HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Seon, A. A. Articles by Amiche, M. Search for Related Content PubMed PubMed Citation Articles by Seon, A. A. Articles by Amiche, M. Social Bookmarking                      What's this?

J Biol Chem, Vol. 275, Issue 8, 5934-5940, February 25, 2000

Isolation, Structure, Synthesis, and Activity of a New Member of the Calcitonin Gene-related Peptide Family from Frog Skin and Molecular Cloning of Its Precursor^*

Aurelia Anne Seon^§, Thierry Nicolas Pierre^§, Virginie Redeker^¶, Claire Lacombe, Antoine Delfour, Pierre Nicolas, and Mohamed Amiche

From the  Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, 2 Place Jussieu, 75251 Paris Cedex 05, France and ^¶ Laboratoire de Neurobiologie, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS UMR 7637, 10 rue Vauquelin, 75005 Paris, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Calcitonin gene-related peptide has been extracted from the skin exudate of a single living specimen of the frog Phyllomedusa bicolor and purified to homogeneity by a two-step protocol. A total volume of 250 µl of exudate yielded 380 µg of purified peptide. Mass spectrometric analysis and gas phase sequencing of the purified peptide as well as chemical synthesis and cDNA analysis were consistent with the structure SCDTSTCATQRLADFLSRSGGIGSPDFVPTDVSANSF amide and the presence of a disulfide bridge linking Cys² and Cys⁷. The skin peptide, named skin calcitonin gene-related peptide, differs significantly from all other members of the calcitonin gene-related peptide family of peptides at nine positions but binds with high affinity to calcitonin gene-related peptide receptors in the rat brain and acts as an agonist in the rat vas deferens bioassay with potencies equal to those of human CGRP. Reverse transcriptase-polymerase chain reaction coupled with cDNA cloning and sequencing demonstrated that skin calcitonin gene-related peptide isolated in the skin is identical to that present in the frog's central and enteric nervous systems. These data, which indicate for the first time the existence of calcitonin gene-related peptide in the frog skin, add further support to the brain-skin-gut triangle hypothesis as a useful tool in the identification and/or isolation of mammalian peptides that are present in the brain and other tissues in only minute quantities.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Calcitonin gene-related peptide (CGRP)¹ (1) is found throughout the brain, in the terminals of sensory nerves, and in tracheal serous cells (2, 3). CGRP is one of the most potent vasodilatory and cardiotonic endogenous peptides discovered so far. The vasodilatation elicited by CGRP in the central coronary and peripheral vasculature has led to its therapeutic evaluation in the treatment of cerebral vasospasm, angina, migraine, Raynaud's disease, and erectile dysfunction of the penis (4). In the skin, CGRP is synthesized and released from capsaicin-sensitive c-fibers and can activate a number of target cells including keratinocytes, melanocytes, Langherans cells, mast cells, and microvascular epithelial cells (5, 6). As such, CGRP can have both protective and pathophysiological effects by participating to the processes that occur during inflammation and wound healing in the skin.

Despite a widespread distribution in virtually all organs, CGRP is present in the tissues in only minute quantities, making it difficult to chemically characterize the CGRP-like species detected by immunological methods. For instance, complete chemical identification of CGRP from the rabbit intestine was only achieved after extraction of 1 kg of intestines that yielded 10 µg (~2.5 nmol) of purified peptide (7). In a similar vein, 30 µg of human CGRP have been extracted from 45 human spinal cords (625 g of frozen tissues), a tissue where this peptide is especially concentrated (8, 9). Likewise, identification and characterization of CGRP in the central nervous system and the gastrointestinal tract of the European frog Rana ridibunda required the handling of 1200 brains and 400 intestines, respectively (10). Therefore, the very low amount of CGRP that can be recovered in a pure form from animal tissues makes mandatory the use of costly synthetic replicates for conducting routine pharmacological and clinical investigations.

On the other hand, the frog dermatous glands synthesize and expel an extraordinarily rich variety of mammalian-like hormones and neuropeptides (11-13). Amphibian peptides are often produced in such enormous quantities that it is possible to isolate enough material from a single specimen to determine the amino acid sequence and establish the pharmacological profile. This paper describes the isolation and characterization of CGRP from the skin exudate of a single living specimen of the arboreal frog Phyllomedusa bicolor, together with the cloning of its precursor and a preliminary evaluation of the pharmacological activity of the peptide.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Breeding of the South American Frog P. bicolor-- Frogs were placed in large wooden cages (120 × 90 × 90 cm; 12 animals/cage), covered on three sides by plastic mosquito net to provide good ventilation. Phyllodendron, Potos, and Dracena were used as perches, and water bowls were made available for nocturnal baths. The frogs were fed crickets. Humidity was kept at 65% by a constantly operating humidifier. Temperature was maintained at 25 ± 1 °C.

