From the Department of Biochemistry,
National Cardiovascular Center Research Institute, Suita, Osaka
565-8565, the ¶ Institute of Life Science, Kurume University,
Kurume, Fukuoka 839-0861, the Departments of
Surgery and
** Post-Genomics and Diseases, Osaka University Graduate
School of Medicine, Suita, Osaka 565-0871, and the
§ Translational Research Center, Kyoto University Hospital,
Kyoto, 606-8507, Japan
Received for publication, May 30, 2002, and in revised form, September 26, 2002
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ABSTRACT |
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Ghrelin, a novel 28-amino acid
peptide with an n-octanoyl modification at
Ser3, was isolated from rat stomach and found to be an
endogenous ligand for the growth-hormone secretagogue receptor (GHS-R).
This octanoyl modification is essential for ghrelin-induced GH release. We report here the purification and identification of human ghrelin from the stomach, as well as structural analysis of the human ghrelin
gene and quantitation of changes in plasma ghrelin concentration before
and after gastrectomy. Human ghrelin was purified from the stomach by
gel filtration and high performance liquid chromatography, using a
ghrelin-specific radioimmunoassay and an intracellular calcium influx
assay on a stable cell line expressing GHS-R to test the fractions. In
the course of purification, we isolated human ghrelin of the expected
size, as well as several other ghrelin-derived molecules. Classified
into four groups by the type of acylation observed at Ser3;
these peptides were found to be non-acylated, octanoylated (C8:0), decanoylated (C10:0), and possibly decenoylated (C10:1). All peptides found were either 27 or 28 amino acids in length, the former lacking the C-terminal Arg28, and are derived from the same ghrelin
precursor through two alternative pathways. The major active form of
human ghrelin is a 28-amino acid peptide octanoylated at
Ser3, as was found for rat ghrelin. Synthetic octanoylated
and decanoylated ghrelins produce intracellular calcium increases in
GHS-R-expressing cells and stimulate GH release in rats to
a similar degree. Both ghrelin and the ghrelin-derived molecules were
found to be present in plasma as well as stomach tissue. Plasma levels
of immunoreactive ghrelin after total gastrectomy in three patients
were reduced to approximately half of their pre-gastrectomy values,
after which they gradually increased. This suggests that the stomach is
the major source of circulating ghrelin and that other tissues
compensate for the loss of ghrelin production after gastrectomy.
Growth hormone (GH)1
secretion from the pituitary gland is regulated by two hypothalamic
hormones, growth hormone-releasing hormone and somatostatin (1, 2). A
third independent pathway responsible for regulation of GH release has
recently emerged from studies of artificial GH secretagogues (GHSs) (3,
4). GHSs are synthetic compounds that are potent stimulators of
pituitary GH release, acting through the GHS receptor (GHS-R) (5-7).
Previously, we identified ghrelin, an endogenous ligand for GHS-R, from
rat stomach (8). Ghrelin, a 28-amino acid peptide capable of
stimulating GH release in vitro and in vivo, has
a unique n-octanoyl modification at its third serine residue
(Ser3), which is essential for this function (9-11).
Subsequently, des-Gln14-ghrelin, also isolated from rat
stomach, was identified as a second endogenous ligand for GHS-R (12).
Des-Gln14-ghrelin is produced from the ghrelin gene by an
alternative splicing mechanism and is also octanoylated at
Ser3.
In the present study, we purified human ghrelin from the stomach, using
a ghrelin-specific radioimmunoassay (RIA) (13) and an intracellular
calcium influx assay on a stable cell line expressing GHS-R (8, 12).
During the course of purification, we noticed several minor peptides
with characteristics different from standard ghrelin that displayed
ghrelin-like activity. We identified these stomach peptides as
ghrelin-derived molecules and examined the levels of ghrelin as well as
these ghrelin-derived molecules in human plasma.
The ghrelin gene is abundantly expressed in rat (8) and human (14)
stomach, and in the rat, no other major sources of ghrelin production
have been observed (13, 15). These results prompted us to question
whether ghrelin should be drastically reduced following gastrectomy. To
address this question, we examined the change in plasma levels of
immunoreactive ghrelin (ir-ghrelin) in humans before and after total gastrectomy.
