ACCELERATED PUBLICATION
Ghrelin Can Bind to a Species of High Density Lipoprotein Associated with Paraoxonase*

Nicholas J. BeaumontDagger §, Vernon O. SkinnerDagger , Tricia M.-M. TanDagger , Bala S. RameshDagger , Dominic J. Byrne||, Gavin S. MacColl**, Jeff N. KeenDagger Dagger , Pierre M. Bouloux**, Dimitri P. Mikhailidis||, K. Richard BruckdorferDagger , Mark P. Vanderpump, and Kaila S. SraiDagger

From the Departments of Dagger  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 Dagger Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 + 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).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha  and beta  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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Fig. 2.   The binding of HDL to ghrelin during affinity chromatography can be inhibited by free ghrelin or paraoxon. A, ghrelin affinity chromatography: inhibition by free ghrelin. SDS-PAGE of the proteins that were eluted from the ghrelin-Sulfolink matrix after incubation with 1 ml of fasted human plasma and stated concentration of free acyl ghrelin are shown. The proteins were eluted by 100 mM glycine, pH 2.5, after washing the matrix with 2 M sodium chloride. Lane 1, control; lane 2, 125 pM ghrelin; lane 3, 250 pM ghrelin; lane 4, 500 pM ghrelin; lane 5, 1 nM ghrelin; lane 6, 2 nM ghrelin; lane 7, 4 nM ghrelin; lane 8, 8 nM ghrelin. B, ghrelin affinity chromatography: inhibition by paraoxon. SDS-PAGE of the proteins that were eluted from the ghrelin-Sulfolink matrix after incubation with 1 ml of fasted human plasma and stated concentration of paraoxon are shown. The proteins were eluted by 100 mM glycine, pH 2.5, after washing the matrix with 2 M sodium chloride. The intensity of apoA-I was measured by Bio-Rad imaging (mean ± S.E. from three experiments).

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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Fig. 3.   Endogenous ghrelin can co-purify with HDL. A, endogenous ghrelin and HDL migrate together during gel-filtration chromatography. HDL isolated from fasted human plasma by density-gradient ultracentrifugation was de-salted by Sephadex G25 gel-filtration chromatography. Total protein content of fraction was measured by absorbance at 280 nm, and the ghrelin content was assessed by RIA (Phoenix Pharmaceuticals). B, dot blot of lipoprotein samples probed by anti-ghrelin antibody. Dot 1, 10 µl of plasma; dot 2, 10 µg of LDL; dot 3, 10 µg of HDL; dot 4, 10 µg of HDL (albumin removed); dot 5, 1 µg of ghrelin (positive control). Membrane was probed by antiserum raised against C3S ghrelin.

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).

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    FOOTNOTES

* 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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