Purification of CGRP from Frog Skin-- 250 µl of peptide exudate were recovered from a single living frog specimen by gentle squeezing of the latero-dorsal portion of the skin and dissolved in 250 µl of 10% acetic acid. The homogenate was sonicated for 1 min and centrifuged for 10 min at 2,000 × g. The supernatant was recovered and fractionated by reverse-phase HPLC on a semipreparative column (Nucleosil 5-µm C18, 250 × 10 mm) using a solvent system composed of water containing 0.1% trifluoroacetic acid as solvent A and acetonitrile containing 0.07% trifluoroacetic acid as solvent B. The column was eluted at 0.75 ml/min with a 0-60% linear gradient of solvent B for 60 min. Aliquots of the fractions were assayed for CGRP-like immunoreactivity using an enzyme immunoassay procedure and a monoclonal anti-CGRP antibody as described previously (14). The immunoreactive fractions were pooled, freeze-dried, solubilized in 0.1 ml of water, and further fractionated by reverse-phase HPLC using an analytical C18 column (Lichrosorb 5-µm C18, 250 × 4.6 mm). Elution was achieved in 60 min with a 0-60% linear gradient of solvent B. Fractions were collected at a flow rate of 0.75 ml/min, from which aliquots were assayed for immunoreactivity using the enzyme immunoassay procedure.

Amino Acid Sequence Analysis-- Sequence determinations were carried out on a gas phase automatic protein sequencer (Applied Biosystems 476 A gas phase peptide sequanator). Phenylthiohydantoin-derivatives were detected with an on-line Applied Biosystems 120A analyzer. Data collection and analysis were performed with an Applied Biosystems 900A module calibrated with 32.5 pmol of phenylthiohydantoin-derivative standards.

Mass Spectrometry-- Mass spectra of positive ions were recorded in reflectron mode with a single stage reflectron matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer (Voyager-STR, PerSeptive Biosystems Inc., Framingham, MA) equipped with a delayed extraction device. Delayed extraction time was set at 200 ns. A saturated solution of 2,5-dihydroxybenzoic acid (Sigma) in 0.1% trifluoroacetic acid was the matrix used for all MALDI experiments. External calibration was performed with a mixture of angiotensin, ACTH (clip 1-17), ACTH (clip 18-39), and ACTH (clip 7-38) with monoisotopic mass-to-charge ratio (m/z) values corresponding to protonated [M + H]⁺ ions of 1296.685, 2093.087, 2465.189, and 3657.93, respectively, and bovine insulin with an average m/z value corresponding to [M + H]⁺ of 5734.59. Spectra were obtained with a resolving power M/M from 6000 to 13,000 at 50% valley.

Carboxypeptidase Y Digestion-- 40 pmol of intact peptide were dissolved in 10 µl of 0.1 M ammonium acetate buffer, pH 5.5. 1 µl of carboxypeptidase Y at a concentration of 2 ng/µl (carboxypeptidase Y sequencing grade; Roche Molecular Biochemicals) was added to the peptide solution. The mixture was left to react for 1, 2, 5, 15, 30, 60, and 120 min at 37 °C. The digestion was stopped at the desired time by the addition of 0.3 µl of 2.5% trifluoroacetic acid. Mass spectrometric analysis of the digestion products was performed by mixing 0.5 µl of the digest with 0.5 µl of matrix.

