at the C terminus (total of 450 residues for the final
recombinant protein). The nucleotide sequences of the polymerase chain
reaction products and the junction regions between the insert and the
vector were verified from both strands using an Applied Biosystems 373 Automated DNA Sequencer (PerkinElmer Life Sciences).
Expression and Purification of Recombinant Human Chromogranin
A--
One liter of a suspension of Escherichia coli,
BL21(DE3) cells (Novagen), was transformed with the pET-(hCgA-6his)
expression vector, and grown in LB broth containing kanamycin (25 µg/ml) at 37 °C until an A590 of 0.4 was
reached. Isopropyl Radioiodination of Recombinant Human CgA--
Recombinant hCgA
was iodinated using a modified chloramine-T procedure as described
previously (22) and dialyzed against 0.01 M sodium
phosphate, pH 7.4, 0.15 M NaCl (phosphate-buffered saline).
Other Proteins--
Human Glu-plasminogen, the native
circulating form of the molecule, was isolated from fresh human blood
collected into 3 mM benzamidine, 3 mM EDTA, 100 units/ml aprotinin (Miles, Kankakee, IL), and 100 µg/ml soybean
trypsin inhibitor (Sigma). The plasma was subjected to affinity
chromatography on lysine-Sepharose (23) in phosphate-buffered saline
with 1 mM benzamidine, 0.02% NaN3, and 3 mM EDTA followed by molecular exclusion chromatography on Ultrogel AcA44 (IBF Biotechnics, Villeneuve-la-Garennem, France). The
plasminogen concentration was determined spectrophotometrically at 280 nM using an extinction coefficient of 16.8 (24). Urokinase (u-PA) was obtained from Calbiochem.
Mass Spectrometric Analysis--
Purified hCgA was
digested with plasmin, and the reaction mixtures were analyzed by
MALDI mass spectrometry (25, 26). The plasmin digests were dissolved in
0.1% trifluoroacetic acid to a concentration of 10 µM. Either
Mass spectra obtained from the MALDI analyses were compared with values
generated from a theoretical digest of mature hCgA by a protease with
specificity for cleavage at the C-terminal side of basic amino acids,
performed using the program "Sherpa: Your Guide to the Peaks" (27).
This program calculates the molecular weight and the charge-state mass
value m/z (mass/charge ratio) for all possible
fragments generated from a theoretical proteolytic digestion and orders
the fragments by molecular mass.
Reverse-phase High Pressure Liquid Chromatography
(RP-HPLC)--
Fifty micrograms of plasmin-digested hCgA were
separated on a Supelcosil LC-318 column (25 cm × 4.6 mm;
Supelco). The column was equilibrated in 0.1% trifluoroacetic
acid/H2O (eluent A), and the peptides were eluted with a
linear gradient of 0-80% acetonitrile, 0.1% trifluoroacetic
acid (eluent B). The effluent was monitored at 214 nm, and fractions
were collected at 0.5-min intervals. Peak fractions were lyophilized
and stored at Protein Sequence Analysis--
Amino-terminal sequence analysis
was performed using automated Edman microsequencing (Applied Biosystems
Precise Sequencer, model 494, PerkinElmer Life Sciences)
Peptide Synthesis--
Peptides were synthesized at
10-100-µmol scale using Fmoc protection chemistry and purified to
>95% homogeneity by RP-HPLC on a C-18 column. Authenticity and purity
of the peptides were verified by electrospray ionization or MALDI mass spectrometry.
Cell Culture--
PC12 cells (28) (at passage number 8) were
grown at 37 °C, 6% CO2, in Dulbecco's modified
Eagle's medium/high glucose medium supplemented with 5% fetal
bovine serum, 10% horse serum, 100 units/ml penicillin, and 100 µg/ml streptomycin as described previously (15, 29).
Secretagogue-stimulated Catecholamine Release--
Catecholamine
secretion studies were performed as described previously (15, 29).
Briefly, PC12 cells were plated on poly-D-lysine-coated polystyrene dishes (Falcon, Franklin Lakes, NJ) and labeled with L-[3H]norepinephrine (PerkinElmer Life
Sciences) at 1 µCi/ml in the PC12 cell culture medium. After a 3-h
incubation, the cells were washed twice with release buffer (150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM HEPES, pH 7.0) and further
incubated in release buffer at 37 °C for 30 min in either the
presence or absence of the secretagogue, nicotine (60 µM). The release buffer was aspirated, and the cells were
lysed in release buffer containing 0.1% Triton X-100. The amounts of
[3H]norepinephrine in the medium and cell lysates were
measured by liquid scintillation counting. Results were expressed as
percentage of secretion (amount released/(amount released + amount in
cell lysate)) × 100. Net release is secretagogue-stimulated
release minus basal release, where basal norepinephrine release is
typically 5.8 ± 0.36% of total cellular
[3H]norepinephrine released over 30 min
(n = 10 separate secretion assays).
