From the Departments of Biochemistry and Molecular
Biology and ** Medicine, Royal Free and University College
Medical School, Rowland Hill Street, London NW3 2PF, United
Kingdom, the Departments of ¶ Endocrinology and
Clinical Biochemistry, Royal Free Hospital NHS Trust, Pond
Street, London NW3 2QG, United Kingdom, and the
School of Biochemistry and Molecular
Biology, University of Leeds, Leeds LS2 9JT, United Kingdom
Received for publication, October 11, 2002, and in revised form, January 10, 2003
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ABSTRACT |
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Ghrelin is a 28-residue peptide hormone that is
principally released from the stomach during fasting and prior to
eating. Two forms are present in human plasma: the unmodified peptide and a less abundant acylated version, in which octanoic acid is attached to the third residue, a serine, via an ester linkage. The
acylated form of ghrelin acts as a ligand for the growth hormone secretagogue receptor and can stimulate the release of growth hormone from the pituitary gland. It also initiates behavioral and
metabolic adaptations to fasting. Here we show that an immobilized form
of ghrelin specifically binds a species of high density lipoprotein associated with the plasma esterase, paraoxonase, and clusterin. Both
free ghrelin and paraoxon, a substrate for paraoxonase, can inhibit
this interaction. An endogenous species of ghrelin is found to
co-purify with high density lipoprotein during density gradient
centrifugation and subsequent gel filtration. This interaction links
the orexigenic peptide hormone ghrelin to lipid transport and
metabolism. Furthermore, the interaction of the esterified hormone
ghrelin with a species of HDL containing an esterase suggests a
possible mechanism for the conversion of ghrelin to des-acyl ghrelin.
Ghrelin is a peptide hormone that was purified from the stomach
that can cause the release of growth hormone from the anterior pituitary gland (1). It has subsequently been found that ghrelin is
predominantly released from the stomach prior to feeding (2), although
other tissues have been shown to express the gene as well (3).
Peripheral injections of ghrelin have been shown to increase feeding in
both rats (4) and humans (5), and a course of injections leads to
increased obesity in rats (4). Therefore, ghrelin can be seen as an
important link between the stomach, appetite, and metabolism as well as
playing a role in growth hormone release.
The aim of this study was to establish whether ghrelin interacts with
any other component of plasma. It was hypothesized that any interaction
might be an important factor in determining the activity or longevity
of ghrelin in plasma. Furthermore, an interaction in the plasma might
be involved in the creation of the two distinct forms of ghrelin (6):
the acylated form, in which octanoic acid is covalently bound to the
peptide, and the more prevalent des-acyl form in which the peptide is
unmodified. The acylated form of ghrelin stimulates the release of
growth hormone from the pituitary gland, therefore any mechanism that
might convert one form of ghrelin into the other could be an important
factor in controlling the activity of ghrelin.
A peptide corresponding to mature human ghrelin, with a cysteine
residue substituted for the serine residue at the third position (S3C
ghrelin), was synthesized by
Fmoc1 chemistry using a
Rainin PS3 automatic peptide synthesizer (Protein Technologies). The
peptide was purified to over 90% homogeneity by reverse-phase
chromatography using a Varian 500LC HPLC. The molecular weight of the
purified peptide was verified by surface-enhanced laser
desorption/ionization (SELDI)-mass spectrometry. The purified peptide
(10 mg) was covalently bound to 2 ml of Sulfolink matrix (Pierce) via the sulfydryl group of the introduced cysteine. Briefly, the peptide was dissolved in 1 ml of PBS and then gently agitated with
the resin for 1 h at room temperature. The slurry was then centrifuged for 1 min at 1,000 × g and the supernatant
removed. Any remaining iodoacetyl binding sites on the matrix were then blocked by incubation with 15 mM free cysteine for 20 min.
A negative control matrix, with no peptide attached, was only blocked
with free cysteine. The two matrices were washed in 100 mM
glycine, pH 2.5, and then neutralized in PBS.
Blood was taken into EDTA from an individual who had not eaten for over
12 h. The plasma was purified from the blood cells by
centrifugation at 1,500 × g for 10 min, and the two
column matrices were each incubated with 10 ml of plasma for 1 h.
Each column was then sequentially washed with 5 ml of PBS, 5 ml of PBS + 1 M sodium chloride, 5 ml of PBS + 2 M sodium
chloride, and then the proteins that had remained bound were eluted
with 5 ml of 100 mM glycine, pH 2.5. All samples were
concentrated into 100 µl of PBS using Amicon Centricon preparators
(cutoff: 10 kDa). Samples were made up in Laemmli buffer, and 10 µl
was used for SDS-PAGE, prior to staining with Coomassie Blue, while 20-µl samples were used for protein sequencing. Gels were blotted onto PVDF membrane (Amersham Biosciences), at 1 mA/cm2 for
1 h, and stained with Ponceau S. Stained protein bands were excised from the PVDF membrane, washed, and de-stained in methanol containing 0.1% (v/v) ammonia, rinsed with methanol, and dried in air
in preparation for sequencing. The N-terminal residues of the protein
bands were identified by automated Edman degradation using liquid-pulse
chemistry on an Applied Biosystems Procise 494 with on-line 140C
narrow-bore HPLC system and 610A data analysis software.