Radioimmunoassays for Ghrelin--
RIAs specific for ghrelin
were performed as previously described (13). In rabbits two types of
polyclonal antibodies were raised against the N-terminal fragment
(Gly1-Lys11 with
O-n-octanoylation at Ser3) and the
C-terminal fragment (Gln13-Arg28) of rat
ghrelin. The RIA incubation mixture consisted of 100 µl of standard
ghrelin or unknown sample, and 200 µl of antiserum diluted with
RIA buffer (50 mM sodium phosphate buffer (pH 7.4), 0.5%
bovine serum albumin, 0.5% Triton X-100, 80 mM NaCl, 25 mM Na2EDTA,A. O. and 0.05%
NaN3) containing 0.5% normal rabbit serum. The anti-rat
ghrelin-(1-11) or anti-rat ghrelin-(13-28) antisera were used at
final dilutions of 1:6,000,000 and 1:20,000, respectively. After
incubation for 12 h, 100 µl of 125I-labeled tracer
(15,000 cpm) was added. Thirty-six hours later, 100 µl of anti-rabbit
IgG goat serum was added. After 24 h of incubation, free and bound
tracers were separated by centrifugation at 3,000 rpm for 30 min.
Pellet radioactivity was counted with a gamma counter (ARC-600, Aloka,
Tokyo). All assay procedures were performed in duplicate at
4 °C.
Both types of antisera exhibited 100% cross-reactivity with human and
rat ghrelins. The anti-rat ghrelin-(1-11) antiserum specifically recognized the n-octanoylated portion at
Ser3 of ghrelin and did not recognize des-acyl ghrelin. The
anti-rat ghrelin-(13-28) antiserum equally recognized
n-octanoyl and des-acyl ghrelins. In the following
sections, the RIA system using antiserum against the N-terminal
fragment of rat ghrelin-(1-11) is termed N-RIA; the RIA system using
antiserum against the C-terminal fragment (13-28) is termed C-RIA.
Detection of Ghrelin Activity by Calcium Mobilization
Assay--
CHO-GHSR62 cells, which stably express rat GHS-R (8, 12),
were plated in flat-bottom, 96-well black-wall plates (Corning Inc.,
Corning, NY) at 4 × 104 cells/well for 12 h prior to the assay. The cells were loaded with 4 µM
Fluo-4-AM fluorescent indicator dye (Molecular Probes, Inc., Eugene,
OR) for 1 h in assay buffer (Hanks' balanced salts solution, 20 mM HEPES, 2.5 mM probenecid, 1% fetal calf
serum) and washed four times in assay buffer without fetal calf serum. Intracellular calcium concentration
([Ca2+]i) changes were measured using a
fluorometric imaging plate reader (FLIPR, Molecular Devices, Sunnyvale,
CA). Maximum changes in fluorescence compared with the baseline were
used to quantitate the agonist responses.
Purification of Human Ghrelins from Stomach--
A human stomach
mucosa (27 g) was minced and boiled for 5 min in 5× volumes of water
to inactivate intrinsic proteases. The solution was adjusted to 1 M acetic acid (AcOH)-20 mM HCl. The stomach
tissue was homogenized with a Polytron mixer. The supernatant of the
extract, obtained after 30-min centrifugation at 11,000 rpm, was
concentrated to ~25 ml by evaporation. The residual concentrate was
subjected to acetone precipitation in 66% acetone. After removal of
the precipitate, the supernatant was evaporated to remove the acetone
and then loaded onto a 10-g cartridge of Sep-Pak C18 (Waters, Milford,
MA), pre-equilibrated in 0.1% trifluoroacetic acid. The Sep-Pak
cartridge was washed with 10% CH3CN/0.1% trifluoroacetic acid, and the peptides were eluted in 60% CH3CN/0.1%
trifluoroacetic acid. The eluate was evaporated and lyophilized. The
lyophilized materials were then redissolved in 1 M AcOH and
applied to a Sephadex G-50 gel-filtration column (1.8 × 130 cm,
Amersham Biosciences, Uppsala, Sweden). Five-milliliter fractions were
collected. A portion of each fraction was subjected to ghrelin-specific
RIA and the intracellular calcium influx assay using CHO-GHSR62 cells. The active fractions (#43-46) were separated by carboxymethyl (CM) ion-exchange high performance liquid chromatography (HPLC) on a
TSK CM-2SW column (4.6 × 250 mm, Tosoh, Tokyo, Japan) using an
ammonium acetate (HCOONH4) (pH 4.8) gradient of 10 mM to 1 M in the presence of 10%
CH3CN and a flow rate of 1 ml/min for 100 min.