Expression and Purification of Recombinant Human CgA--
We
sought to obtain highly purified CgA in order to precisely characterize
the peptide products generated by plasmin proteolysis. Results of
expression and purification of recombinant hCgA using the construct
pET-(hCgA-6his) are shown in Fig. 1.
Aliquots from cultures of E. coli strain BL21(DE3) harboring
plasmid pET-(hCgA-6his) were removed at the indicated times following
IPTG induction, and total cellular proteins were examined by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A protein band, with
an apparent molecular mass of 70 kDa, corresponding to the recombinant
hCgA was observed 30 min after IPTG induction (Fig. 1, lane
2).2 The
recombinant hCgA was estimated to comprise ~20-25% of the total
cellular protein 3 h after IPTG induction (Fig. 1, lane 5) based on the Coomassie Blue-stained gels.
E. coli cell lysates were subjected to affinity
chromatography using the Ni2+-nitrilotriacetic
acid-Sepharose column as described under "Experimental Procedures."
The peak fraction containing purified recombinant hCgA (Fig. 1,
lane 6) was electrophoresed, and the 70-kDa band was cut out and electroeluted as described under "Experimental Procedures." Based on Coomassie Blue staining, the human CgA (Fig. 1,
lane 7) was 99.9% pure. When the recombinant
hCgA was analyzed by MALDI mass spectrometry, the entire spectra
encompassing the mass range of 1000-80,000 Da showed only singly
charged and doubly charged signals at m/z of
50,976 and 25,495 (see below; Fig. 3, A-C). The observed
mass value of 50,976 × 1 for the singly charged signal (or
25,495 × 2 for the doubly charged signal) is consistent with the
molecular mass calculated from the primary sequence of the 450-residue
recombinant hCgA.3 These
results (Fig. 3, A-C) demonstrated that the hCgA substrate obtained was highly purified (without contaminating small peptides) for
subsequent proteolytic studies.
Proteolysis of Recombinant hCgA by Plasmin--
We examined
whether recombinant hCgA could be processed by plasmin. Plasmin readily
digested the recombinant hCgA, and the extent of the digestion was
dose-dependent (Fig. 2).
Major products were sequentially produced with apparent molecular
masses of 65, 55, and 43 kDa, as detected using SDS-PAGE.
Identification of a 1546 m/z CgA Peptide Derived from Plasmin
Proteolysis--
To identify the peptides produced by plasmin
proteolysis of hCgA, mass spectrometric analysis was applied. The
mixture of proteolytic products of hCgA after digestion with plasmin
for 4 h was analyzed by MALDI mass spectrometry. The resulting
mass spectra are shown in Fig. 3
(D-F). The most prominent peak was observed at
m/z = 1546. A time course of proteolysis of
hCgA by plasmin was performed, and the proteolytic products were
analyzed on the mass spectrometer (Fig.
4). The 1546 m/z
peptide was the predominant species observed at 15 min (Fig.
4A) and was still the predominant species with an even
stronger signal after incubation for 45 and 120 min (Fig. 4,
B and C). Additional time course experiments demonstrated the appearance of this peptide at even earlier time points
(5 min) and also demonstrated that this peptide was stable to further
cleavage even after prolonged incubation for 8 h (data not shown).
In control experiments, MALDI analysis of plasminogen plus urokinase
alone incubated for 2 h at 37 °C and incubation of hCgA alone
for 2 h at 37 °C gave no peaks in the m/z
range of 500-5000, showing that neither of these control incubations generated any detectable peptides (data not shown). Thus, the observed 1546 m/z peak was specifically generated
by the interaction between plasmin and hCgA.
To investigate the identity of the 1546 m/z hCgA
peptide produced by the interaction of plasmin and hCgA, these data
were analyzed and compared against specific mass information generated from a theoretical digest, using the program Sherpa (27). This program
calculates molecular weight and the charge state
m/z values for all possible fragments generated
from a theoretical proteolytic digest and orders them by molecular
mass. Based on the known primary sequence of mature hCgA, a theoretical
digest was carried out, cleaving at basic (arginine or lysine) residues
(known recognition sites for plasmin (30)) within hCgA and allowing for
partial digests (with up to 7 basic residue skips). This analysis
generated 403 possible peptides ranging in size from 75.04 m/z (a single glycine residue) to 13,519.21 m/z. Among these peptides within the primary
sequence of hCgA, the observed 1546 m/z peak
corresponded uniquely to the 14-mer, hCgA-(360-373) (ARAYGFRGPGPQLR)
(calculated molecular mass = 1545.77 Da).
