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J. Biol. Chem., Vol. 276, Issue 44, 40441-40448, November 2, 2001

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Bullfrog Ghrelin Is Modified by n-Octanoic Acid at Its Third Threonine Residue^*

Hiroyuki Kaiya^§, Masayasu Kojima, Hiroshi Hosoda, Aya Koda^¶, Kazutoshi Yamamoto^¶, Yasuo Kitajima, Masaru Matsumoto, Yoshiharu Minamitake, Sakae Kikuyama^¶, and Kenji Kangawa

From the  Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan, the ^¶ Department of Biology, School of Education, Waseda University, 1-6-1 Nishiwaseda, Shinjuku-ku Tokyo 169-8050, Japan, and the  Suntory Institute for Medicinal Research and Development, 2716-1 Kurakake, Akaiwa, Chiyoda-machi, Ora-gun, Gunma 370-0503, Japan

Received for publication, June 6, 2001, and in revised form, August 20, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have identified the amphibian ghrelin from the stomach of the bullfrog. We also examined growth hormone (GH)-releasing activity of this novel peptide in both the rat and bullfrog. The three forms of ghrelin identified, each comprised of 27 or 28 amino acids, possessed 29% sequence identity to the mammalian ghrelins. A unique threonine at amino acid position 3 (Thr³) in bullfrog ghrelin differs from the serine present in the mammalian ghrelins; this Thr³ is acylated by either n-octanoic or n-decanoic acid. The frog ghrelin-28 has a complete structure of GLT (O-n-octanoyl)FLSPADMQKIAERQSQNKLRHGNM; the structure of frog ghrelin-27 was determined to be GLT(O-n-octanoyl)FLSPADMQKIAERQSQNKLRHGN; frog ghelin-27-C10 possessed a structure of GLT(O-n-decanoyl)FLSPADMQKIAERQSQNKLRHGN. Northern blot analysis demonstrated that ghrelin mRNA is predominantly expressed in the stomach. Low levels of gene expression were observed in the heart, lung, small intestine, gall bladder, pancreas, and testes, as revealed by reverse transcription polymerase chain reaction analysis. Bullfrog ghrelin stimulated the secretion of both GH and prolactin in dispersed bullfrog pituitary cells with potency 2-3 orders of magnitude greater than that of rat ghrelin. Bullfrog ghrelin, however, was only minimally effective in elevating plasma GH levels following intravenous injection into rats. These results indicate that although the regulatory mechanism of ghrelin to induce GH secretion is evolutionary conserved, the structural changes in the different ghrelins result in species-specific receptor binding.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Growth hormone (GH)¹ secretion from the pituitary gland is regulated by hypothalamic hormones; growth hormone-releasing hormone (GHRH) stimulates GH secretion, whereas somatostatin is inhibitory (1). Derivatives of Met-enkephalin stimulate GH release (2), the first demonstration that small synthetic peptides and nonpeptide molecules, dubbed growth hormone secretagogues (GHSs), can mediate GH release through a receptor distinct from that of GHRH (3-5). The G-protein-coupled GHS receptor (GHS-R) was subsequently identified in swine, rat, and human (6, 7), suggesting that one or more unknown ligands for this receptor are endogenously present.

We recently discovered an endogenous ligand for GHS-R from the rat stomach, using an intracellular calcium influx assay in stable cell lines expressing rat GHS-R (8). This novel molecule, a 28-amino acid peptide named ghrelin (from "ghre," the Proto-Indo-European root of "grow"), possesses a unique serine residue at the third N-terminal position (Ser³) that is n-octanoylated (8, 9). Acylation of Ser³ is essential for ghrelin bioactivity. cDNA analysis revealed that the rat ghrelin sequence follows the 23-residue signal sequence within the 117-residue prepro-ghrelin. Ghrelin stimulates GH secretion both in vivo and in vitro. Accumulating evidence in mammals suggests that, in addition to regulating GH release, ghrelin also influences feeding behavior (10, 11), gastrointestinal function (12, 13), and energy metabolism (14).

Little is known about the activity of GHS in non-mammalian vertebrates. Limited studies in these organisms, however, suggest the involvement of GHS homologues in GH regulation. In the chicken, a growth hormone-releasing peptide (GHRP)-6 induces GH secretion in vivo (15). A nonpeptidyl GHS, L-692,429, also stimulated GH secretion in the chicken in vivo and in vitro (16). Palyha et al. (17) isolated three GHS-R homologues from the pufferfish; one of these GHS-R homologues (78B7), sharing 58% identity with human GHS-R, is activated by three kinds of GHS. Intraperitoneally injected GHRP, KP-102, also stimulates GH secretion in a teleost, the tilapia (18). These findings suggest that GHS-Rs and their natural ligands are necessary for the function to regulate GH secretion in non-mammalian vertebrates.