One-milliliter fractions were collected and subjected to
ghrelin-specific RIAs and intracellular calcium influx assays. The six
active fractions (fractions A-F) separated by CM-HPLC were finally
individually purified using C18 reverse-phase HPLC (RP-HPLC) columns
(Symmetry 300, 3.9 × 150 mm, Waters). The amino acid sequences of
the purified peptides were analyzed with a protein sequencer (494, Applied Biosystems, Foster City, CA).
Mass Spectrometric Analysis of Human Ghrelins--
Electrospray
ionization mass spectrometry (ESI-MS) was performed on a quadrupole
mass spectrometer SSQ7000 (Finnigan, San Jose, CA) equipped with a
Finnigan ESI source. A needle capillary was heated to 150 °C to
evaporate the samples. Samples (~20 pmol) were dissolved in 50%
(v/v) methanol, 1% AcOH and introduced into the +4.5 kV (positive
ionization) ion source at a flow rate of 5 µl/min by direct infusion
with a syringe pump. Molecular masses of the purified peptides were
calculated using ICIS software Bioworks provided by Finnigan.
Human Ghrelin Peptide Synthesis--
Peptide synthesis of
acylated human ghrelin was performed as previously described for rat
ghrelin (8, 12). Fully protected 27- and 28-amino acid peptides (with
the exception of the exposed hydroxyl group of Ser3) were
synthesized by the Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase method using a peptide synthesizer (433A, Applied Biosystems). The Ser3 hydroxyl groups were acylated with
n-octanoic acid or n-decanoic acid by the action
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of
4-(dimethylamino)pyridine.
Cloning of Human Prepro-ghrelin cDNA--
Based on the amino
acid sequence determined for purified rat ghrelin, we searched the
GenBankTM expressed sequence tag data base. One rat
expressed sequence tag sequence (accession number AI549172) contained
the rat ghrelin sequence. Based on this expressed sequence tag
sequence, we designed sense and antisense primers
(5'-TTGAGCCCAGAGCACCAGAAA-3' and 5'-AGTTGCAGAGGAGGCAGAAGCT-3',
respectively) and performed PCR on a rat stomach cDNA library. The
conditions for the PCR involved 35 cycles of 98 °C for 10 s,
55 °C for 30 s, and 72 °C for 1 min. A rat ghrelin cDNA
of 501 bp was obtained. The amplified fragment was labeled with
[32P]dCTP and used as a screening probe for a human
stomach cDNA library, which was constructed from 1 µg of human
stomach poly(A)+ RNA (Clontech, Palo
Alto, CA) using a cDNA synthesis kit (Amersham Biosciences). By
this method, a full-length human ghrelin cDNA was isolated. Several
positive phages were isolated and subcloned into the plasmid pBS. Both
strands of cloned cDNAs were sequenced.
In Vivo Assay of Growth Hormone-releasing Activity--
Male
Wister rats (270-300 g) were prepared with a single indwelling jugular
catheter under sodium pentobarbital. Each rat received a 0.5- or 2-nmol
injection of synthetic human ghrelins. Blood samples were collected at
0, 5, 10, 15, 20, 30, and 60 min after injection. All samples were
centrifuged immediately, and the plasma samples were assayed for GH
using the Biotrak rat GH enzyme immunoassay system (Amersham
Biosciences, Buckinghamshire, UK).
Human Blood Sample Analysis--
To study the changes in plasma
levels of ir-ghrelin after gastrectomy, we followed three patients (one
male and two females), 50-61 years of age, who underwent total
gastrectomy due to gastric cancer. Blood samples were obtained before
and within 30 min after gastrectomy, and then 3, 7, 14, 30, 150, and
240 days after gastrectomy for measurement of plasma ir-ghrelin levels.
Four control males (60-70 years of age) who underwent standard partial
colectomy due to cancer, were also examined. Their blood samples were
obtained before and 1 day after surgical operation.