We next synthesized this 14-mer peptide, using Fmoc protection
chemistry, and purified the peptide to >95% homogeneity by reverse
phase-HPLC on a C-18 column. Mass spectrometric analysis of the peptide
resulting from this synthesis using both MALDI-MS (see below; Fig.
6A) and electrospray (data not shown) revealed an observed
m/z of 1546, coinciding with its theoretical mass and identical to the experimental mass (determined by MALDI-MS) of the
peptide recovered from our plasmin digest of hCgA (Figs. 3 and 4).
To confirm the identity of the 1546 m/z peptide
generated from the interaction between plasmin and hCgA, we applied
RP-HPLC in combination with MALDI mass spectrometry and
microsequencing. The plasmin-digested hCgA mixture was first separated
by RP-HPLC on a Supelcosil LC-318 column. A major peak (marked with an
arrowhead in Fig.
5A) was identified at a
retention time of 19.5-20 min, a retention time that corresponded to
that of the synthetic 14-mer, hCgA-(360-373), determined in parallel
experiments. The peak fraction from the plasmin digest was collected
and analyzed by MALDI mass spectrometry. The detected molecular mass of
the major peptide peak in this HPLC fraction was 1546 (Fig.
5B). This same eluted RP-HPLC fraction was also subjected to
amino-terminal microsequencing. Using Edman degradation microsequencing
over 7-residue cycles, the first 7 amino-terminal residues were
determined to be ARAYGFR, precisely matching the N-terminal sequence of
the 14-mer, hCgA-(360-373).
Incubation of hCgA Synthetic Peptides, hCgA-(352-372) and
hCgA-(360-373), with Plasmin--
In a previous study, we screened
synthetic peptides spanning ~80% of the length of bovine CgA (11)
and identified a 21-mer, bovine chromogranin A-(344-364)
(RSMRLSFRARGYGFRGPGLQL), which demonstrated a pronounced inhibitory
effect on catecholamine secretion ("catestatin") (Table
I). Its human homolog, hCgA-(352-372)
(SSMKLSFRARAYGFRGPGPQL), also significantly suppressed catecholamine
secretion by pheochromocytoma and adrenal chromaffin cells (11). We
tested whether human "catestatin," hCgA-(352-372), or the 14-mer
hCgA-(360-373), ARAYGFRGPGPQLR, could be further processed by plasmin.
Both peptides were subjected to MALDI-MS analyses before and after
incubation with plasmin. Before plasmin treatment, each spectrum showed
a clear peak, with m/z of either 1546 or 2326, corresponding to the 1546 peptide (Fig.
6A) or the hCgA-(352-372)
(21-mer) (Fig. 6C), respectively. After plasmin digestion,
the spectrum in Fig. 6B showed no difference from that in
Fig. 6A (single peak at m/z = 1546), whereas the spectrum in Fig. 6D contained a new peak
at an m/z of 1389. By analysis of the mass
spectrometric data using the Sherpa program, the 1389 m/z peak was identified as ARAYGFRGPGPQL,
corresponding to hCgA-(360-372). Thus, hCgA-(352-372) was
selectively cleaved by plasmin at Arg359 within the 21-mer
hCgA sequence (hCgA-(352-372),
SSMKLSFR359 Effect of the 14-Mer, hCgA-(360-373), on Secretagogue-stimulated
Catecholamine Secretion from PC12 Cells--
To test whether the
14-mer, hCgA360-373 (ARAYGFRGPGPQLR), could inhibit
secretagogue-stimulated catecholamine release, the effect of synthetic
hCgA-(360-373) was evaluated on nicotine-induced norepinephrine
secretion from PC12 pheochromocytoma cells (Fig. 7). (Nicotine acts at nicotinic
cholinergic receptors, as does acetylcholine, the major physiologic
stimulus mediating catecholamine release from chromaffin cells.) The
peptide, ARAYGFRGPGPQLR (hCgA-(360-373)), inhibited nicotine-induced
catecholamine release in a dose-dependent fashion with
marked inhibition of catecholamine release in the micromolar range
(IC50 = 3.0 ± 0.046 µM). In control
experiments with the reverse peptide, RLQPGPGRFGYARA, no inhibition
was detected (Fig. 7).