In this study, we have identified ghrelin and the precursor cDNA in an amphibian, the bullfrog. The structure of bullfrog ghrelin contains an n-octanoylated threonine at amino acid position 3, differing from the serine present in the mammalian forms. Bullfrog ghrelin stimulates GH and prolactin (PRL) secretion in bullfrog but is unable to mediate this effect in the rat.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fluorometric Imaging Plate Reader (FLIPR) Assay-- Through the purification process, ghrelin activity was measured by changes in intracellular calcium concentrations ([Ca²⁺]_i) using a FLIPR system (Molecular Devices, Sunnyvale, CA) in CHO-GHSR62. These cells, stably expressing rat GHS-R as previously described (8, 9), were plated onto black wall 96-well microplates at 5 × 10⁴ cells/well. Cells were cultured in a humidified environment of 95% air:5% CO₂ for 20 h prior to the assay. Cells were incubated for 1 h with 4 mM Fluo-4 AM (Molecular Probes Inc., Eugene, OR), dissolved in assay buffer (Hanks' balanced salt solution, 20 mM HEPES, 2.5 mM probenecid) containing 1% fetal calf serum. The cells were then washed four times by an automatic cell washer in assay buffer without fetal calf serum. Lyophilized test samples were dissolved in 120 µl of assay buffer containing 0.01% bovine serum albumin and 0.001% Triton X-100. One hundred microliters of each sample were added to the CHO-GHSR62 cells. The maximum [Ca²⁺]_i change was then determined as a response. We utilized this assay to study the dose-response relationships for synthetic rat ghrelin, frog ghrelin-27, and frog ghrelin-28, all of which were modified by n-octanoic acid.

Purification of Bullfrog Ghrelin from Stomach Tissue-- Bullfrogs, Rana catesbeiana, were purchased from a commercial supplier (Ohuchi, Saitama, Japan). A frozen bullfrog stomach (10.75 g) was pulverized and boiled for 10 min in 5 volumes of water to inactivate intrinsic proteases (19, 20). The sample was then chilled on ice and adjusted to 1 M acetic acid (AcOH) by the addition of glacial AcOH. The boiled stomach tissue was homogenized using a Polytron mixer. The homogenate was then centrifuged for 30 min at 10,000 × g. The supernatant, diluted in an equal volume of distilled water, was loaded onto a Sep-Pak Plus C18 environmental cartridge (Waters, Milford, MA) pre-equilibrated with 0.5 M AcOH. The cartridge was washed with a 10% acetonitrile (CH₃CN)/0.1% trifluoroacetic acid (TFA) solution. The peptide was sequentially eluted with 10 ml of 25, 40, and 60% CH₃CN/0.1% TFA. A 50-mg tissue equivalent of each fraction was subjected to the FLIPR assay. The active fractions (40 and 60% CH₃CN/0.1% TFA fractions) were combined and lyophilized. The lyophilized material was then dissolved in 10 mM ammonium formate (pH 4.8), containing 10% CH₃CN (solvent A). We subjected the samples to carboxymethyl (CM)-ion exchange high-performance liquid chromatography (HPLC) at a flow rate of 1 ml/min; this procedure utilized a TSK-gel CM-2SW column (4.6 × 250 mm, Tosoh, Japan) with a two-step gradient profile, first from solvent A to 25% solvent B (1 M ammonium formate containing 10% CH₃CN, pH 4.8) for 10 min and then to 55% solvent B for 90 min. The eluate was collected in 1-ml fractions. The activity of each fraction was determined by subjecting a 50-mg tissue equivalent to FLIPR analysis. Active peak, P1 (fraction 25-29), was diluted in an equal volume of 0.1% TFA. The lyophilized sample was subsequently dissolved in 500 µl of 100 mM phosphate buffer (pH 7.4). This active solution was purified by anti-rat ghrelin (1-11) immunoglobulin G (IgG) immunoaffinity chromatography. Absorbed substances were eluted in 1 ml of 60% CH₃CN/0.1% TFA. The eluate was concentrated by evaporation and then subjected to reversed-phase (RP-) HPLC using a µBondasphare C18 column (2.1 × 150 mm, Waters) at a flow rate of 0.2 ml/min under a linear gradient from 10 to 60% CH₃CN/0.1% TFA for 40 min. The eluate corresponding to each absorption peak was collected. A part of each fraction (~100-mg tissue equivalent) was assayed for activity by FLIPR. The active fractions (numbers 9-11) were combined and further purified by RP-HPLC using a diphenyl column (2.1 × 150 mm, 219TP5215, Vydac, Hesperia, CA) at a flow rate of 0.2 ml/min with a linear gradient from 10 to 60% CH₃CN/0.1% TFA for 80 min. Each absorption peak was collected; a part of each fraction (~100-mg tissue equivalent) was assayed for activity by FLIPR. The active fraction was purified using a CHEMCOSRORB 3-ODS-H column (Chemco, Osaka, Japan) at a flow rate of 0.2 ml/min with a linear gradient from 10 to 60% CH₃CN/0.1% TFA for 40 min. Approximately 10 pmol of the purified peptide was analyzed by a protein sequencer (model 494, Applied Biosystems, Foster City, CA). Five pmol of the purified peptide was redissolved in 5 µl of 50% (v/v) methanol containing 1% AcOH. We determined the molecular weight using electrospray ionization mass spectrometry (ESI/MA) (SSQ 7000; Finnigan, San Jose, CA) as previously described (9).