Preparation of Human Plasma Samples--
Whole blood samples
were collected with Na2EDTA (2 mg/ml) and aprotinin (500 KIU/ml). Plasma, after centrifugation at 4 °C, was diluted with an
equal volume of 0.9% saline. The samples were loaded onto a Sep-Pak
C18 cartridge (Waters) pre-equilibrated with 0.9% NaCl, washed with
0.9% NaCl and 5% CH3CN/0.1% trifluoroacetic acid, and
then eluted with 60% CH3CN/0.1% trifluoroacetic acid. After lyophilization of the eluates, they were subjected to RIAs for
ghrelin as described above.
Purification of Human Ghrelins--
We purified human ghrelin from
stomach tissue extracts by gel-filtration chromatography and HPLC,
using an intracellular calcium influx assay with a stable cell line
expressing rat GHS-R (CHO-GHSR62) and two ghrelin-specific RIAs to
screen fractions for the presence of ghrelin. Ir-ghrelin obtained by
N-RIA specifically represents active acylated ghrelin, whereas
ir-ghrelin acquired by C-RIA represents the total immunoreactivity of
both acylated and des-acyl ghrelin. Fig.
1a depicts the gel-filtration
chromatographic separation of stomach peptide extracts. Fractions
possessing ghrelin immunoreactivity and promoting intracellular calcium
influx were eluted at a molecular weight of roughly 3000. Active
gel-filtration fractions were further separated by CM ion-exchange HPLC
into six fractions, A-F (Fig. 1b). Fractions B-F induced
intracellular calcium influxes and possessed ir-ghrelin as assessed by
both C-RIA and N-RIA. Fraction A possessed only C-RIA ir-ghrelin and
did not induce intracellular calcium influx. Each of the five factions,
B-F, were separately purified to homogeneity by RP-HPLC and subjected
to the calcium-mobilization assay (Fig. 1c). Each of the
five fractions contained one active peak, except for fraction E, which
had two (peaks E-I and E-II). Fraction A was also
separated by RP-HPLC into two peaks (peaks A-I and
A-II), which were found to possess ir-ghrelin by C-RIA (Fig.
1d). Each of the eight resulting purified active peaks
contained a single peptide, and these peptides were then subjected to
further analysis.
Structural Analyses of Human Ghrelins--
The eight purified
peptides were subjected to protein sequencer, showing that peaks C,
E-I, E-II, and F shared the 28-amino acid sequence
GSXFLSPEHQRVQQRKESKKPPAKLQPR, whereas peaks B and D
were of the sequence GSX'FLSPEHQRVQQRKESKKPPAKLQP, identical except for the lacked of the C-terminal arginine. Complementary DNA
analysis of human ghrelin indicated that the third X and
X' residues should be serine, and the serine residues were
acyl-modified as described below. Moreover, the amino acid sequences of
peak A-I and A-II were GSSFLSPEHQRVQQRKESKKPPAKLQPR and
GSSFLSPEHQRVQQRKESKKPPAKLQP, respectively, the same as the
acyl-modified ghrelin peptides. We did not detect
des-Gln14-ghrelin in this human stomach tissue.
To determine whether the purified peptides were also modified by
n-octanoic acid at Ser3 as is rat ghrelin, we
subjected the peptides to ESI-MS and measured their molecular masses
(Table I). The measured molecular mass of
peak C, the major active peptide, was 3371.3 ± 0.1, and the calculated molecular mass of the 28-amino acid sequence is 3244.6. The
discrepancy, 126.7 mass units, strongly suggests that the hydroxyl
group of the Ser3 in this peptide is indeed replaced by an
n-octanoyl moiety (C8:0). The same was found for peak B,
which had a measured mass (3214.6 ± 0.6) that was 126.2 mass
units higher than the calculated molecular mass of the 27-amino acid
peptide (3088.4), indicating modification by n-octanoic
acid. The measured molecular masses of the peptides from peaks D and F
were ~154 molecular mass units higher than the calculated molecular
masses, indicating that these two peptides were modified by
n-decanoic acid (C10:0). Peaks E-I and E-II were both 152.6 molecular mass units higher than the calculated molecular masses, 2 mass units smaller than what would be expected for decanoyl modification. Based on this result and the fact that peaks E-I and E-II
were eluted at a time between the octanoyl (peaks C and E) and
decanoyl-modified ghrelins (peaks D and F) by RP-HPLC, it is most
likely that the peptides from peaks E-I and E-II are modified by
decenoic acid (C10:1). The amounts of peptide purified from peaks E-I
and E-II were very low, preventing a determination of the double-bond
site of the decenoic acid. In conclusion, we were able to divide the
collected ghrelins into four groups on the basis of acyl modification
at Ser3: non-acylated, octanoylated, decanoylated, and
possibly decenoylated.