Solid Phase Peptide Synthesis-- Skin calcitonin gene-related peptide was prepared by stepwise solid phase synthesis using Fmoc polyamide active ester chemistry on a Milligen 9050 Pepsynthesizer. Fmoc-amino acids and PAL-PEG-PS resin were from Milligen. Side chain protections were O-tert-butyl ester for aspartic acid, O-tert-butyl ether for threonine, and trityl for asparagine and glutamine. Synthesis was carried out using a triple coupling protocol: N-Fmoc-amino acids (4.4 M excess) were coupled for 30-60 min with 0.23 M diisopropylcarbodiimide in a mixture of dimethylformamide and dichloromethane (60:40, v/v). Acylation was checked after each coupling step by the Kaiser test. Cleavage of the peptidyl resins and side chain deprotection were carried out at a concentration of 40 mg of peptidyl resin in 1 ml of a mixture composed of trifluoroacetic acid, phenol, thioanisole, water, and ethyl methyl sulfide (82.5:5:5:5:2.5, v/v/v/v/v) for 2 h at room temperature. After filtering to remove the resin and ether precipitation at 20 °C, the crude peptide was recovered by centrifugation at 5000 × g for 10 min, washed three times with cold ether, dried under nitrogen, dissolved in 20% acetic acid, and lyophilized. After lyophilization, the crude peptide was purified by preparative reverse-phase HPLC on a Waters RCM compact preparative cartridge Deltapak C 18 (300 Å; 25 × 100 mm) eluted at a flow rate of 8 ml/min by a multistep linear gradient of acetonitrile in 0.1% trifluoroacetic acid in water (5 min; wash at 5% acetonitrile followed by a 5-60% linear gradient of acetonitrile at 0.5%/min). Homogeneity of the synthetic peptide was assessed by gas phase sequence analysis, mass spectrum analysis, and analytical HPLC on a Lichrosorb C18 column (5 µm; 4.6 × 250 mm) eluted at a flow rate of 0.75 ml/min by a linear gradient of acetonitrile in 0.07% trifluoroacetic acid/water.

Receptor Binding Assay-- Decerebellated whole brains of male Harlan Sprague Dawley rats weighing 200-300 g (Dépré; Saint Doulchard, France) were homogenized by four cycles of Polytron and centrifugation (20 min, 12,000 × g) at 4 °C (15). The buffer was 50 mM Tris-Cl, pH 7.4. The yield of washed membranes equivalent to 10 brains was dispersed in 100 ml of 50 mM Tris-Cl, pH 7.4, 20% glycerol and stored at 80 °C. The final protein concentration of this extract was 9.25 mg/ml as determined by the method of Lowry et al. (16) using bovine serum albumin as standard. Binding assays were performed at 24 °C in 50 mM Tris-Cl, pH 7.4, containing 0.1 M NaCl, 4 mM MgCl₂, and 2% bovine serum albumin. Each assay contained, in a final volume of 500 µl, the membrane preparation (30 µl) and 32 pM human (2-[¹²⁵I]iodohistidyl 10)-CGRP (~2000 Ci/mmol; Amersham Pharmacia Biotech) with or without unlabeled ligand. The tubes were incubated for 60 min. The binding reaction was terminated by rapid vacuum filtration through 0.1% polyethyleneimine-coated Whatman glass fiber filters (GF/B). The filters were washed five times with 3 ml of ice-cold 50 mM Tris-Cl, pH 7.4, containing 0.1 M NaCl, 4 mM MgCl₂, and 2% bovine serum albumin. Specific binding was defined as total binding minus nonspecific binding determined in the presence of 1 µM unlabeled human CGRP. The specific binding represented about 80% of total binding when using 32 pM ¹²⁵I-labeled human CGRP. All determinations were performed in duplicate. The 50% inhibitory concentration values (IC₅₀) were obtained from nonlinear least-squares regression to a two-parameter logistic equation of the percentage of specific binding versus log (dose) curves. The inhibitory constant (K_i) of the various unlabeled ligands was calculated from the relation K_i= IC₅₀/(1 + (L/K_d)) (17), where L represents the concentration of the labeled ligand, and K_d is its equilibrum dissociation constant determined by saturation binding analysis.

Bioassay-- Synthetic and natural S-CGRPs were tested for their effectiveness in depressing electrically evoked contractions of the isolated vas deferens of the rat as described (18). Human CGRP was used as an internal standard. Briefly, one adult male Harlan Sprague Dawley rat (180-200 g), obtained from Dépré, was sacrificed by decapitation. The rat vas deferens were dissected carefully and immediately placed in oxygenated (95% O₂, 5% CO₂) Krebs-Ringer buffer solution (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.6 mM KH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, and 11 mM glucose maintained at 37 °C). The rat vas deferens were mounted on platinum electrodes in a bath containing 3 ml of oxygenated Krebs-Ringer buffer. The tissues were equilibrated for 1 h at a tension of 1 g. The tissues were stimulated with square electrical pulses using a Grass stimulator (model S88). The stimulation parameters were as follows: amplitude, 60 V; duration, 1 ms; frequency, 0.2 Hz. The responses were recorded on a Grass polygraph (model 79D) using a Grass force displacement transducer, model FT03 (Quincy, MA). Complete dose-response curves were constructed for each of the tested peptides (six different concentrations; six assays for each). Potencies were expressed as IC₅₀ values (nM) determined by regression analysis.