3'-Rapid Amplification of the cDNA Ends (RACE)-- Total RNA was extracted from 2.5 g of bullfrog stomach using TRIzol reagent (Life Technologies, Inc.). Poly(A)⁺ RNA was isolated using a mRNA purification kit (TaKaRa, Kyoto, Japan). First strand cDNAs were synthesized from 500 ng of poly(A)⁺ RNA using an adaptor primer supplied by the 3'-RACE system (Life Technologies, Inc.). The reaction mixture was purified utilizing a Wizard PCR preps DNA purification system (Promega, Madison, WI) and eluted in 50 µl of sterilized water. One tenth of this purified cDNA served as a template for polymerase chain reaction (PCR) using four degenerate sense-primers based on the N-terminal 7-mino acid sequence of mammalian ghrelin (GSSFLSP). The sense-primer sequences of ghrelin (GRL)-s7: 5'-GGGTCGAG(C/T)TTCTT- (A/G)TC(A/G/T/C)CC-3'; GRL-s8: 5'-GGGTCGAG(C/T)TTCTT- (A/G)AG(C/T)CC-3'; GRL-s9: 5'-GGGTCGAG(C/T)TTCCT(A/G/T/C)TC(A/G/T/C)CC-3'; and GRL-s10: 5'-GGGTCGAG(C/T)TTCCT(A/G/T/C)AG(C/T)CC-3' were used to amplify the desired sequences. Primary PCR was performed using these degenerate sense-primers, a 3'-universal amplification primer supplied by the 3'-RACE kit, and Ex Taq DNA polymerase (TaKaRa). Sequences were amplified at 94 °C for 1 min with 35 subsequent cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min. We then performed nested PCR with one tenth of the purified PCR product. We designed two degenerate nested sense-primers based on the amino acid sequence of purified bullfrog ghrelin (8-14) (ADMQKIA). The nested sense-primer sequences contained sequences of 5'-GC(A/G)GA(C/T)ATGCA(A/G)AA(A/G)AT(A/C/T)GC-3' for the frog (f) GRL-nest 1 primer and 5'-GC(C/T)GA(C/T)ATGCA(A/G)AA(A/G) AT(A/C/T)GC-3' for the fGRL-nest 2 primer. The nested PCR was performed using conditions of 94 °C for 1 min and 30 subsequent cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 1 min. The candidate PCR product was subcloned using a TOPO TA cloning kit (pCR II-TOPO vector, Invitrogen, Carlsbad, CA). The nucleotide sequence was determined by DNA sequencer (model 373, Applied Biosystems) according to the Thermosequence II dye terminator cycle sequencing kit protocol (Amersham Pharmacia Biotech Inc.) using the M13 forward and reverse primers. The obtained bullfrog ghrelin cDNA EcoRI fragment (395 base pairs (bp)) was used as a probe to screen a bullfrog cDNA library.

Construction of Bullfrog Stomach cDNA Library-- A double-stranded cDNA was synthesized from 3 µg of poly(A)⁺ RNA using a cDNA synthesis kit (Amersham Pharmacia Biotech Inc.) with SuperScript II reverse transcriptase (Life Technologies, Inc.). cDNA ligated to EcoRI/NotI adapters. Following size-fractionation on a Sephacryl S-500 HR column (Life Technologies, Inc.), the cDNA was ligated into the EcoRI site of ZAP II vector arms. Phages were packaged in vitro using Gigapack III gold (Stratagene, La Jolla, CA), according to the manufacturer's protocol. The titer of the cDNA library was 1 × 10⁶ plaque-forming unit/ml.

Cloning of Bullfrog Prepro-ghrelin cDNA-- The phage DNA was transferred onto BIODYNE B nylon membranes (PALL, East Hills, NY), prehybridized with a hybridization buffer (5 × SSPE (750 mM NaCl, 50 mM NaH₂PO₄, and 5 mM EDTA, pH 7.4), 5 × Denhardt's solution, 50% formamide, 0.5% SDS, and 100 ng/ml calf thymus DNA) at 37 °C for 2 h. A 395-bp bullfrog ghrelin cDNA fragment was labeled by [-³²P]dCTP (Amersham Pharmacia Biotech Inc.) using a multiprime DNA labeling kit (Amersham Pharmacia Biotech Inc.) at 37 °C for 24 h. This labeled probe was then hybridized to the phage sequences. Following two washes with 2× SSC/0.1% SDS at 55 °C for 30 min, hybridized membranes were subjected to autoradiography with x-ray films (Kodak, Tokyo, Japan) at 80 °C for 24 h. Ten positive plaques were isolated and subjected to a secondary screening. Eight positive plaques, re-isolated in the second screening, infected XL 1-Blue MRF' in the presence of a helper phage. Following in vivo excision, we sequenced the resultant plasmid containing the bullfrog ghrelin cDNA.

Northern Blot Analysis-- Poly(A)⁺ RNA was isolated from the total RNA of 13 bullfrog tissues using a mRNA purification kit (TaKaRa). Poly(A)⁺ RNA (2 µg) was electrophoresed on a denaturing 1% agarose-formamide gel for 2 h under 50 volts. RNA was then transferred onto a nylon membrane (Zeta-Probe, Bio-Rad, Hercules, CA) and fixed by a UV-cross-linker. A ³²P-labeled, full-length bullfrog ghrelin cDNA was hybridized to the membrane. Conditions of hybridization and wash followed the procedure described above. The intensity of hybridization was analyzed using a BAS-5000 bioimaging analyzer (Fuji Film, Tokyo, Japan).

Gene Expression Analysis by RT-PCR-- Template cDNAs were made from 100 ng of poly(A)⁺ RNA derived from 13 bullfrog tissues using SuperScript II reverse transcriptase. RT-PCR reaction mixtures consisted of 10 ng of purified cDNA, a sense-primer (fGRL-full (FL)-s; 5'-CTTGTTCTGCCTGCTGTGGACG-3', nucleotides 82-103), an antisense-primer (fGRL-FL-as; 5'-GGATTTTCATTCTTGTCTTCT-3', nucleotides 358-378), and Ex Taq polymerase. The reactions were performed at 94 °C for 1 min with 40 subsequent cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. The PCR product (297-bp) was electrophoresed on a 2% agarose gel.