In this study, we have demonstrated that proteolysis of CgA with
the major fibrinolytic enzyme, plasmin, results in the production of a
discrete and stable peptide, ARAYGFRGPGPQLR, corresponding to human
CgA-(360-373), which markedly inhibits secretagogue-stimulated catecholamine release from catecholaminergic cells. These results suggest important interactions between catecholaminergic and
fibrinolytic systems that may have a profound influence on
cardiovascular regulation.
In earlier studies done to demonstrate that CgA may function as a
prohormone, catecholamine release-inhibitory activity was generated by
incubation of CgA with the serine proteases, trypsin (9), and the
bacterial enzyme, endoproteinase Lys C (10), proteases that are not
present in the environment of the chromaffin cell. More recently, we
demonstrated that CgA is readily processed by plasmin, which also
results in generation of a peptide mixture with catecholamine
release-inhibitory activity, significantly inhibiting
nicotine-mediated catecholamine release from PC12 cells and
primary bovine adrenal chromaffin cells (16). However, the specific
peptide(s) that inhibited catecholamine release had not been
identified. In a previous study (11), we screened synthetic peptides
spanning ~80% of the linear sequence of mature bovine CgA. Using
this screening approach, we identified a peptide domain approximated by
the region corresponding to bovine CgA-(344-364) (human
CgA-(352-372)), referred to as "catestatin," which inhibited secretagogue-stimulated catecholamine release. Interestingly, the
peptide ARAYGFRGPGPQLR, produced by plasminolytic cleavage of human CgA
and identified in the current study, is contained within the catestatin
sequence with the addition of an Arg at the carboxyl terminus of the
peptide (Table I). Thus, using two independent approaches, this region
of the CgA molecule has been demonstrated to harbor catecholamine
secretion-inhibitory activity.
We investigated processing of CgA by plasmin because plasminogen
circulates at a high concentration (2 µM (13)) and is
present in the local chromaffin cell environment. In addition, our
previous studies revealed that chromaffin cells express the major
activator of this system, t-PA, which is targeted to catecholamine
storage vesicles and is co-released along with catecholamines and the substrate CgA, from this subcellular storage pool in response to
chromaffin cell stimulation (15). Moreover, these cells express high
affinity, high capacity cell surface binding sites for plasminogen (16)
as a means for localizing and concentrating the activity of this
protease system at the cell surface, where plasminogen is rapidly
activated to plasmin by t-PA released from intracellular secretory
vesicular stores during chromaffin cell stimulation. These results,
then, suggested the presence of a local, chromaffin cell,
plasminogen/t-PA system, through which chromaffin cells have the
ability to concentrate and spatially organize plasmin activity in the
local environment into which CgA is secreted, providing a logical
microanatomic and physiologic rationale for a mechanism for local
extracellular proteolytic processing of CgA.
In other previous experiments, overexpression of t-PA in PC12 cells
resulted in markedly diminished secretagoguestimulated catecholamine release. Conversely, when plasmin activity was inhibited using a monoclonal antibody against the catalytic domain of
plasmin, nicotine-stimulated catecholamine secretion was
substantially increased in these cells (16). Thus, both positive and
negative modulation of the local chromaffin cell plasminogen/t-PA
system resulted in marked alterations in secretagogue-mediated
exocytotic release of catecholamines, suggesting a major role for
fibrinolytic molecules in the regulation of catecholamine secretion. We
therefore sought to identify regulatory peptide(s) produced when CgA
interacts with plasmin, in particular focusing on regulatory CgA
fragments with catecholamine release-inhibitory activity, as a
mechanism through which the effects of the plasminogen/t-PA system
might mediate regulatory effects on catecholamine secretion.
To examine the proteolytic effects of plasmin on CgA, we prepared
highly purified recombinant human CgA and exposed this highly purified
substrate to plasmin in controlled proteolytic studies. Recombinant
human CgA was readily processed by plasmin in a
dose-dependent fashion. We used a combined approach that
included mass spectrometry, RP-HPLC, and amino-terminal microsequencing
to confirm the identity of the specific peptides formed from the
interaction of CgA with plasmin. This approach utilizes specific mass
information of the peptides generated and the known primary sequence of
the CgA substrate. Such an approach has been shown to be highly
effective in the identification of peptide fragments from complex,
heterogeneous samples, including those generated from proteolytic
digests (25, 26, 31-37). Examination of the peptide mixture produced
by plasmin-mediated cleavage of CgA by MALDI mass spectrometry
identified a major peptide peak at 1546 m/z.