Cloning Procedure-- One adult specimen of Phyllomedusa bicolor was anesthetized with pentobarbital and immersed in liquid nitrogen and kept deep frozen until further processing. The skin was dissected on dry ice, and the tissues, approximately 7 g, were homogenized. Total RNAs were extracted as described by Chomczynski and Sacchi (19). Poly(A)⁺ RNAs were purified over an affinity oligo(dT)-cellulose spun column supplied by Amersham Pharmacia Biotech, and a cDNA library was constructed from skin poly(A⁺) RNA as described (20) using a standard procedure (21). Recombinant plasmids of the library were extracted from bacteria grown at 37 °C for 16 h in LB medium containing 100 mg/ml ampicillin and linearized by BamHI or EcoRI. An aliquot of the cDNA linearized by BamHI was used for PCR. The reaction was performed using a sense primer T7 (primer T7: 5'-AATACGACTCACTATAGGG-3') and an antisense degenerated primer corresponding to amino acids 25-31 of S-CGRP (primer 1: 5'-CKGTKGGKACRAARTCKGG-3') under the following conditions: 94 °C for 240 s, followed by 25 cycles of 94 °C for 40 s, 54 °C for 30 s, and 72 °C for 60 s. At the end of the last cycle, the sample was further incubated at 72 °C for 5 min and electrophoresed in 1.2% agarose gel. DNA fragments were excised from the gel and purified by the Qiaquick gel extraction kit protocol (Qiagen). The PCR product was cloned in the pGEMT-Easy vector system (Promega). Nucleotide sequencing analysis was performed by the dideoxy chain termination technique (22) in double-stranded pGEMT-Easy vector. We used the fluorescence-labeled dye terminator method and an ABI 377 automatic sequencer. To determine the sequence of the 3'-end of the prepro-S-CGRP, we used cDNA cleaved by EcoRI as template with an antisense universal primer (primer PU: 5'-GTAAAACGACGGCCAGTG-3') and a 3' sense gene-specific primer deducted from the 5' cDNA prepro-S-CGRP (primer 2: 5'-CTCTCTCCTGGCTGTCCT-3'). The following thermal cycle profile was used for the rapid amplification of cDNA end PCRs: 94 °C for 240 s, 25 cycles of 94 °C for 40 s, annealing at 54 °C for 30 s and 72 °C for 60 s, and a final extension step of 72 °C for 5 min. PCR products were purified by the Qiaquick kit (Qiagen), cloned in pGEMT-Easy vector (Promega), and sequenced as mentioned above.

RT-PCR-- mRNAs from frog brain and intestine were prepared using the Micro-FastTrack kit (Invitrogen). RT-PCR was performed using the SuperScript One-Step RT-PCR System (Life Technologies, Inc.). Both cDNA synthesis and PCR were performed in a single tube using specific primers deduced from the skin prepro-SCGRP (primer 3 sense: 5'-GGGTCACAGAGGCGCACA-3'; primer 4 antisense: 5'-GCAGTCCCGCCAGAAGCA-3') and mRNA from frog brain or intestine. The following thermal cycle profile was used for the PCR: 94 °C for 240 s, 25 cycles of 94 °C for 40 s, annealing at 56 °C for 30 s and 72 °C for 60 s, and a final extension step of 72 °C for 5 min. PCR products were purified by Qiaquick Kit (Qiagen), cloned in pGEMT-Easy vector (Promega), and sequenced as mentioned above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation and Purification of Calcitonin Gene-related Peptide from Frog Skin-- CGRP was purified to homogeneity from P. bicolor skin exudate by a two-step protocol. 250 µl of skin exudate recovered by gentle squeezing of the latero-dorsal portion of the skin of a single living frog were first fractionated on a reverse-phase HPLC semipreparative column. As shown in Fig. 1, the CGRP enzyme immunoassay revealed a single zone of immunoreactivity. The immunoreactive material was pooled and further fractionated on a reverse-phase HPLC analytical column. As depicted in Fig. 2, the initial immunoreactive material was recovered under a single symmetrical sharp peak accounting for >95% of the eluted material. The amount of purified peptide recovered was 380 µg starting from 250 µl of exudate. This purified material was used for further chemical and biological analysis.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Fractionation profile of the frog skin exudate on a reverse-phase HPLC semipreparative column (Nucleosil 5-µm C18, 250 × 10 mm) using a solvent system composed of water containing 0.1% trifluoroacetic acid as solvent A and acetonitrile containing 0.07% trifluoroacetic acid as solvent B. The column was eluted at 0.75 ml/min with a 0-60% linear gradient (dashed line) of solvent B for 60 min. The arrow points to the elution position of CGRP-like immunoreactive species as measured by an enzyme immunoassay. The absorbance at 220 nm is represented as a solid line.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Reverse-phase HPLC separation of the immunoreactive fraction recovered from Fig. 1 using an analytical C18 column (Lichrosorb 5-µm C18, 250 × 4.6 mm). Elution was achieved in 60 min with a 0-60% linear gradient (dashed line) of solvent B. Fractions were collected at a flow rate of 0.75 ml/min. The arrow points to the elution position of synthetic S-CGRP under the same conditions. The absorbance at 220 nm is represented as a solid line.