Intravenous Injection of Ghrelin into Rats and Plasma GH Measurement-- Under pentobarbital sodium anesthesia, male Sprague-Dawley rats (250-300 g) were cannulated in the femoral artery and vein. After sampling untreated blood from the femoral artery, a bolus of 20 ng/g of either synthetic rat ghrelin, bullfrog ghrelin-27, or bullfrog ghrelin-28 was injected into the femoral vein. Blood (150 µl) was collected in a syringe containing EDTA (1 mg/ml blood) 5, 10, 15, 20, 30, and 60 min after injection; isolated blood was then centrifuged at 5,000 rpm for 5 min. Plasma was stored at 30 °C until use. Plasma GH levels were measured using a rat GH enzyme immunoassay system (BIOTRAK, Amersham Pharmacia Biotech Inc.).

Effect of Ghrelin on Adenohypophyseal Hormone Secretion in Bullfrog Pituitary Cells-- We examined the effect of ghrelin on the secretion of adenohypophyseal hormones, such as GH, PRL, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), in dispersed bullfrog anterior pituitary cells (21). Bullfrogs purchased from a commercial supplier (Ohuchi, Saitama, Japan) in July and October were sacrificed by decapitation. The anterior pituitary glands were rapidly dissected out under sterile conditions. The pituitaries were diced in Ca²⁺-/Mg²⁺-free frog Ringer solution (50.4 mM NaCl, 0.7 mM KCl, 9.2 mM Na₂HPO₄, 0.9 mM KH₂PO₄, 2.4 mM NaHCO₃ and 2.4 mM EDTA) and transferred to 70% Medium 199 (M199, Nissui Pharmaceutical, Tokyo, Japan) containing 0.2% collagenase (Wako Pure Chemicals, Osaka, Japan) and 0.001% DNase (Sigma). After mechanical and enzymatic disruption, the suspension was centrifuged at 100 × g for 5 min. Following removal of the supernatant, the dispersed cells were resuspended in M199 containing 0.1% bovine serum albumin (Fraction V; Sigma) and plated in 96-multiwell plates (Corning Costar Japan, Tokyo, Japan) at 60,000 cells/well. Plates were precultured for 24 h at 23 °C in a humidified incubator with 95% air:5% CO₂. Test substances (200 µl/well) dissolved in cultured medium were added to the precultured cells for a 24-h incubation. The resulting media (150 µl) were collected in V-bottom 96-well microplates (Iwaki, Tokyo, Japan) following centrifugation at 100 × g for 5 min to remove cell debris. The collected medium (100 µl) was stored at 20 °C until use in a homologous radioimmunoassay for GH (22), PRL (23), FSH (24), and LH (25).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation and Sequence Determination of the Bullfrog Ghrelin-- Crude stomach extracts were eluted using a Sep-Pak C18 cartridge; the fraction eluted by 40-60% CH₃CN/0.1% TFA contained ghrelin activity. The lyophilized sample was then subjected to CM-ion exchange HPLC (Fig. 1A). Ghrelin activity was observed in 16 sequential fractions (numbers 18-32). A portion of the active peak, P1 (fraction 25-29), contained the highest activity. These pooled fractions were subjected to immunoaffinity chromatography using an IgG antiserum raised against rat ghrelin (1-11). The peptides, absorbed on the immunoaffinity column, were separated by RP-HPLC on a µBondasphare C18 column (Fig. 1B). Although many peaks were observed after the immunoaffinity chromatography, ghrelin activity was observed in peak numbers 9-11. These active peaks were combined and further purified by RP-HPLC on a diphenyl column (Fig. 1C). The three resultant active peaks (peaks I, II, and III) were purified by RP-HPLC on an ODS column (data not shown). Based on the peak height, the isolated peptide yield was estimated to be 20 pmol for peak I, 53 pmol for peak II, and 33 pmol for peak III. Ten pmol of each purified peptide was sequenced using a protein sequencer. Peak I contained a 28-amino acid residue peptide with a sequence of GLXFLSPADMQKIAERQSQNKLRHGNMN (X, unidentified) (Fig. 1D). Peaks II and III consisted of 27 amino acid residues with a sequence identical to that of peak I except for the deletion of the asparagine (N) residue at the C terminus: GLXFLSPADMQKIAERQSQNKLRHGNM (X, unidentified) (Fig. 1D). As the N-terminal amino acid sequence of the purified peptides, GLXFLSP, demonstrated a high similarity to the mammalian ghrelins (GSSFLSP), these purified peptides have been designated the bullfrog ghrelins. The unidentified X residue at position 3 was predicted to contain an acyl modification, as seen in rat ghrelin.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Successive purification of bullfrog ghrelin. Black bars indicate the fluorescence change of [Ca2+]i in CHO-GHSR62 cells. A, CM-ion exchange HPLC (pH 4.8) of Sep-Pak fraction eluted with 40 and 60% CH3CN/0.1% TFA. Column: TSK-gel CM-2SW (4.6 × 250 mm). Solvent: (a) 10 mM ammonium formate (pH 4.8):CH3CN (90:10 (v/v)), (b) 1 M ammonium formate (pH 4.8):CH3CN (90:10 (v/v)). Elution: linear gradient from a:b = 100:0 to a:b = 75:25 for 10 min, followed by a second gradient from a:b = 75:25 to a:b = 45:55 for 90 min. Flow rate: 1 ml/min. Fraction size: 1 ml/tube. The active peak, P1 (fraction 25-29), indicated by a solid bar, was purified by anti-rat ghrelin (1-11) IgG immunoaffinity chromatography. B, PR-HPLC of immunoaffinity chromatography adsorbed fraction. Column: µBondasphare C18 (2.1 × 150 mm). Elution: linear gradient from 10% CH3CN/0.1% TFA to 60% CH3CN/0.1% TFA for 40 min. Flow rate: 0.2 ml/min. Fraction size: each peak. The active fraction (numbers 9-11), indicated by solid bar, was further purified by an additional RP-HPLC. C, final purification of bullfrog ghrelin by RP-HPLC. Column: 219TP5215 diphenyl (2.1 × 150 mm). Elution: linear gradient from 10% CH3CN/0.1% TFA to 60% CH3CN/0.1% TFA for 80 min. Flow rate: 0.2 ml/min. Fraction size: full width of each peak. D, structures of bullfrog ghrelin, determined from peaks I, II, and III. The identity of the third residue as a threonine was determined by cDNA analysis. The modification of Thr3 by n-octanoic acid in peaks I and II and that with n-decanoic acid for peak III were analyzed by ESI/MS and confirmed by co-chromatography with synthetic and native peptides.

