Proc. Natl. Acad. Sci. USAVol. 91, pp. 10295-10299, October 1994Neurobiology

Skin peptide tyrosine-tyrosine, a member of the pancreaticpolypeptide family: Isolation, structure, synthesis, andendocrine activity

(peptide YY/neuropeptlde Y/melanostatin/frog skin peptides/melanotropin secretion)

A. MOR*, N. CHARTRELt, H. VAUDRYt*, AND P. NICOLAS**Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, Universitd de Paris 7, 2 place Jussieu, 75251 Paris, France; and tLaboratoire deNeuroendocrinologie Cellulaire et Mol6culaire, Unit6 413 de l'Institut National de la Sante et de la Recherche M6dicale, Unitd AffMliie au Centre National dela Recherche Scientifique, Institut F6d&ratif de Recherches Multidisciplinaires sur les Peptides, Universit6 de Rouen, 76821 Mont-Saint-Aignan, France

Communicated by Vittorio Erspamer, July 13, 1994 (received for review April 10, 1994)

ABSTRACT Pancreatic polypeptide, peptide tyrosine-tyrosine (PYY), and neuropeptide tyrosine (NPY), three mem-bers of a family of structurally related peptides, are mainlyexpressed in the endocrine pancreas, in endocrine cells of thegut, and in the brain, respectively. In the present study, wehave isolated a peptide of the pancreatic polypeptide familyfrom the skin of the South American arboreal frg Phylome-dusa bicolor. The primary structure of the peptide was estab-lished as Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly-Glu'O-Asp-Ala-Ser-Pro-Glu-Glu-Met-Asn-Lys-Tyr2O-Leu-Thr-Ala-Leu-Arg-His-Tyr-Be-Asn-Leu3O-Val-Thr-Arg-Gln-Arg-Tyr-NH2.This unusual peptide, named skin peptide tyrosine-tyrosine(SPYY), exhibits 94% sia with PYY from the frog Ranaridibunda. A synthetic replicate ofSPYY inhibits m tpirelease from perifused frog neurointermediate lobes in verymuch the same way as NPY. These results demonstrate theoccurrence of a PYY-llke peptide in frog skin. Our data alsosuggest the existence of a pituitary-skin regulatory loop inamphibians.

The poison glands in the skin of amphibians are an excep-tionally rich source of biologically active substances includ-ing alkaloids, biogenic amines, and peptides (1). To date,>110 different regulatory peptides have been characterizedfrom amphibian skin extracts (2). These peptides generallybelong to families of biologically active peptides, which havetheir counterparts in mammals, such as tachykinins (3, 4),bradykinins (5, 6), angiotensin (7), caerulein/cholecystokinin(8), bombesin/gastrin-releasing peptide (9), opioid peptides(10), antimicrobial peptides (11, 12), and hypophysiotropicneuropeptides (13, 14). Although the physiological role of thevarious peptides synthesized and released by dermatousgranular glands remains largely unknown, most of thesepeptides are produced in such enormous quantities that it isoften possible to isolate enough material from a single skin todetermine the amino acid sequence (15, 16).The pancreatic polypeptide (PP) family consists of three

members of structurally related peptides, which all possess36 amino acid residues, exhibit a C-terminal tyrosine amidemoiety, and share common features of tertiary structure: (i)PP, originally discovered in the chicken pancreas (17), isexclusively localized in endocrine cells of pancreatic islets(18); (ii) peptide tyrosine-tyrosine (PYY), initially isolatedfrom pig intestine (19), is primarily synthesized in gut endo-crine cells (20) as well as in brainstem neurons (18); (iii)neuropeptide tyrosine (NPY), first characterized from the pigbrain (21), is expressed in neurons of the central and periph-eral nervous system (22). Phylogenic studies indicate that

NPY and PYY are both present in primitive vertebrates(23-29), suggesting that the genes encoding these two pep-tides arose from early duplication of an ancestral gene (30).In contrast, PP-like peptide is absent in fish and, therefore,it has been proposed that the PP gene arose from duplicationof the PYY gene at the time ofemergence ofamphibians (30).The primary structures of all three members of the PP familyhave recently been determined in the frogs Rana catesbeiana(31) and Rana ridibunda (32, 33). However, the occurrenceof PP-related peptides has never been reported in the skin ofamphibians.We have recently undertaken the purification ofa series of

unusual polypeptides from the skin of the frog Phyllomedusabicolor, using their antifungal activity as a biological test (34,35). We report here the isolation, characterization, and totalsynthesis of a 36-residue polypeptide, which shows 94%identity with R. ridibunda PYY (33). We also show that thesynthetic replicate inhibits melanotropin (a-melanocyte-stimulating hormone, a-MSH) release from frog pars inter-media in very much the same way as NPY (32, 36, 37). Thispeptide has been called skin peptide tyrosine-tyrosine(SPYY).