Covalent Structure of Skin Calcitonin Gene-related Peptide-- The sequence of the purified peptide was determined up to the 34th residue as SXDTSTXATQRLADFLSRSGGIGSPDFVPTDVSA by automated Edman degradation with a gas phase sequencer. Since no phenylthiohydantoin signals were obtained in positions 2 and 7, an aliquot of the purified peptide was reduced with dithiothreitol, alkylated with 4-vinylpyridine, and subjected to Edman degradation. After this treatment, two S-pyridylethylated cysteine derivatives were obtained, at cycles 2 and 7, respectively. No alkylated cysteines were observed at these positions when the reduction step was omitted, suggesting that the two cysteines were forming an intramolecular disulfide bridge in the native peptide. The purified peptide was also subjected to mass spectrometric analysis using MALDI-TOF mass spectrometry (Fig. 3A). The monoisotopic mass-to-charge ratio obtained for the protonated peptide (3806.77) suggested that we did not have the full sequence of the molecule. To gain additional information about the sequence, the molecule was subjected to carboxypeptidase Y digestion, and the resulting digestion fragments were analyzed by MALDI-TOF mass spectrometry (Fig. 3, B and C). Using this procedure, the C-terminal amino acid sequence of the peptide was unambiguously identified as TDVSANSF amide. From the results of the above experiments, a sequence of 37 residues could be proposed for the skin peptide as SCDTSTCATQRLADFLSRSGGIGSPDFVPTDVSANSF amide, with the 2 cysteines forming an intramolecular disulfide bridge. The experimental monoisotopic mass of the protonated peptide, measured by MALDI-TOF mass spectrometry (3806.77), corresponds to the theoretical mass calculated from this proposed sequence (3806.74). This suggested first that we had the full sequence of the peptide and second that the peptide is carboxyamidated and possesses a disulfide bridge.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   MALDI-TOF mass spectra of the S-CGRP peptide. MALDI-TOF mass spectrometric analysis were performed in the reflectron positive ion mode. A, the mass spectrum of the purified S-CGRP peptide reveals a monoisotopic mass to charge ratio of the protonated molecular ion [M + H]+ of 3806.77. B and C correspond to the mass spectrometric analysis of the digestion products obtained after carboxypeptidase Y treatment of the S-CGRP peptide during 5 and 15 min, respectively. B, the mass spectrum performed after 5 min of carboxypeptidase Y digestion indicates the presence of a phenylalanine-amide residue (corresponding to a loss of 146.05 atomic mass units) in the carboxyl-terminal position of the peptide. C, after 15 min of carboxypeptidase Y digestion, the mass spectrum identifies the following carboxyl-terminal sequence: TDSANF-NH2. The asterisks indicate ions corresponding to a loss of H2O.

A computer search comparing the skin peptide sequence to protein sequences contained in the Swiss-Prot protein data base revealed 50-80% amino acid positional identity with the calcitonin gene-related peptide family of peptides and 40-45% identity with the amylin family of peptides (Fig. 4). Identities with peptides of the calcitonin and adrenomedulin families were much lower. Accordingly, the novel frog skin peptide was named skin calcitonin gene- related peptide (S-CGRP).