To determine the complete sequence of the purified peptides, we isolated a cDNA encoding the precursor protein from a bullfrog stomach cDNA library. As the peptide sequence of bullfrog ghrelin was quite different from the mammalian ghrelins, a mammalian ghrelin cDNA was unlikely to hybridize to the bullfrog ghrelin cDNA. Therefore, we used a partial bullfrog ghrelin cDNA fragment obtained by 3'-RACE-PCR as a screening probe. The cDNA fragment was ~400 bp long containing a known sequence as determined by peptide sequence analysis. This cDNA (395 bp, s8-n1-1) was used to screen a bullfrog stomach cDNA library. The isolated full-length bullfrog ghrelin cDNA was 485 bp long, containing 46 bp in the 5'-untranslated region, 345 bp of coding region, and 94 bp in the 3'-untranslated region. An AATAAA polyadenylation signal was identified in the 3'-non-coding region (Fig. 2). The predicted initiation methionine was located at nucleotide 47-49. The deduced amino acid sequence of the coding region indicated that the bullfrog ghrelin precursor is composed of 114 amino acid residues, two to four amino acids shorter than the mammalian ghrelin precursors (Fig. 3). The bullfrog prepro- and mature-ghrelins were only 18 and 29% identical, respectively, as compared with the mammalian counterparts. Computer analysis using the SignalP server (26) predicted the presence of a signal sequence comprised of the N-terminal 24 residues of the precursor in agreement with the determined sequence of the purified peptide. The mature-ghrelin peptide, therefore, directly follows the signal peptide. Furthermore, a typical dibasic processing sequence, Arg-Arg, flanks the mature-ghrelin peptide at nucleotides 203-208 at the C terminus (Fig. 2). The unidentified third residue of the purified peptide was determined from the nucleotide sequence to be threonine. This is a difference from those of the highly conserved serine present in the mammalian ghrelins.

View larger version (53K): [in this window] [in a new window]   Fig. 2.   Nucleotide sequences and deduced amino acid sequence of the bullfrog ghrelin. The bullfrog ghrelin cDNA contains a 485-base pair sequence. Prepro-ghrelin is composed of 114 amino acids. The frog ghrelin-28 mature sequence is underlined. A typical dibasic processing sequence, Arg-Arg, is boxed. Doubled underline indicates the polyadenylation signal (AATAAA).

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Comparison of amino acid sequence of the bullfrog ghrelin precursor to those of mammalian ghrelin precursors. Asterisks indicate the amino acids that are identical in all species. Amino acid sequences are available from the DDBJ/EMBL/GenBankTM data bases (accession number AB058510 for frog; AB029434 for human; AB092433 for rat; AB03571 for mouse; AF350329 for bovine; AF308930 for pig; AJ298295 for dog).

To determine the structure of the purified frog ghrelins, we determined the molecular weights of the purified peptides. Analysis by ESI/MS demonstrated that the molecular weights of the peak I (3308.5 ± 0.9) and peak II (3196.1 ± 0.9) ghrelins were ~126 mass units greater than the theoretical mass calculated from the 28-residue peptide sequence (3183.6) and the 27-residue of peak II (3069.6), respectively. These results indicate that the The³ hydroxyl groups for the peak I and peak II ghrelins are n-octanoylated, as seen for mammalian ghrelins. We designated these octanoylated 28- and 27-residue peptides as frog ghrelin-28 and frog ghrelin-27, respectively. The molecular weight of peak III (3225.3 ± 1.7) was 154 mass units greater than the theoretical mass (3069.6). This result suggests that the The³ residue is modified by n-decanoic acid. We designated this decanoylated 27-residue peptide as frog ghrelin-27-C10. These structures were confirmed by co-chromatography of the purified peptide with synthetic peptides by RP-HPLC (data not shown).