MATERIALS AND METHODSPurification of SPYY from Frog Skin Extract. The early

fractionation steps of the skin extract have been describedelsewhere (35). Briefly, the skin of a specimen of the SouthAmerican arboreal frog P. bicolor was extracted in 10%acetic acid and chromatographed on a Sephadex G-50 col-umn; aliquots from these fractions were assayed for antifun-gal activity. Active fractions (fractions 80-90) were subjectedto reversed-phase HPLC on a preparative C18 column (RCM10 x 25). Fractions that eluted between 62 and 68 min werepooled, freeze dried, and solubilized in 0.5 ml of 20%oacetonitrile/water containing 0.1% trifluoroacetic acid(TFA). An aliquot (50 ,ul) from the soluble material waschromatographed on a Nucleosyl C4 analytical HPLC col-umn (Millipore; 300 A, 5 ,um, 250 x 4.6 mm) using a solventsystem composed of water containing 0.1% TFA and 0.2%tetraethylammonium as solvent A and pure acetonitrile assolvent B. Elution was achieved with a 20-60% linear gra-dient of solvent B for 40 min at a flow rate of0.5 ml/min. Thefinal purification step was performed on an analytical Delta-Pak HPLC column (Waters; C18, 5 g&m, 150 x 2 mm). Aftera 5-min wash with 0.1% TFA/water the column was eluted

Abbreviations: a-MSH, a-melanocyte-stimulating hormone; NPY,neuropeptide tyrosine; PP, pancreatic polypeptide; PYY, peptidetyrosine-tyrosine; SPYY, skin peptide tyrosine-tyrosine; TFA, tri-fluoroacetic acid; Fmoc, fluoren-9-ylmethoxycarbonyl; NIL, neu-rointermediate lobe.4To whom reprint requests should be addressed.

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with a linear gradient of acetonitrile (0-60%) containing0.07% TFA at a flow rate of 0.2 ml/min.

Sequence Analysis. Amino acid sequence was determined onan Applied Biosystems model 477A gas-phase protein se-quencer and on a MilliGen 6600 solid-phase sequencer aftercovalent binding of the sample to a Sequelon arylamine mem-brane. Phenylthiohydantoin-derivatized amino acids were de-tected with an on-line HPLC column (WatersMS HPLC; SequaTag C-18 PTH analysis column, 350 x 3.9 mm) developed withammonium acetate (pH 4.8) and acetonitrile. The column wascalibrated with 15 pmol of phenylthiohydantoin-derivatizedamino acid standards. Data collection and analysis were per-formed with a Maxima-PTH chromatography analysis softwarepackage [Dynamic Solution (Waters)].

Electrospray Ionization Mass Spectroscopy. Mass spectralanalysis was performed with a quadrupole coupled electro-spray mass spectrometer (model R10-10; Nermag, Houston)associated with a source produced by Analytica (Branford,CT). The sample was dissolved in a water/methanol (1:1)mixture and injected via a metal capillary (stainless steel) atatmospheric pressure at a rate of 1 Al/min with a microsy-ringe installed in a syringe infusion pump (Harvard Appara-tus; model 11). An electrospray voltage of -5 kV was appliedto the internal wall of the source at the origin of the liquiddispersion for electrospray formation and ion extraction.Ions were detected and analyzed in the positive mode as afunction of their m/z ratio.

Peptide Synthesis. Peptides were prepared by stepwisesolid-phase synthesis using fluoren-9-methoxycarbonyl(Fmoc) polyamide active ester chemistry on a MilliGenmodel 9050 Pepsynthesizer. All Fmoc amino acids were fromMilliGen. 4-Hydroxymethylphenoxyacetic acid (HMP)-linked polyamide/kieselguhr resin and Fmoc amino acidpentafluorophenyl and 3-hydroxy-2,3-dehydro4oxobenzot-riazine esters were from MilliGen/Biosearch. Side-chainprotections were t-butyl for tyrosine, serine, and threonine;pentamethylchromansulfonyl (Pmc) for arginine; trityl (Trt)for asparagine and histidine; t-butyloxycarbonyl for lysine;t-butoxy for glutamic acid and aspartic acid. Cleavage ofpeptidyl resin and side-chain deprotection were carried out ata concentration of 5 mg of peptidyl resin in 1 ml of a mixturecomposed of TFA, p-cresol, thioanisole, water, and ethylmethyl sulfide (82.5%, 5%, 5%, 5%, and 2.5%; vol/vol) for 2h at room temperature. After filtering to remove the resin andether extraction, the crude peptides were purified by a