View larger version (40K): [in this window] [in a new window]   Fig. 4.   A comparison of the deduced or chemically determined primary structure of calcitonin gene-related peptides from different vertebrate species: bovine (34), ovine (35), rat (1, 29), pig (36), rabbit (7), human (8, 28, 30, 37), Rana (10), chicken (38), cod (39), and salmon (40, 41). Alignments were performed by using CLUSTAL V Multiple Sequence Alignment software. Identical amino acid sequences are in the black box.

Solid Phase Synthesis of Skin Calcitonin Gene-related Peptide-- S-CGRP was synthesized by the solid phase method to confirm the proposed structure and to demonstrate that biological activities of the purified natural peptide reflected intrinsic properties. After purification by HPLC on a reverse-phase C18 column, synthetic CGRP was shown to be indistinguishable from the natural product by the following criteria. HPLC analysis revealed that synthetic CGRP eluted exactly at the same position as the natural corresponding product (Fig. 2); coinjection of the native and synthetic peptides resulted in only one symmetrical peak; mass spectrometric analysis of the synthetic peptide gave a monoisotopic mass to charge ratio of 3806.58 for the protonated peptide; the natural and the synthetic peptides have almost identical receptor binding activities.

Identification of cDNA Clones Encoding Skin Calcitonin Gene-related Peptide-- To allocate definitively S-CGRP to the calcitonin gene-related peptide family of peptides, we used polymerase chain reaction to amplify the nucleotide sequence encoding prepro-S-CGRP from skin poly(A⁺) mRNA. We have cloned and sequenced an 825-bp cDNA revealing an open reading frame encoding a 115-amino acid prepropeptide (Fig. 5). The deduced amino acid sequence begins with a putative signal peptide rich in hydrophobic amino acid residues. The peptide bond between Ala²⁵ and Ala²⁶ is the best point for cleavage by a signal peptidase, as determined by the method of Von Heijne (23). The putative signal sequence (residues 1-25) is immediately followed by a proregion (residues 26-67) comprising 42 residues with a pair of basic residues Lys⁶⁸-Arg⁶⁹ at its carboxyl terminus. The S-CGRP progenitor sequence, which is directly C-terminally flanked to the proregion, terminates by a Gly residue, which serves as an amide donor and a tetrabasic cleavage site. Cleavage of the precursors at the carboxyl side of these proteolytic signals by endoproteases and removal of the basic residues by carboxypeptidases would also liberate the mature frog skin CGRP. The cDNA sequence clearly confirmed the amino acid sequence of S-CGRP obtained by biochemical methods. Furthermore, the extensive sequence identities that are present between the CGRP precursors originating from various animal species and that of the novel skin peptide (51-70% at the amino acid level; 46-53% at the nucleotide level) are not found with the amylin precursors. It is thus likely that the 37-residue skin peptide isolated herein represents a novel member of the CGRP family of peptides.

View larger version (46K): [in this window] [in a new window]   Fig. 5.   Nucleic and amino acid sequences of cDNA encoding S-CGRP from the skin, brain, and intestine of P. bicolor. The deduced amino acid sequence is indicated above the nucleotide sequence. The 37-amino acid S-CGRP peptide is between the KR cleavage site and the GRRRR cleavage-amidation site. The numbers indicate the positions of the nucleotides (right) and amino acids (above the peptide sequence). Nucleotides are numbered positively from the 5'- to 3'-end of the cDNA. Amino acids are numbered starting with position 1 in the open reading frame. A solid line is drawn under the polyadenylation signal. Sense and antisense primers used in RT-PCR experiments are underlined by arrows.

The distribution of mRNA for prepro-S-CGRP was examined by RT-PCR using specific oligonucleotide primers (see "Materials and Methods"). A single strong amplification signal was observed at the expected size in the skin, the intestine, and the brain (Fig. 6). To certify that the amplified products correspond to prepro-S-CGRP mRNA, the purified PCR products were cloned in pGEM-Easy vector and sequenced. As shown in Fig. 5, isolated clones from the intestine and the brain have the same sequence as the skin prepro-S-CGRP except for one neutral third base change in the mature S-CGRP sequence, one change in the tetrabasic cleavage signal, and three nucleotide mutations in the 3'-noncoding region.

View larger version (92K): [in this window] [in a new window]   Fig. 6.   Amplification products generated by RT-PCR from frog skin, brain, and intestine mRNAs with specific primers 3 and 4 of prepro-S-CGRP.