Gene Expression of the Bullfrog Ghrelin-- To examine the frog ghrelin gene expression pattern, we performed Northern blot analysis using poly(A)⁺ RNA isolated from 13 bullfrog tissues. A strong signal derived from ghrelin mRNA (~0.5 kilobase) was observed in the stomach; no signal could be detected in other tissues (Fig. 4A). To detect lower levels of gene expression, we performed RT-PCR analysis for the same tissues. We observed the expected PCR product (297 bp) at high levels in stomach, with moderate levels in small intestine, pancreas, and testes and low levels in the heart, lung, and gall bladder (Fig. 4B).

View larger version (114K): [in this window] [in a new window]   Fig. 4.   Gene expression of the bullfrog ghrelin in various tissues. A, Northern blot analysis. Each lane contains 2 µg of poly(A)+ RNA. B, RT-PCR analysis. Poly(A)+ RNA (100 ng) was subjected to reverse transcription; one tenth of the resultant cDNA was used as a template for specific amplification. Each lane contains one fourth of the reacted solution. Lane 1, brain; lane 2, heart; lane 3, lung; lane 4, liver; lane 5, stomach; lane 6, small intestine; lane 7, large intestine; lane 8, gall bladder; lane 9, spleen; lane 10, pancreas; lane 11, kidney; lane 12, adrenal gland; lane 13, testes.

Biological Activity of Bullfrog Ghrelin-- Frog ghrelin-27 and frog ghrelin-28 increased [Ca²⁺]_i in CHO-GHSR62 cells in a dose-dependent manner (Fig. 5) with a potency ~1/20 that of rat ghrelin. A similar stimulatory response was observed, altering plasma GH concentrations when either frog or rat ghrelin were injected intravenously into rats (Fig. 6). Rat ghrelin potently stimulated GH secretion, whereas the frog ghrelins had weak effects only.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Dose-response relationships of change in intracellular calcium concentrations by bullfrog and rat ghrelins in CHO-GHSR62 cells. CHO-GHSR62 cells (5 × 104 cells/well) were cultured in black 96-well plates for 20 h. After the addition of reagents, fluorescence changes were automatically measured by a FLIPR system. The maximum value of the response was used for data calculation. Values represent the mean ± S.E. (n = 3).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Time-course of changes in plasma GH concentration after intravenous injection of bullfrog and rat ghrelins into rats. Either synthetic bullfrog or rat ghrelin (20 ng/g body weight) was injected into the femoral vein of male Sprague-Dawley rats (250-300 g) anesthetized with pentobarbital sodium. Blood (150 µl) was collected from the femoral artery at time points up to 60 min after injection. Values (mean ± S.E., n = 5) are expressed in terms of the ratio of each time point to the initial level due to variations in the initial levels (mean ± S.E., frog ghrelin-27, 144.1 ± 11.1 ng/ml; frog ghrelin-28, 184.2 ± 28.0 ng/ml; rat ghrelin, 130.4 ± 15.5 ng/ml; saline, 273.3 ± 26.9 ng/ml).

The assessment of frog ghrelin activity throughout the purification was performed using CHO-GHSR62 cells expressing rat GHS-R. To examine the effect of frog ghrelin on cells expressing bullfrog GHS-R, we examined GH-releasing activity in dispersed bullfrog adenohypophyseal cells. The effects on other adenohypophyseal hormones such as PRL, LH, and FSH were examined simultaneously. Frog ghrelin-27 stimulated GH and PRL secretion in a dose-dependent manner (Fig. 7, A and B). Similar results were observed for frog ghrelin-28 (data not shown). The minimum effective concentration of frog ghrelin enhancing GH release was 0.1 nM; the value needed to enhance PRL release was 0.01 nM. Rat ghrelin stimulated GH and PRL secretion in this system, but the potency was reduced more than 3 orders of magnitude from frog ghrelin. We also examined the effect of GHRP-6 on GH and PRL secretion. Compared with the control value, 10 nM GHRP-6 stimulated GH secretion (32.82 ± 2.56 versus 26.39 ± 0.71 ng/10,000 cells/24 h, mean ± S.E. (n = 6); p < 0.05) and PRL secretion (61.95 ± 2.86 versus 46.24 ± 3.53 ng/10,000 cells/24 h, mean ± S.E. (n = 6); p < 0.001). The GH-releasing potency of GHRP-6 was reduced 2 orders of magnitude from frog ghrelin. No effects were observed on FSH and LH secretion (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Effect of bullfrog and rat ghrelins on the secretion of GH and PRL from dispersed bullfrog adenohypophyseal cells. Left and right panels show the secretion of GH (A) and PRL (B), respectively. Dispersed bullfrog adenohypophyseal cells were plated in 96-multiwell plates at 60,000 cells/well and precultured for 24 h at 23 °C in a humidified incubator with 95% air:5% CO2. Either frog ghrelin-27 or rat ghrelin (200 µl/well) was added to the precultured cells and incubated for 24 h. We measured the GH and PRL concentrations in the incubated media by a homologous radioimmunoassay designed to detect bullfrog GH or PRL. Values represent mean ± S.E. (n = 5-6). *p < 0.05 as compared with controls (concentration 0) by Student's t test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We isolated an amphibian ghrelin using a stable cell line expressing rat GHS-R (CHO-GHSR62) binding ghrelin. Bullfrog ghrelin was a potent ligand for the rat GHS-R, clearly demonstrating the GH-releasing activity of ghrelin in this system and in bullfrog adenohypophyseal cells.