These data were compared against specific mass information generated
from a theoretical digest (with cleavages at basic residues) of the
known primary sequence of mature human CgA. This analysis revealed that
the observed 1546 m/z peak corresponded uniquely to the mass of the peptide, ARAYGFRGPGPQLR, a 14-mer corresponding to
hCgA-(360-373), results that were hence consistent with proteolytic cleavage on the C-terminal side of residues Arg359 and
Arg373 and also consistent with the known proteolytic
substrate specificity of plasmin (cleavage at the C terminus of basic
residues) (30).
The identity of the peptide produced from the interaction of CgA with
plasmin was further established by several independent criteria. First,
when the digestion mixture of CgA and plasmin was separated by RP-HPLC,
a major peak was noted at a retention time corresponding to the
retention time of the synthetic peptide, ARAYGFRGPGPQL (corresponding
to hCgA-(360-373)). Second, MALDI mass spectrometric
analysis of the peak HPLC fraction and of the synthetic peptide,
ARAYGFRGPGPQLR, yielded identical molecular mass determinations of 1546 m/z (identical to the experimental mass,
determined by MALDI-MS, of the peptide recovered from plasmin digests
of CgA). Third, amino-terminal microsequencing of the peak HPLC
fraction, revealed an amino-terminal sequence of ARAYGFR, precisely
matching the amino-terminal sequence of the 14-mer, hCgA-(360-373). The observed 1546 m/z peptide was specifically liberated by the
interaction between hCgA and plasmin and not observed in control
experiments, including experiments with hCgA substrate alone or with
plasminogen plus plasminogen activator without substrate.
We found that ARAYGFRGPGPQLR (corresponding to hCgA-(360-373))
exhibited bioactivity. In functional secretagogue-mediated release
studies, hCgA-(360-373) markedly inhibited nicotine-induced catecholamine secretion from pheochromocytoma cells. In control experiments, the reverse peptide, RLQPGPGRFGYARA, had no effect on
nicotine-mediated catecholamine release, demonstrating the specificity
of this effect for hCgA-(360-373). These results suggest that the
interaction between CgA and plasmin is sufficient to liberate a
specific peptide from the CgA catestatin domain, which is bioactive,
with pronounced effects on catecholamine secretion.
Our results also demonstrated that cleavage of CgA by plasmin occurred
in a selective fashion, with processing at specific internal cleavage
sites within the CgA sequence. Multiple digestion experiments and time
course studies revealed that the 14-mer, hCgA-(360-373), was
consistently and selectively liberated from CgA. In addition, the
peptide was released from the parent CgA protein at early time points,
and the peptide fragment formed was quite stable, with no evidence of
further internal cleavages (for example, at Arg361 or
Arg366) within the hCgA-(360-373) sequence, even after
prolonged exposure to plasmin. The stability of hCgA-(360-373) and the
selectivity of the cleavage site at Arg359 were also
demonstrated in experiments in which plasmin was incubated with the
synthetic peptides, hCgA-(360-373) and hCgA-(352-372) (Fig. 6). These
findings (i.e. early, robust cleavage of the peptide from
the CgA substrate and stability (resistance to further internal cleavages by the protease responsible for its liberation)) are also
consistent with an important physiological role for this peptide
fragment. Thus, taken together, these results suggest that plasmin may
indeed act upon CgA in a highly selective fashion (cleaving CgA at the
basic residues Arg359 and Arg373 but not
cleaving at Arg361 and Arg366) to liberate a
specific bioactive peptide, hCgA-(360-373), with catestatin activity.