To investigate whether the interaction between the ghrelin matrix and
the species of HDL could be inhibited, 1-ml samples of plasma, from a
fasted individual, were incubated with 0.1 ml of
ghrelin-Sulfolink matrix and 100 µl of PBS or an equivalent volume containing either free ghrelin or paraoxon. The matrices were
then sequentially washed with 1 ml of PBS, 1 ml of PBS + 1 M sodium chloride, 1 ml of PBS + 2 M sodium
chloride, and then the remaining bound proteins were eluted with 1 ml
of 100 mM glycine, pH 2.5. The glycine-eluted samples were
then washed extensively and concentrated into 100 µl of PBS using
Amicon Centricon preparators. Samples of 10 µl were made up in
Laemmli buffer and run on SDS-PAGE for staining with Coomassie Blue.
Human low (LDL) and high (HDL) density lipoproteins were isolated from
fresh blood, donated by healthy fasted volunteers, by a modification of
the method of Chung et al. (7). Whole plasma was loaded into
a discontinuous gradient (1.3 and 1.006 g/ml), which formed into a
linear gradient between upper and lower density limits during
centrifugation for 150 min in a Beckman Ti-70 rotor at 160,000 × g. The density ranges of the low and high density
lipoprotein classes are 1.019-1.063 and 1.063-1.21 g/ml,
respectively. Serum albumin was removed from HDL using blue Sepharose
chromatography (Amersham Biosciences). The lipoproteins were
de-salted on a G25 column, fractions were monitored for absorbance at
280 nm, and samples were analyzed for immunoreactivity by RIA (Phoenix Pharmaceuticals).
Lipoprotein fractions, along with a positive control of acylated
ghrelin (Phoenix Pharmaceuticals), were spotted onto methanol-whetted PVDF membrane in triplicate. The membrane was blocked in 5% milk powder in PBS containing 0.1% Tween 20 for 1 h and then probed for 16 h with 1% milk powder in PBS-T (phosphate-buffered
saline with 0.1% (v/v) Tween 20) containing 1/1,000 antiserum that had been raised against S3C ghrelin coupled to keyhole limpet
hemocyanin. The membrane was then probed with 1/5,000 goat
anti-rabbit IgG conjugated to horseradish peroxidase (Sigma) in 1%
milk powder in PBS-T for 1 h and then extensively washed in PBS-T
and visualized by enhanced chemiluminescence (Amersham Biosciences).
Affinity chromatography, using an engineered form of ghrelin, was
used to identify the moieties in plasma that could bind to ghrelin. The
samples that were eluted by the acid glycine were separated by SDS-PAGE
and then electroblotted onto PVDF membrane and stained for protein. All
the stained bands in the sample on the PVDF membrane were subjected to
N-terminal sequencing, and proteins were identified by at least eight
cycles of Edman degradation.
The most abundant protein that was purified by the ghrelin affinity
chromatography (Fig. 1) was identified as
apolipoprotein A-I (apoA-I). It was eluted from the ghrelin
column as a band of ~30 kDa, which is consistent with the predicted
molecular mass of 27 kDa for the mature protein. Serum
paraoxonase (PON I) was detected at ~40 kDa. The residues identified
at the N terminus correspond to the signal sequence, which, unusually,
is not cleaved from the mature protein (8). Both the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
chains
of clusterin (apolipoprotein J) were detected as a band below 50 kDa,
and as a dimer, which is consistent with the predicted
molecular weight for the mature protein. Clusterin, PON I, and apoA-I
associate on a species of HDL particle (9) that comprises ~1% of the HDL particles in plasma. Therefore, it is consistent that these proteins were identified eluting from the column together. Serum albumin and vitronectin were identified as a single band at ~65 kDa;
however, serum albumin was identified as binding to both the ghrelin
column and the control column, suggesting that this was not specific
binding.
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Fig. 1.
Ghrelin affinity chromatography of proteins
from human plasma. A Coomassie Blue-stained SDS-PAGE showing the
proteins that were eluted from the control and ghrelin affinity matrix
after incubation with 10 ml of human plasma is shown.
Lanes 1-4 were eluted after incubating 10 ml of fasted
human plasma with a 2-ml column without peptide (control). Lanes
5-8 were eluted, after incubating 10 ml of fasted human plasma,
from a 2-ml column of the immobilized ghrelin. Lanes 1 and
5 were washed from the columns by PBS. Lanes 2 and 6 were eluted by PBS + 1 M sodium chloride.
Lanes 3 and 7 were eluted by PBS + 2 M sodium chloride. Lanes 4 and 8 were
eluted by 100 mM glycine, pH 2.5.
The specificity of the interaction between this form of HDL
and ghrelin was investigated by repeating the affinity chromatography in the presence of free ghrelin to assess whether it would inhibit the
binding of the HDL species with immobilized ghrelin. Virtually all
binding of the HDL to the column of immobilized ghrelin was inhibited
by 4 nM ghrelin (Fig.