The Ser³ hydroxyl group of the mammalian ghrelins is acylated by n-octanoic acid. Des-n-octanoyl form of ghrelin, designated as des-acyl ghrelin, has no effect on [Ca²⁺]_i in CHO-GHSR62 cells (8). This acylation, therefore, is essential for the biological activity of ghrelin (8, 9). The present study revealed that bullfrog ghrelin changes the acylated amino acid from Ser to Thr, the first report of an amino acid residue other than Ser of the ghrelin molecule that is n-octanoylated. This amino acid alteration at position 3 occurs by a point mutation of codon from AGC to ACC. Both Ser and Thr contain a hydroxyl group, creating the acylation site. These results suggest that the n-octanoyl modification of the position 3 amino acid hydroxyl group is the general structure of ghrelin across multiple species. As bullfrog ghrelin increases [Ca²⁺]_i in CHO-GHSR62 cells and stimulates GH secretion from the dispersed bullfrog adenohypophyseal cells, this acylation may mediate the biological activity of ghrelin.

We also identified a bullfrog ghrelin acylated by n-decanoic acid, the first report of acylation of ghrelin by a modifier other than n-octanoic acid. We have also observed n-decanoic acid acylation of a ghrelin present in human stomach, comprising 23% of the isolated ghrelin.² Approximately 35 pmol of decanoyl-modified ghrelin was isolated from bullfrog stomach, 33% of the isolated bullfrog ghrelin (105 pmol). A decanoyl-modified ghrelin has not been identified in the rat.² These differences may be species-specific. The mechanisms governing peptide acylation in the posttranslational processing of ghrelin, however, are still unknown.

Two lengths of bullfrog ghrelin were isolated in this study, one of 28 residues and the other of 27 residues. The later has a residue deleted from the C terminus of the 28-residue ghrelin. Analysis of the bullfrog ghrelin cDNA revealed that a typical dibasic processing sequence, Arg-Arg, follows the C terminus of the 28-residue ghrelin. The yield of 28-residue ghrelin (20 pmol) was less than that of the 27-residue ghrelin (86 pmol) in this purification. The mechanism governing the greater abundance of the 27-residue bullfrog ghrelin is unclear; it is likely, however, that an unusual endoproteolytic processing mechanism controls the maturation of bullfrog ghrelin.

The sequence of bullfrog ghrelin differs substantially from the mammalian counterparts (30% identity). The N-terminal amino acid residues (1-7), however, are highly conserved. The amino acids at positions 1, 4, 5, 6, and 7 are identical in all the species examined. Bullfrog ghrelin elicited increases in [Ca²⁺]_i in CHO-GHSR62 cells expressing rat GHS-R with a reduced potency from rat ghrelin. The Gly-Ser-Ser (n-octanoyl)-Phe-Leu segment at the N terminus constitutes the "active core" required for agonist potency at GHS-R (29); des-acyl ghrelin has no effect on [Ca²⁺]_i (8). Des-acyl ghrelin does not bind to GHS-R (28). In addition to the N-terminal amino acid sequence, the acylated amino acid at position 3 is involved in receptor binding and subsequent signal transduction.

Bullfrog ghrelin demonstrates a potent GH-releasing activity in bullfrog adenohypophyseal cells, with only a weak GH-releasing activity in in vivo experiment of rat. This result indicates the species specificity of the ligand binding to the receptor, suggesting that the structure of GHS-R ligand recognition is different between the bullfrog and rat. Although the bullfrog GHS-R has not been defined, key amino acid residues essential for ligand binding and activation have been defined in transmembrane regions 3 and 4 in human GHS-R (30). The N-terminal sequence of ghrelin is believed to contain the key amino acids for binding to the receptor. Amino acids at positions 2 and 3 in bullfrog ghrelin, however, are altered to Leu²-Thr³, instead of Ser²-Ser³ present in the mammalian ghrelins. Amino acids at positions 2 and 3 of the bullfrog ghrelin are likely to be important in the recognition of the ligand by bullfrog GHS-R.

Bullfrog ghrelin potently stimulated GH secretion in bullfrog adenohypophyseal cells. Hypothalamic GHRH primarily stimulates GH secretion in mammals (1). Although a bullfrog GHRH has not been identified, GH-releasing activity by human GHRH has been observed in the bullfrog in vitro (31). A GHRH-like peptide in the frog (Rana ridibunda) stimulates GH secretion from the bullfrog pituitary cell (32-34). These results indicate that the regulatory mechanism governing GH secretion by GHRH exists in the frog. In addition, thyrotropin-releasing hormone (27), proopiomelanocortin-derived peptides (27), pituitary adenylate cyclase-activating polypeptide (34), and endothelin (35) are involved in GH release in the frog. These facts indicate that the stimulation of GH secretion in frog is governed by a complicated hypothalamic hormonal regulation.

Ghrelin is also synthesized in the brain of rat (8). The few ghrelin-producing cells are located in a specific hypothalamic region, the hypothalamic arcuate nucleus (8), participating in the regulation of GH secretion from the pituitary (36). The presence of hypothalamic ghrelin in the bullfrog has not been confirmed. The gene expression of ghrelin in the bullfrog brain was not detectable by either Northern blot or RT-PCR analyses. In bullfrog, the expression of the ghrelin gene or the number of ghrelin-producing cells may be too low for detection, even by RT-PCR. As hypothalamic ghrelin may still play a role in the stimulation of GH secretion (36), it will be important to identify the expression of ghrelin in the bullfrog brain.