The cleavage sites at Arg359 and Arg373 are
particularly noteworthy in light of recent studies utilizing substrate
phage display and peptide substrates, which revealed that several
primary sequence determinants in the P2P1 Also of note, interspecies comparison of the primary sequence of CgA
(including human (20), bovine (19), porcine (39), rat (21), and mouse
(40) sequences) reveals that the cleavage sites at Arg359
and Arg373 (numbering according to the human sequence) are
completely conserved, and the additional preferential substrate
P2P1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside (IPTG) was
then added to a final concentration of 1 mM. Three hours
following IPTG induction, the cells were harvested by centrifugation at
7500 × g for 5 min at 4 °C. The cells were lysed in
100 ml of buffer A (6 M guanidine hydrochloride, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 8.0) by
stirring at 4 °C for 18 h. The lysate was centrifuged at
27,000 × g for 30 min at 4 °C. The supernatant was
loaded onto a Ni2+-nitrilotriacetic acid-Sepharose column
(1.5 × 4 cm), preequilibrated with 5 column volumes (25 ml) of
buffer A. The column was washed at a flow rate of 15 ml/h, with 30 ml
of buffer A, followed by 20 ml of buffer B (8 M urea, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 8.0)
until the effluent had an A280 of <0.01. The
bound proteins were eluted with a pH gradient of 5.9-4.5 (composed of
8 ml of buffer B adjusted to pH 5.9 and 4 ml of buffer B adjusted to pH 4.5) according to the manufacturer's instructions (Qiagen, Santa Clarita, CA). Fractions of 1 ml were collected, and 5 µl of each fraction were electrophoresed on SDS-polyacrylamide gels under reducing
conditions followed by Coomassie Blue staining. The affinity-purified recombinant hCgA was identified based on the reported apparent molecular mass for native CgA, 70 kDa (17-21). The peak fractions were
pooled and electrophoresed on SDS-polyacrylamide gels under reducing
conditions. The band migrating with an apparent molecular mass of 70 kDa was cut out and placed into a sample trap with a 10,000 molecular
weight cut-off dialysis membrane. The gel slices were soaked in 10 mM Tris acetate, 1 mM EDTA, pH 7.6. The
proteins were eluted at 4 °C in electrophoresis buffer (40 mM Tris acetate, 1 mM EDTA, pH 7.6) following
the manufacturer's instructions (ISCO, Inc., Lincoln, NE). After
electroelution, hCgA was dialyzed into 0.01 M Tris-HCl, pH
8.0, 1 mM EDTA. The purified hCgA was concentrated, and its
concentration was determined using the Bradford dye-binding assay
(Bio-Rad protein assay).
-cyano-4-hydroxycinnamic acid or sinapinic
acid was used as the UV laser desorption matrix depending on the mass
ranges included in the analysis. Samples of 5-10 pmol were loaded onto
the mass spectrometer sample probe and dried at ambient temperature.
Mass spectra were acquired using a Voyager-Elite mass spectrometer
(Perseptive Biosystems, Houston, TX).
20 °C.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression and purification of hCgA.
E. coli BL21 (DE3) cells transformed with recombinant
plasmid pET-(hCgA-6his) were cultured in the presence of IPTG for
various times. Samples were taken at 0 min (lane
1), 30 min (lane 2), 90 min
(lane 3), 120 min (lane 4),
and 180 min (lane 5) after IPTG induction. The
samples were lysed, total cellular proteins were analyzed on SDS-PAGE
under reducing conditions, and gels were stained with Coomassie Blue.
Three hours after IPTG induction, cell pellets were solubilized in 6 M guanidine HCl, pH 8.0, and applied to a
Ni2+-nitrilotriacetic acid-Sepharose column, and the bound
proteins were eluted with a pH gradient as described under
"Experimental Procedures." Lane 6 shows 50 µg of the peak fraction containing purified recombinant hCgA.
Lane 7 shows 2.5 µg of purified recombinant
hCgA after electroelution.
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Fig. 2.
Proteolysis of 125I-hCgA by
plasmin. 125I-hCgA (5 nM) was incubated at
37 °C with either buffer alone (0.01 M Tris Cl, pH 8.0, 0.15 M NaCl) or buffer plus increasing concentrations of
plasmin for 1 h. Samples were analyzed by SDS-PAGE on 10% gels.
The gels were dried and exposed at 80 °C to Kodak X-Omat AR film
with an image-intensifying screen.
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Fig. 3.
MALDI-MS of purified recombinant hCgA and its
proteolytic products detected after plasmin digestion. Five pmoles
of the recombinant hCgA dissolved in 0.1% trifluoroacetic acid were
analyzed on a Voyager-Elite mass spectrometer using
-cyano-4-hydroxycinnamic acid (A and B) and
sinapinic acid (C) as matrices. The entire spectra from mass
range of 1000 to 80,000 Da (A
C) showed only
[M + H]+ and [M + 2H]2+ signals at
m/z of 50,976 and 25,495. Human CgA (at 10 µM) was incubated with plasminogen (2 µM)
and urokinase (10 nM) at 37 °C for 4 h. The
reaction was stopped by the addition of aprotinin (2.5 µM), 5 pmol of the reaction mixture were loaded onto the
mass spectrometer sample probe, and the same mass range was scanned
using matrices of
-cyano-4-hydroxycinnamic acid (D and
E) and sinapinic acid (F). A prominent peak was
observed at an m/z of 1546.