0cm

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0

combination of Sephadex gel filtration, ion-exchange chro-matography, and preparative HPLC. Homogeneity of thesynthetic peptides was assessed by analytical HPLC, aminoacid analysis, solid-phase sequence analysis, and mass spec-trometry as described (15).a-MSH Rease-Inhibiting Activity ofSPYY and SPYY-(14-

36). The biological activity of SPYY and its C-terminalfragment SPYY-(14-36) was tested on the neurointermediatelobe (NIL) of the frog R. ridibunda. Adult male frogs werepurchased from a commercial supplier (Coudtard, St. Hilairede Riez, France) and housed in a temperature-controlledroom (8°C ± 1°C) under an established photoperiod of 12 h oflight per day (light on from 06:00 to 18:00 h) for at least 1 weekbefore sacrifice. The frogs were killed by decapitation be-tween 08:00 and 09:00 and the NILs were removed under amicroscope. The glands were preincubated for 15 min atroom temperature in a Krebs-Ringer solution (15 mM Hepesbuffer/112mM NaCl/2mM KCl/2mM CaCl2) supplementedwith glucose (2 mg/ml) and bovine serum albumin (0.3mg/ml). The Krebs-Ringer solution was gassed for 30 minwith 02/CO2 (95:5) before use and thepH was adjusted to 7.4.The perifusion system used to evaluate the effect of SPYY

and SPYY-(14-36) on a-MSH release from frog NILs hasbeen described in detail (38). Briefly, intact NILs were mixedwith Bio-Gel P2 beads and transferred into plastic columns(internal diameter, 0.9 cm; three NILs per column). Thetissues were perifused with the Krebs-Ringer solution at aconstant flow rate (0.3 ml/min) and temperature (28°C)throughout the experiment. The effluent perifusate was col-lected as 7.5-min fractions during the stabilization period oras 2.5-min fractions during infusion of the peptide solutions.The fractions were immediately chilled on ice until radioim-munoassay. The concentration of a-MSH was measured ineach fraction, on the same day as the perifusion experiment,by a double-antibody radioimmunoassay procedure (39).The perifusion profiles were calculated and expressed as a

percentage of the basal secretory rate. The basal values werecalculated as the means offour consecutive fractions (7.5 mineach) collected just before the infusion of the peptide solu-tions. Each figure represents the mean (±SEM) of at leastfour independent perifusion experiments.