2a). This suggests that there is a limited number of binding sites on the HDL species for ghrelin. It
has been reported that ghrelin levels fluctuate diurnally, but can
reach levels as high as 0.5 nM (2).
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The interaction between the immobilized form of ghrelin and the HDL was also inhibited by paraoxon, a substrate for the esterase PON I. The binding of HDL to the column of immobilized ghrelin was inhibited by ~50% by 1 mM paraoxon (Fig. 2b), which is close to the Km of PON I for paraoxon. This suggests that either ghrelin binds to the same active site as paraoxon or that the binding of paraoxon causes an alteration to an allosteric site where ghrelin interacts.
To establish whether any endogenous ghrelin is bound to HDL or LDL in
human plasma, the lipoproteins were purified by density-gradient ultracentrifugation. When the sodium bromide was removed from the HDL
fraction by gel filtration, the ghrelin immunoreactive species
co-migrated with the lipoprotein (Fig.
3A). This shows that ghrelin
migrated as a high molecular weight species during density-gradient
ultracentrifugation and gel filtration and co-migrated with HDL under
both of these purification processes.
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In Fig. 3B, the fractions of lipoprotein were spotted in triplicate onto PVDF membrane and probed by an antibody raised against ghrelin. The antibody recognized ghrelin in human plasma, but not in low density lipoprotein. The antibody bound ghrelin in the HDL fraction, and this was not reduced by the removal of serum albumin from this fraction.
Ghrelin affinity chromatography was also performed on serum from a rabbit that had been fed ad libidum. Only PON I and apoA-I were identified after acid-glycine elution from the column (data not shown). It could be that the difference in these results is due to different properties of the proteins from different species or that the calcium in the serum, or the EDTA in the plasma, alters this interaction. It is also possible that satiety alters this interaction, therefore further work is being undertaken to establish what factors are important.
PON I, an esterase whose physiological substrate has not been
established, is part of the species of HDL that binds the immobilized form of ghrelin. However, there is no inhibition of the enzymatic breakdown of paraoxon by PON I, by either 1 mM ghrelin or
des-acyl ghrelin (data not shown).
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DISCUSSION |
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PON I has been shown to possess esterase activity, and there is an ester bond in ghrelin that links the octanoic acid to the third residue, a serine (1). However, the present affinity chromatography data were obtained using a matrix that has the peptide bound via a thioether bond at the point at which the octanoic acid would be attached to the peptide via an ester bond. The purified peptide was immobilized via the sulfydryl group to Sulfolink matrix, which has a spacer group containing a 12-carbon chain between the sulfydryl-specific iodoacetyl group and the cross-linked agarose matrix. Therefore, this engineered form of ghrelin has an aliphatic chain attached to the peptide at the correct position, but via a thioether linkage, rather than the ester linkage in natural ghrelin.
Engineered forms of ghrelin, in which the vulnerable ester linkage is exchanged for a more robust bond, may alter the biostability and activity of ghrelin. If PON I breaks ghrelin down to the des-acyl form, then PON I may be a suitable target for developing drugs to control the biostability, and therefore activity, of endogenous ghrelin. This interaction may offer opportunities for therapeutic intervention in the control of either appetite or lipoprotein metabolism.
No previous data have suggested a link between ghrelin and a subtype of HDL. However, both ghrelin and this species of HDL are present in plasma during fasting, although they are released from different tissues; therefore, it should be possible for these two serum components to interact.
The relationship between growth hormone (GH), ghrelin, and HDL is not fully understood. Both GH deficiency and excess are associated with an increased risk of atherosclerosis. Acromegaly is associated with both low HDL levels (11) and low ghrelin levels (12). GH deficiency is associated with low ghrelin levels (13), but it is unclear whether there are changes in HDL levels in these patients (10, 14). The present data suggest a significant link between HDL and ghrelin, but it is unclear whether the changes in ghrelin and HDL levels in these pathophysiological states are causally linked and whether low ghrelin levels might influence the course of atherosclerosis in these patients or any other groups.
This study raises some questions about the physiology of ghrelin and HDL. Further investigations will be required to distinguish which form of ghrelin can interact with the species of HDL and to measure the proportions of bound and free ghrelin in plasma. The interaction may affect the biological activities of these molecules, so this ratio could be an important factor in health and disease. This ratio may vary between individuals, or fluctuate with ghrelin levels, or in specific disease states. It is also possible that ghrelin interacts with other moieties in the plasma under different conditions, and further work will be required to fully characterize the interactions and physiology of ghrelin in the blood.
In conclusion, the interaction between ghrelin and this species of HDL
is a link between hunger, growth hormone release, and lipid transport,
as part of the response to fasting. Further research may establish the
importance of this interaction for health and disease.
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FOOTNOTES |
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* 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. Tel.: 44-207-794-0500 (ext. 3703); Fax: 44-207-830-2917; E-mail: n.beaumont@rfc.ucl.ac.uk.
Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.C200575200
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ABBREVIATIONS |
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The abbreviations used are: Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; LDL, low density lipoprotein; HDL, high density lipoprotein; RIA, radioimmunoassay; PON I, serum paraoxonase; GH, growth hormone.
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REFERENCES |
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