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Fig. 4.
MALDI-MS of recombinant hCgA digested with
plasmin for various times. Purified recombinant hCgA (10 µM/reaction) was incubated at 37 °C with plasminogen
(2 µM) and urokinase (10 nM) in buffer (0.01 M Tris-Cl, pH 8.0, 0.15 M NaCl) for various
times, and the reaction was stopped by the addition of aprotinin (2.5 µM). Five pmoles of digested recombinant hCgA were
analyzed on the mass spectrometer using -cyano-4-hydroxycinnamic
acid as matrix. A predominant 1546 m/z peak was
observed after a 15-min incubation with plasminogen and u-PA
(A) and was present with a stronger signal after longer
incubations of 45 min (B) and 120 min (C).
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Fig. 5.
Isolation and identification of the major
peptide from the plasmin digestion of hCgA by reverse phase HPLC,
MALDI-MS, and amino-terminal sequence analysis. Fifty micrograms
of hCgA (10 µM) were incubated at 37 °C with
plasminogen (2 µM) and u-PA (10 nM) in 0.01 M Tris-Cl, pH 8.0, 0.15 M NaCl for 2 h,
and the reaction was stopped by the addition of aprotinin (2.5 µM). The reaction mixture was chromatographed on a
Supelcosil LC-318 column (25 cm × 4.6 mm). The column was
equilibrated in 0.1% trifluoroacetic acid/H2O (eluent A),
and the peptides were eluted in a linear gradient of 0-80%
acetonitrile, 0.1% trifluoroacetic acid (eluent B). The effluent was
monitored at 214 nm, and fractions were collected at 0.5-min intervals
(see A). Approximately 5 pmol of recovered peptide from
fraction 40 (retention time 19.5-20 min, marked with an
arrowhead in A) was analyzed by MALDI-MS
(B), which revealed a single peptide peak consistent with
the 14-mer peptide, ARAYGFRGPGPQLR. Amino-terminal sequence analysis of
the same HPLC fraction revealed that the first 7 residues were
ARAYGFR, matching the amino-terminal sequence of hCgA-(360-373).
ARAYGFRGPGPQL) to yield hCgA-(360-372),
ARAYGFRGPGPQL (see Fig. 6). No cleavage at either Arg361 or
Arg366 within the 21-mer catestatin peptide was
observed.
Relationship of human CgA-(360-373) to human and bovine synthetic
catestatin
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Fig. 6.
MALDI-MS of synthetic CgA peptides,
hCgA-(360-373) (14-mer) and hCgA-(352-372) (21-mer), before and after
incubation with plasmin. Synthetic peptides were dissolved in
0.1% trifluoroacetic acid to a final concentration of 10 µM and analyzed on a Voyage-Elite mass spectrometer using
-cyano-4-hydroxycinnamic acid as matrix (A and
C). Each spectrum showed a clear peak at
m/z of 1546 or 2326, representing the 14-mer
(A) or 21-mer (C), respectively. After incubation
of the peptides (10 µM) with plasminogen (2 µM) and urokinase (10 nM) for 2 h in
0.01 M Tris-Cl, pH 8.0, 2 mM CaCl2,
reactions were stopped by the addition of 2.5 µM
aprotinin. Samples were loaded onto the mass spectrometer sample probe
and scanned over the mass range of 500 to 3500 Da (B and
D). The spectrum in B showed no difference from
that in A, whereas the spectrum in D generated a
new peak at m/z of 1389, demonstrating processing
of the 21-mer at the Arg359 residue within the hCgA
sequence (hCgA-(352-372),
SSMKLSFR359
ARAYGFRGPGPQL to hCgA-(360-372),
ARAYGFRGPGPQL).
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Fig. 7.
Effect of the 14-mer, hCgA-(360-373), on
secretagogue-stimulated norepinephrine secretion by PC12 cells.