To verify the deduced structures, we synthesized four peptides,
[O-n-octanoyl-Ser3]-human ghrelin,
[O-n-octanoyl-Ser3]-human
ghrelin-(1-27),
[O-n-decanoyl-Ser3]-human ghrelin,
and [O-n-decanoyl-Ser3]-human
ghrelin-(1-27) and compared their characteristics with those of the
purified peptides. The natural and synthetic peptides showed identical
retention times by RP-HPLC and identical molecular masses. Moreover,
the synthetic acyl-modified peptides had the same effects as purified
ghrelin peptides on cells expressing GHS-R. These results confirmed our
structural predictions for human ghrelin and the ghrelin-derived
molecules. We designate the newly purified peptides as follows:
[O-n-decanoyl-Ser3]-human ghrelin
as "human decanoyl ghrelin,"
[O-n-octanoyl-Ser3]-human
ghrelin-(1-27) as "human ghrelin-(1-27)," and
[O-n-decanoyl-Ser3]-human
ghrelin-(1-27) as "human decanoyl ghrelin-(1-27)." The yield of purified human ghrelin was ~300 pmol from 27 g of
stomach mucosa, and the molar ratio of the various subsets are shown in Table II.
Pharmacological Characterization of Ghrelins Using GHS-R-expressing
Cells--
Fig. 2 shows the
dose-response relationships of the synthetic human ghrelin and the
ghrelin-derived molecules on [Ca2+]i
changes in GHS-R-expressing cells. Four synthetic ghrelins potently
induced increases in [Ca2+]i in CHO-GHSR62 cells.
Ghrelin, ghrelin-(1-27), decanoyl ghrelin, and decanoyl
ghrelin-(1-27) had EC50 values of 2.7 × 10 In Vivo Effects of Human Ghrelins on GH Secretion--
To confirm
that human ghrelins possessed GH-releasing activity, we intravenously
injected synthetic ghrelins into anesthetized rats and measured plasma
GH concentrations. After injection of the each of the four synthetic
ghrelins, plasma GH concentrations increased and reached a maximum
within 10-15 min (Fig. 3). Each of the
peptides displayed nearly identical dose-response relationships, confirming that the newly identified human ghrelin-derived molecules are endogenous GH-releasing peptides with similar potency to human ghrelin.
Structure of the Human Prepro-ghrelin cDNA--
Using a rat
ghrelin cDNA, we screened a human stomach cDNA library under
low stringency conditions and obtained positive phages. Analysis of
these clones yielded a deduced amino acid sequence for human
prepro-ghrelin (a 117-amino acid precursor) (GenBankTM
accession number AB029434), depicted in Fig.
4. The putative initiation codon ATG is
located at nucleotides 34-36, preceded by the consensus initiation
sequence, whereas a terminal codon TAG is found 117 codons downstream
at position 385-387. A typical polyadenylation signal, AATAAA, is
found at position 494-499.
Although nearly all of the cDNA clones isolated from human stomach
encoded the prepro-ghrelin precursor, a few cDNA clones encoded the
prepro-des-Gln14-ghrelin precursor. Also, although we were
not able to isolate des-Gln14-ghrelin from the stomach
extracts during this study, this result indicates that
des-Gln14-ghrelin is indeed present in very low amounts in
the human stomach.
Characterization of Human Plasma Ghrelin Immunoreactivity--
To
confirm the presence of multiple molecular forms of ghrelin in human
plasma in addition to the stomach, Sep-pak extracts of normal human
plasma were fractionated by CM ion-exchange HPLC in exactly the same
manner as those from the stomach. The HPLC pattern of plasma extracts
in terms of the presence of ir-ghrelin was observed to be similar to
that of the stomach extracts (Fig. 5),
with peaks a-f emerging at positions identical to that of peaks A-F in
Fig. 1b. Thus, it can be concluded that both ghrelin and the
ghrelin-derived molecules circulate in human blood.