Pharmacological Assays-- The receptor binding profiles of natural and synthetic S-CGRP for CGRP sites of the rat brain were determined by competition experiments using ¹²⁵I-labeled human CGRP as prototypical radiolabeled ligand. As expected, both peptides competitively inhibited the high affinity specific ¹²⁵I-labeled human CGRP binding in a concentration-dependent manner, with identical 50% inhibitory concentrations (K_i = 0.13 and 0.12 nM, respectively, for natural and synthetic skin CGRP) and quasi-Hill coefficients close to unity (Fig. 7). The inhibitory constant K_i of the natural and synthetic S-CGRP toward the sites labeled by ¹²⁵I-labeled human CGRP were almost identical to that determined for unlabeled human CGRP under similar experimental conditions (K_i = 0.25 nM). The displacement curves for these unlabeled ligands could all be fit to a simple competitive model assuming only one homogeneous population of binding sites. No evidence for a more complicated model was observed.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Competition curves for human (2-[125I]iodohistidyl 10)-CGRP binding sites by human CGRP (), frog S-CGRP natural () and frog S-CGRP synthetic (). Results are expressed as the percentage of maximal specific binding in the absence of unlabeled peptides. The data shown are from a single representative experiment. Values for IC50 were determined by regression analysis based upon three independent experiments carried out in duplicate.

Since binding assays do not determine whether S-CGRP acts as an agonist or an antagonist, we have tested synthetic and natural S-CGRPs for their effectiveness in depressing electrically evoked contractions of the isolated vas deferens of the rat (24). Human CGRP was used as an internal standard. As shown in Fig. 8, human CGRP (IC₅₀ = 38.8 nM), synthetic S-CGRP (IC₅₀ = 26.2 nM), and natural S-CGRP (IC₅₀ = 26.9 nM) were equiactive in the vas deferens bioassay. The inhibitory effects of these peptides were fully reversed by the prototypical antagonist human CGRP-(8-37) (Fig. 8) but remained unaffected by the addition of peptidase inhibitors (not shown).

View larger version (95K): [in this window] [in a new window]   Fig. 8.   Original tracing showing the effects of natural (25 nM) and synthetic (25 nM) SCGRP in comparison with the effect of human -CGRP (25 nM) on the electrically evoked contractions of the isolated rat vas deferens. These effects were reversed by human -CGRP-(8-37) (1 µM). Peptides were added in the bath at the arrows.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

One of the most fascinating stories in comparative biochemistry is represented by the studies of Erspamer and colleagues on amphibian skin secretions (12). They discovered in skin extracts a host of novel, biologically active peptides bearing close structural and pharmacological similarities to mammalian peptides that interact primarily with receptors of the central and peripheral nervous system and the gastrointestinal tract. As a result of the repeated discovery of structural correspondence between frog skin peptides and mammalian neuropeptides and hormones, Erspamer et al. (25) predicted that each peptide discovered in the skin would have a counterpart in the brain and the gut of mammals (i.e. the so-called brain-skin-gut triangle hypothesis). So far, more than 200 different biologically active peptides have been isolated from skin extracts of various frog species at a still increasing pace and with no signs yet of exhaustion (13). Most of these peptides belong to families that have their counterparts in mammals. Examples are many and include bradykinins, angiotensins, tachykinins, bombesin/gastrin-releasing peptide, hypophysiotropic neuropeptides, pancreatic polypeptide/peptide tyrosine-tyrosine/neuropeptide tyrosine, and the opioid peptides dermorphin/deltorphins. The discovery of calcitonin gene-related peptide in the skin of P. bicolor adds further support to the brain-skin-gut triangle hypothesis and suggests that other members of the superfamily may also be present in the skin.

In the present study, 380 µg of S-CGRP have been purified to homogeneity from 250 µl of the milky exudate obtained by gentle squeezing of the latero-dorsal portion of the skin of a single living frog. This procedure can be repeated every 10 days to allow the complete replenishment of the dermatous glands. Therefore, frog skin provides us with an inexhaustible and low cost source of natural CGRP that can be easily purified in great quantities by a two-step purification scheme.