PC12 cells were labeled with
L-[3H]norepinephrine and treated with 60 µM nicotine, either alone or in combination with varying
concentrations of either the 14-mer hCgA-(360-373) (ARAYGFRGPGPQLR)
(filled circles) or the reverse peptide
(hCgA-(373-360), RLQPGPGRFGYARA) (filled
triangles). After a 30-min incubation, cells and media were
examined for norepinephrine secretion as described under
"Experimental Procedures." Control (100%) net norepinephrine
release is release in the presence of 60 µM nicotine
alone without peptides and was 29.5 ± 0.8% (n = 6) of the total cellular
L-[3H]norepinephrine.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
P1'P2' positions may
enhance substrate cleavage specificity of plasmin. These include a
basic residue (Arg or Lys) at the cleavage site (at the P1 position);
an aromatic residue, in particular Phe or Tyr, at the P2 position;
residues Arg, Ser > Lys, Gly at P1'; and Arg, Lys, or Gly at the P2'
position (38). Both of the observed cleavage sites, at hCgA residue 359 (FR359
AR) and hCgA residue 373 (LR373
RG),
exhibit three out of four of these preferential sequence characteristics, which may thus contribute to the selective nature of
the proteolytic action of plasmin upon CgA.
P1'P2' sequence characteristics are also well conserved across
species (Table II). In addition, the
amino acid sequence of the bioactive 14-mer hCgA-(360-373), liberated
from CgA by plasmin, is highly conserved with observed sequence
homologies of 86-93% (12/14 residues for bovine/human, porcine/human,
or mouse/human comparisons and 13/14 residues comparing the rat with
the human sequence) (Table II).
Interspecies comparison of the human CgA-(360-373) peptide and its
flanking residues
Finally, we examined a range of substrate and enzyme concentrations likely to occur physiologically in the milieu of the chromaffin cell (nanomolar to micromolar range) and noted extensive processing of CgA by plasmin. In addition, the concentration of CgA within the chromaffin vesicle is extremely high, ~4 mM, so that following exocytosis, the concentration of CgA in the local extracellular space may be as high as 0.4 mM (41), much greater than the concentrations examined here. In addition, plasminogen circulates at high concentration (2 µM (13)) and, as we have recently demonstrated, is concentrated through specific plasminogen binding sites at the cell surface (16). Thus, the local increase in both CgA substrate concentration and plasminogen concentration at the cell surface brought about through these mechanisms would further enhance processing of CgA in the environment of the cell after the exocytotic release reaction.
In summary, the results of this study demonstrate that processing of
CgA by plasmin is sufficient to selectively liberate a discrete and
stable CgA fragment with a pronounced inhibitory effect on
catecholamine secretion. These results suggest that the
plasminogen/t-PA system through its interaction with CgA may play a
major role in catecholaminergic function and suggest a specific
mechanism as well as a discrete CgA peptide fragment through which this
effect is mediated. These interactions between catecholaminergic and
fibrinolytic pathways may have important implications for
cardiovascular regulation under normal and pathophysiologic conditions,
including regulation of catecholamine release during stress.
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ACKNOWLEDGEMENTS |
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We are grateful to Lee Helman for providing the hCgA cDNA plasmid, pGEM-hCgA. We appreciate protein sequencing assistance by Matthew Williamson in the laboratory of Dr. Paul Price (Department of Biology, University of California, San Diego) and mass spectrometry expertise by Ken Harris and Dr. Gary Siuzdak (The Scripps Research Institute, La Jolla, CA).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL-50398, HL-58120, HL-38272, HL-07261, and DK-07671 and by the Department of Veterans Affairs.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.
§ To whom correspondence should be addressed: Nephrology/Hypertension (9111-H), University of California, San Diego, 3350 La Jolla Village Dr., San Diego, CA 92161. Tel.: 858-552-8585 (ext. 7356); Fax: 858-552-7549; E-mail: rparmer@ucsd.edu.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M101545200
2 CgA has a highly anomolous electrophoretic mobility in SDS-polyacrylamide gels. Whereas the predicted molecular mass of CgA is 50 kDa, the apparent molecular mass by SDS-PAGE is 70-75 kDa (17-21).
3 m/z, the mass/charge ratio, depends on the number of charges on the protein generated by ionization, such that a given protein generates more than one ion signal detected by the mass spectrometer. Thus, the detected m/z ratios of 50,976 and 25,495 represent the 450-residue recombinant hCgA with charge states of 1+ and 2+, respectively.
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ABBREVIATIONS |
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The abbreviations used are:
CgA, chromogranin A;
hCgA, human chromogranin A;
MALDI, matrix-assisted laser
desorption/ionization;
MS, mass spectrometry;
IPTG, isopropyl
-D-thiogalactopyranoside;
t-PA, tissue plasminogen
activator;
u-PA, urokinase plasminogen activator;
PAGE, polyacrylamide
gel electrophoresis;
Fmoc, N-(9-fluor-enyl)methoxycarbonyl;
HPLC, high pressure
liquid chromatography;
RP-HPLC, reverse phase HPLC.
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