A minor unknown peak of ir-ghrelin (peak g) detected only by C-RIA was
observed in human plasma. This peak accounted for ~15% of all
ir-ghrelin by C-RIA. By RP-HPLC, this ir-ghrelin peak was eluted
earlier than that of des-acyl ghrelin (data not shown), suggesting that
the unknown ir-ghrelin in peak g results from digested ghrelin.
Plasma Ghrelin Levels in Gastrectomized Patients--
To
clarify whether circulating ghrelin is indeed drastically reduced
after gastrectomy as would be expected, plasma ir-ghrelin was measured
before and after total gastrectomy in three patients (Fig.
6). Within 30 min after gastrectomy,
plasma ir-ghrelin was found to decrease to approximately half of its
pre-surgery levels. The levels remained depressed for roughly a
week, but after that they began to increase. By the end of the day 240, two of the patients had ir-ghrelin levels that were two-thirds of their
original levels, and one patient's ghrelin levels had completely
normalized. In contrast, the subjects who underwent partial colectomy
showed no change in plasma ir-ghrelin before (75.4 ± 22.0 fmol/ml, mean ± S.D.) and 1 day after operation (69.6 ± 17.8 fmol/ml). These results suggest that the stomach is the major
source of circulating ghrelin, and the other tissues compensate to
maintain circulating ghrelin levels after gastrectomy.
Here, we reported the purification and characterization of human
ghrelin and the other minor ghrelin-derived molecules from the stomach.
The major active form of human ghrelin is a 28-amino acid peptide with
an n-octanoyl modification at Ser3. This peptide
is identical to rat ghrelin with the exception of two residues
(Arg11-Val12). The ghrelin-derived molecules we
observed include octanoyl ghrelin-(1-27), decanoyl ghrelin, decanoyl
ghrelin-(1-27), and decenoyl ghrelin. Moreover, the non-active forms
des-acyl ghrelin and des-acyl ghrelin-(1-27) were also present in the
human stomach. As described, we were able to classify human ghrelin and
the ghrelin-derived molecules into two groups on the basis of amino
acid length and into four groups by type of acylation at
Ser3. Furthermore, all of these molecular forms of ghrelin
were found in human plasma as well as in the stomach. In human stomach,
the processing product ratio of 27-amino acid to 28-amino acid ghrelins was observed to be ~1:3.
It is likely that the 27- and 28-amino acid ghrelin molecules isolated
in this study are produced through alternative C-terminal processing of
the same ghrelin precursor. It is well known that peptide hormones are
cleaved by processing proteases to product multiple forms, such as the
enkephalins (16), endorphins, dynorphins (17), corticotropins, and
The human prepro-ghrelin we isolated is predicted to encoded a
117-residue precursor peptide. We previously reported that there
are two types of ghrelin precursors from rat stomach cDNA analysis,
a 117-amino acid precursor (prepro-ghrelin) and a 116-amino acid
precursor (prepro-des-Gln14-ghrelin) (12).
Des-Gln14-ghrelin, a splice variant of ghrelin, is the
second endogenous ligand for the GHS-R. Only a small percentage of the
ghrelin clones isolated from the human stomach library encoded the
des-Gln14-ghrelin precursor, and the
des-Gln14-ghrelin peptide was not identified in human
stomach. The ratios observed between the two precursor
populations, ghrelin and des-Gln14- ghrelin, was 5 to 1 in rat stomach, and 6 to 5 in mouse stomach (24). These differences are
likely species-specific.
Ghrelin was the first example discovered of a bioactive peptide
modified by an n-octanoic acid moiety. Although acyl
modification of many proteins has been observed, including
G-proteins and some G-protein-coupled receptors, the modifications
are most often myristoylations (25) and palmitoylations (26). In this
study, we further showed that ghrelin can be modified by
n-decanoic acid. All of the acyl-modified ghrelins and
ghrelin-derived molecules studied here have the same potency to
induce an increase of [Ca2+]i in the
GHS-R-expressing cells and stimulate GH release in anesthetized rats,
and de novo synthesis of molecules concretely demonstrated
that octanoic acid is not the only Ser3 modification that
will confer full activity to ghrelin (27, 28). We recently reported the
identification of bullfrog ghrelin acylated with n-decanoic
acid and found that this ghrelin species comprises 33% of total
isolated bullfrog ghrelin (29). In the human stomach, the ratio of
octanoylated to decanoylated ghrelin was found to be roughly 3:1.