Bullfrog ghrelin is predominantly synthesized in the stomach, as seen in the rat. In both the bullfrog and the rat, ghrelin produced and secreted from the stomach is likely to act on the pituitary gland through the systemic circulation. This novel regulatory mechanism controlling GH secretion by a mechanism independent from hypothalamic hormonal control is present in mammals and in amphibians.

Bullfrog ghrelin also stimulated PRL secretion in bullfrog adenohypophyseal cells. The effect is more potent than that observed for GH secretion. In the rat, PRL secretion is not stimulated by rat ghrelin either in vivo or in vitro (8). In the bullfrog, GH and PRL are produced and secreted by different cells (22), suggesting that the coordinate GHS-R may be expressed on both GH- and PRL-producing cells.

In addition to the stomach, bullfrog ghrelin is synthesized in the heart, lung, pancreas, gall bladder, small intestine, and testes at low levels. In mammals, the GHS-R is also expressed peripherally at low levels in various tissues, such as the heart, lung, pancreas, intestine, adrenal, ovary, testes, skeletal muscle, and adipose tissue (4, 37, 38). In accordance with the receptor distribution, a cardiovascular function of GHRP has been observed (39). The distribution of GHS-R in the bullfrog has not yet been examined, but ghrelin may also act on peripheral tissues as an endocrine, paracrine, and/or autocrine factor through the GHS-R.

The discovery of amphibian ghrelin demonstrates the relevance of ghrelin in non-mammalian systems. In previous functional studies of the GHS-R in non-mammalian species, artificial ligands such as GHRPs and nonpeptidyl GHSs have been utilized. A GHS-R homologue identified in the pufferfish shares 58% identity to human GHS-R and is activated by GHRP-6 and nonpeptidyl GHSs (17), suggesting that GHS binding to the GHS-R is not species-specific. These GHS species, however, bind relatively weakly to pufferfish, compared with human GHS-R, possibly due to structural differences between the two receptors. GHS is a more potent agonist of human and/or rat GHS-R. GHSs can stimulate GH secretion in chicken (15, 16) and teleost fish such as tilapia (18), suggesting that GHSs bind to functional GHS-Rs in non-mammals. The present study demonstrates that GHRP-6 can stimulate GH release from the bullfrog pituitary. However, its potency is weak compared with frog ghrelin even though they both bind to the coordinate GHS-R. This may be due to different affinities of the natural and artificial ligands, an issue that should be resolved upon identification of the cognate bullfrog GHS-R. GHRP-6 stimulated PRL secretion from the bullfrog pituitary with a comparable potency to frog ghrelin. Little is known about the PRL-releasing activity of GHS in non-mammalian species. In tilapia, GHS tends to increase the plasma PRL level on a different time scale than GH levels are affected (18). Ghrelin does not stimulate PRL secretion in rats either in vitro or in vitro (8). These results suggest that ghrelin and GHS function as PRL secretagogues in non-mammals. This effect may be species-specific, seen especially in lower vertebrates. The evolutionary distribution of the GHS-R on cells producing GH (somatotrophs), PRL (lactotrophs) or both GH and PRL (mammosomatotrophs) may have influenced the GH- and PRL-releasing activities of ghrelin. It would be worthwhile, then, to examine the PRL-releasing activity of ghrelin in fish or chicken.

Our research suggests that an endogenous ligand for the GHS receptor, ghrelin, is widely present in vertebrates. The ligand has similar physiological functions across species, governing the release of GH.

	ACKNOWLEDGEMENTS

We would like to thank Dr. Yuta Arai (Waseda University, Tokyo, Japan) for collecting the bullfrog stomachs and Dr. Christopher A. Loretz (State University of New York at Buffalo) for careful reading of the manuscript.

	FOOTNOTES

^* This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, a grant-in-aid for Scientific Research from the Science and Technology Agency of Japan, and a grant-in-aid for the Promotion of Fundamental Studies in Health Science from the Organization for Pharmaceutical Safety and Research (OPSR) of Japan.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 DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB058510.

^§ To whom correspondence should be addressed. Tel.: 81-6-6833-5012 ext. 2479; Fax: 81-6-6835-5402; E-mail: kaiya@ri.ncvc.go.jp.

Published, JBC Papers in Press, August 23, 2001, DOI 10.1074/jbc.M105212200

² H. Hosoda, personal communication.

	ABBREVIATIONS

The abbreviations used are: GH, growth hormone; GHRH, growth hormone-releasing hormone; GHS(s), growth hormone secretagogue(s); GHS-R(s) or GHSR, GHS receptor(s); GHRP, growth hormone-releasing peptide; PRL, prolactin; FLIPR, fluorometric imaging plate reader; [Ca²⁺]_i, intracellular calcium concentration; CHO, Chinese hamster ovary; AcOH, acetic acid; CH₃CN, acetonitrile; TFA, trifluoroacetic acid; CM, carboxymethyl; HPLC, high-performance liquid chromatography; RP, reverse-phase; ESI/MA, electrospray ionization mass spectrometry; ODS, octadecyl silica; RACE, rapid amplification of the cDNA ends; PCR, polymerase chain reaction; RT, reverse transcription; bp, base pair(s); FSH, follicle-stimulating hormones LH, luteinizing hormone.

	REFERENCES

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