The CGRP family of peptides includes the 37-amino acid peptide CGRP and the 37-residue peptide amylin, which is found in pancreatic islet -cells (26, 27). There is considerable conservation of sequences between CGRP and amylin, including the N-terminal disulfide bridge, the C-terminal amide, and adjacent regions. Hand alignments of the novel skin peptide with CGRP originating from various animal species showed 50-80% amino acid positional identities (Fig. 4). There is rather less identity (40-45% identity) with members of the amylin family. Furthermore, the extensive sequence similarities that are present between the precursors for CGRP and that of the novel skin peptide (51-70% at the amino acid level; 46-53% at the nucleotide level) are not found with the amylin precursors. These findings make it likely that the 37-residue skin peptide isolated herein, named S-CGRP, represents a novel member of the CGRP family of peptides. Although the same peptide is also present in the brain and the intestine of the frog, the physiological significance of its presence in huge amounts in the skin is unclear. Besides -CGRP, another form, -CGRP, is expressed in human and rat, which is encoded by a different gene (8, 28-30). In the rat, -CGRP differs from -CGRP by 1 residue in position 35. In humans, the amino acid sequences of - and -CGRP differ in the midregion of the peptides, at positions 3, 22, and 25. Therefore, there are no specific positions that may indicate whether S-CGRP resembles more closely - or -CGRP.

To completely reverse the actions of CGRP would suggest that CGRP-(8-37) has a pA2 of 7 or greater in this system. Although the antagonist concentration used in these experiments is high, this may suggest that the rat vas deferens is expressing something more like a CGRP 1 receptor subtype, not the CGRP 2 subtype that would be expected in this tissue (24). On the basis of pharmacological evidence, the existence of at least two CGRP receptor subtypes has been proposed as well as independent binding sites for amylin (31, 32). However, consolidation of this hypothesis is awaiting the development of potent subtype-selective agonists and antagonists. S-CGRP has Ser, Ala, Gln, Ser, Asp, Ser, and Ser at positions 5, 8, 10, 24, 26, 33, and 36, whereas all other members of the CGRP family have Ala, Val, His, Lys, Asn, Gly, and Ala at the corresponding positions (Fig. 4). In addition, the skin peptide is slightly acidic, with a net charge of 1, as compared with the other CGRPs, which have 3-6 positive charges. The changes at the C terminus are particularly striking, since this has been proposed as a crucial determinant for high affinity binding and activity (33), yet S-CGRP is a potent agonist, being as active as human CGRP in displacing radiolabeled human CGRP from rat brain receptors and in inhibiting the electrically evoked contractions in the rat vas deferens preparations. This indicates that the changes to the sequence have not any real significance for binding and activity in these assays. By taking advantage of these sequence differences, the evaluation of the structure-activity relationships of S-CGRP in various tissues and pharmacological model systems may provide a starting point for the design of agonists and antagonists with high selectivity for CGRP receptor subtypes.

	ACKNOWLEDGEMENTS

The expert assistance of J. J. Montagne is deeply appreciated. We gratefully acknowledge Dr. C. Creminon for the generous gift of the anti-rat -CGRP monoclonal antibody. We thank the referees for helpful comments.

	FOOTNOTES

^* This work was supported by the CNRS and the Fondation pour la Recherche Médicale.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 GenBank^TM/EMBL Data Bank with accession number(s) Y18495 (for prepro-skin calcitonin gene-related peptide) and P81564 (for skin calcitonin gene-related peptide).

^§ These two authors contributed equally to this work.

To whom correspondence should be addressed: Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, UMR 7592 CNRS/Université Paris 6 et Université Paris 7, 2 Place Jussieu, 75251 Paris Cedex 05, France. Fax: 01 44 27 59 94; E-mail: amiche@ijm.jussieu.fr.

	ABBREVIATIONS

The abbreviations used are: CGRP, calcitonin gene-related peptide; S-CGRP, skin calcitonin gene-related peptide; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; ACTH, adrenocorticotropic hormone; Fmoc, N-(9-fluorenyl)methoxycarbonyl.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. Orivel, V. Redeker, J.-P. Le Caer, F. Krier, A.-M. Revol-Junelles, A. Longeon, A. Chaffotte, A. Dejean, and J. Rossier Ponericins, New Antibacterial and Insecticidal Peptides from the Venom of the Ant Pachycondyla goeldii J. Biol. Chem., May 18, 2001; 276(21): 17823 - 17829. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Seon, A. A. Articles by Amiche, M. Search for Related Content PubMed PubMed Citation Articles by Seon, A. A. Articles by Amiche, M. Social Bookmarking                      What's this?