Because acylation of ghrelin is essential for its activity, the enzyme
that catalyzes this modification step should be an important regulator
of ghrelin biosynthesis. However, the mechanism by which ghrelin is
acylated during post-translational processing is still unclear.
We also analyzed the change in human plasma ir-ghrelin levels
before and after total gastrectomy. We already reported that plasma
levels of ir-ghrelin in totally gastrectomized patients were reduced to
35% of those in normal controls (14). In this study, C-RIA was used
due to the instability of acylated ghrelin relative to its
des-acylated counterpart, making its measurement from stored
plasma samples unreliable. Plasma levels of ir-ghrelin were promptly
reduced by approximately half within 30 min after total gastrectomy.
Half-lives after intravenous administration of human ghrelin was about
10 min (30). Interestingly, the levels then began to increase in these
cases, and in one patient even completely normalized. Significant
amounts of ir-ghrelin were detected in the rat duodenum, jejunum,
ileum, and colon (13), suggesting that these organs may be responsible
for this compensation.
Further work will be required to determine the physiological
significance of the various different forms of human ghrelin and
ghrelin-related peptides discovered during the course of this study.
Although all of these molecules displayed similar dose-response profiles from the tests performed in this study, it is possible that
the various ghrelin forms have different signaling properties or stability.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification of human ghrelin from
stomach. Black bars indicate ir-ghrelin content, and
open bars indicate the fluorescence changes due to
[Ca2+]i increase in CHO-GHSR62 cells. The
gradient profiles are indicated by the dotted lines.
a, gel-filtration chromatography of human stomach extracts
(27 g). Active fractions 43-46 were eluted at roughly
Mr 3000 (3 K). Vo, void
volume; Vt, total volume. An aliquot from each fraction (5 mg of wet tissue equivalent) was subjected to C-RIA. b, CM
ion-exchange HPLC (pH 4.8) of gel-filtration-derived active fractions
monitored by C-RIA (upper), N-RIA (middle), and
calcium-mobilization assay (bottom) for ghrelin. Active
fractions, indicated by solid bars, were separated into six
fractions (fractions A-F). A portion of each fraction (5 mg
of wet tissue equivalent) was subjected to RIAs for ghrelin.
c, final purification of the each active fractions B-F
derived from CM ion-exchange HPLC by RP-HPLC. d, final
purification of fraction A derived from CM ion-exchange HPLC by
RP-HPLC.
Structural analyses of human ghrelin and ghrelin derived molecules
The molar ratio of purified human ghrelin and ghrelin derived molecules
9, 2.8 × 10
9, 2.5 × 10
9, and 2.5 × 10
9 M,
respectively, and displayed similar potency upon application to
GHS-R-expressing cells.
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Fig. 2.
Pharmacological characterization of synthetic
ghrelin and ghrelin-derived molecules using the GHS-R-expressing
cells. Dose-response relationships of
[Ca2+]i in CHO-GHSR62 cells, in response to
treatment with human ghrelin, ghrelin-(1-27), decanoyl ghrelin, and
decanoyl ghrelin-(1-27). Data points are means ± S.D. of three
independent experiments.
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Fig. 3.
Growth hormone releasing activity of
different synthetic forms of ghrelin in vivo.
Time courses of plasma growth hormone concentrations after intravenous
injections of synthetic ghrelin and ghrelin-derived molecules into male
rats. Each of the synthetic ghrelins was injected intravenously into
male Wistar rats (270-300 g) anesthetized with pentobarbital, and
blood samples were collected from the cervical artery. GH
concentrations were measured by enzyme immunoassay. Solid
lines and broken lines indicate doses of 2 and 0.5 nmol, respectively. Data represent the means of three
experiments.
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Fig. 4.
Nucleotide and deduced amino acid sequence of
human prepro-ghrelin cDNA (GenBankTM accession
number AB029434). The predicted amino acid sequence of
prepro-ghrelin is denoted below the nucleotide sequence. The
dotted line indicates the signal peptide. The human
ghrelin-(1-28) sequence is double-underlined. The
circled S indicates an n-acyl-modified serine.
The termination codon is marked with an asterisk. The AATAAA
sequence, a polyadenylation signal, is underlined. The
boxed AG of Gln14 may be used as a splicing
acceptor site at the 3'-end of the intron to produce
des-Gln14-ghrelin.
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Fig. 5.
CM ion-exchange HPLC of human plasma extract
monitored by radioimmunoassays for ghrelin. Human plasma extracts
from a Sep-Pak C18 cartridge were fractionated by CM ion-exchange HPLC
(pH 4.8) in an identical manner as the stomach extracts. Each sample
(2.5-ml plasma volume equivalent) was monitored by C-RIA
(upper) and N-RIA (lower). The ir-ghrelin
recovery of this CM ion-exchange HPLC step was ~90%. Active
fractions were separated into seven fractions. Fractions
a-f indicated by solid bars correspond to the
active fractions A-F of the first CM ion-exchange HPLC step
from the stomach extracts (Fig. 1b). Fraction g
indicated by the open bar probably contained a C-terminal
fragment of ghrelin and des-acyl ghrelin to be cleaved by
proteases.
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Fig. 6.
Time course of plasma ghrelin levels before
and after total gastrectomy. Individual changes in plasma ghrelin
levels in three patients before and after total gastrectomy. Plasma
levels of ir-ghrelin were measured by C-RIA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lipotropins (18). Many of the known proteolytic precursor cleavage
events occur at pairs of basic amino acid residues (Lys or Arg), and
both basic residues are usually absent from the resultant products
(19). However, some proteolytic cleavages, as in the case of
cholecystokinin, occur immediately after a C-terminal single basic
residue (especially Arg) (20). Ghrelin-(1-28) may fit into this
category, because cleavage to produce this peptide occurs following the
C-terminal Pro27-Arg28. Interestingly, this
basic arginine residue remains at the C terminus of ghrelin-(1-28) but
is removed from ghrelin-(1-27) (which terminates in proline). A
similar cleavage profile is seen in the case of
-neo-endorphin
(YGGFLRKY-Pro-Lys) (21) and
-neo-endorphin (YGGFLRKY-Pro) (22),
whose precursor possesses the Lys-Arg basic pair followed by a
C-terminal proline. It is thought that production of these peptides
occurs through cleavage at the C terminus of paired basic Lys-Arg
residues, followed by removal of the C-terminal basic residue by a
carboxypeptidase B-like enzyme (23). Peptide bonds involving proline
are resistant to common proteases such as this. However, the removal of
C-terminal lysine in
-neo-endorphin occurs partially to generate
-neo-endorphin. In a similar manner, ghrelin-(1-27) may be produced
by removal of the C-terminal arginine of ghrelin-(1-28) by a
carboxypeptidase B-like enzyme. Although ghrelin-(1-27) was present
only at a very low level in rat stomach, it is likely that these
processing mechanisms control of the maturation of human ghrelin.
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ACKNOWLEDGEMENTS |
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We thank K. Mori for technical advice and H. Mondo and M. Miyazaki for their technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, the Ministry of Health, Labor and Welfare of Japan, the Promotion of Fundamental Studies in Health Science from the Organization for Pharmaceutical Safety and Research of Japan, and the Takeda Science Foundation.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB029434 and AB035700.
To whom correspondence should be addressed: Dept. of
Biochemistry, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. Tel.: 81-6-6833-5012; Fax:
81-6-6835-5402; E-mail: kangawa@ri.ncvc.go.jp.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M205366200
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ABBREVIATIONS |
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The abbreviations used are: GH, growth hormone; GHS, growth hormone secretagogue; GHS-R, growth hormone secretagogue receptor; RIA, radioimmunoassay; ir, immunoreactive; CHO, Chinese hamster ovary; [Ca2+]i, intracellular calcium concentration; AcOH, acetic acid; CM, carboxymethyl; RP, reverse-phase; HPLC, high-performance liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; N-RIA, N-terminal fragment of rat ghrelin-(1-11); C-RIA, C-terminal fragment of rat gherlin-(13-28).
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REFERENCES |
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