RESULTSPeptide Isoation. As reported previously (35), size frac-

tionation ofaP. bicolor skin extract on a G-50 gel-permeation

~~~-~ 502- A _ 50

0 10 20 30 6C0

0 20 40Time, min

60

FIG. 1. Purification of SPYY from P. bicolor skin. (A) Reversed-phase HPLC analysis of an aliquot (50 A1) from the soluble materialfractionated on a Nucleosyl C4 analytical HPLC column (300 A, 5 pm, 250 x 4.6 mm) equilibrated with water containing 0.1% TFA and 0.2%tetraethylammonium and eluted with a 20-60%6 linear gradient of acetonitrile for 40 min (dashed line) at a flow rate of 0.5 ml/min. (B) Finalpurification step of the material eluting at 23 min in chromatogram A, performed on an analytical Delta-Pak HPLC column (Waters; C18, 5 PM,150 x 2 mm). After a 5-min wash with 0.1% TFA/water, the column was eluted with a 0-60%o linear gradient of acetonitrile containing 0.07%TFA (dashed line) at a flow rate of 0.2 ml/min. Arrow indicates elution position of synthetic SPYY. (Inset) UV absorbance spectrum of theeluting peak at 50.03 min. Solid line, absorbance at 200 nm.

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column revealed antifungal activity eluting at 4-5 kDa.During the second step of purification of these fractionsachieved by HPLC, the initial antifungal activity from theG-50 column was recovered as three distinct active peakseluting at 41, 64, and 71 min. The latest eluting materialexhibiting antifungal activity was identified as a dermaseptin-related peptide (35). In the present study, the material elutingat 64 min was further purified. As shown in Fig. 1, after aseries of HPLC runs, a well-separated UV-absorbing peakwas obtained that contained a homogeneous peptide (>95%purity as determined by microbore HPLC). Inspection of thenear UV spectra of the peak indicated the presence of theclassical tyrosine UV signature (Fig. 1 Inset). This purifiedpeptide (final yield, =100 ug per g oforiginal dry extract) wasdirectly subjected to amino acid sequence analysis.Amino Acid Sequence Analysis and Mass Spectral Analysis.

In a first attempt to elucidate the primary structure of thepurified peptide, a peptide sequence could be determined upto the 16th residue as YPPKPESPGEDASPEE by automatedEdman degradation on a solid-phase sequencer after carbox-ylic covalent binding of the sample (150 pmol) to a Sequelonarylamine membrane. The purified peptide was then sub-jected to gas-phase sequence analysis and the sequence couldbe determined up to the .36th residue as YPPKPESPGE-DASPEEMNKYLTALRHYINLVTRQRY. This suggestedthe occurrence of a carboxamidated C-terminal residue, inwhich case Edman degradation of the carboxylic-linkedpeptide would indeed allow the determination of the se-quence only up to Glu16 anchored through its side chain,whereas the C-terminal extension of the peptide, being de-void of a free acidic group, would be undetectable by thismethod. Confirmation of this proposal was obtained by massspectral analysis of the peptide using electrospray ionizationmass spectrometry. As shown in Fig. 2, unequivocal pseudo-molecular ions [M + nH]l+ were observed at m/z corre-sponding to n = 3, 4, and 5 protonated species whoseaveraged molecular weight was 4259.1. This value corre-sponded precisely to that expected theoretically for theexperimentally determined amino acid sequence of 36 resi-dues with a carboxamidated C terminus.The search for similarity between the amino acid sequence

of this peptide and that of various related peptides wascarried out with CLUSTAL V multiple sequence alignmentsoftware (40). As shown in Table 1, pairwise alignment of thesequences of various PYY-related peptides with that of thenewly sequenced peptide revealed high similarity levelsranging between 70%6 and 94% amino acid positional identity.In particular, the sequence of the peptide most closelyresembled that ofPYY isolated from the intestine of the frogR. ridibunda (33) with only two amino acid substitutions-i.e., Ser/Asn at position 7 and Asn/Thr at position 18. Based

100-

0

ca

lw

ca0

U0.5

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0-

852.73

413.03

400

1065.94

1420.66

900 1400

mlz

FIG. 2. Electrospray ionization mass spectrum of SPYY showingpseudo-molecular ions [M + nH]n+ at m/z values of 1420.66,1065.94, and 852.73. These multiple ions correspond, respectively, ton = 3, 4, and 5 protonated species whose calculated averagemolecular weight was 4259.1.

on the high level of sequence similarity with these PYY-related peptides, this peptide was designated skin peptide YY(SPYY).

Solid-Phase Synthesis ofSPYY. A definitive confirmation ofthe structure proposed for SPYY was obtained throughsolid-phase synthesis of the peptide by Fmoc methodology.Purification of the synthetic peptide was performed by gelfiltration, ion-exchange chromatography, and reversed-phase HPLC as described (15). After purification, syntheticSPYY was shown to be indistinguishable from natural SPYYby the following chemical and physical criteria: (i) HPLCanalysis, under both gradient or isocratic conditions, re-vealed that synthetic SPYY eluted exactly at the sameposition as the natural peptide (Fig. 1B); coinjection of thenative and synthetic peptides resulted in only one symmet-rical peak. (ii) The sequence of synthetic SPYY could bedetermined only up to Glul6 by automated Edman degrada-tion after covalent binding of the peptide to a Sequelonarylamine membrane. (iii) Mass spectrometry of a sample ofsynthetic SPYY gave pseudo-molecular ions [M + nHfl+ atm/z values corresponding to n = 3, 4, and 5 protonatedspecies whose averaged value was 4259.1, identical to thatobtained with the natural peptide.

Effect of SPYY and SPYY-(14-36) on a-MSH Release.Administration of graded doses of synthetic SPYY (5 x10-1o-10-6 M) induced a dose-related inhibition of a-MSHrelease from perifused frog NILs (Fig. 3). For each dosetested, the maximal inhibitory effect occurred between 25

Table 1. Maximized pairwise sequence alignment of SPYY and various PYYs, NPYs, or PP

Similarity score,Peptide (origin) Amino acid sequence % Ref.SPYY YPPKPESPGEDASPEEMNKYLTALRHYINLVTRQRYa

PYY (frog) YPPKPENPGEDASPEEMTKYLTALRHYINLVTRQRYa 94 33****** * ****** ** * ********** ** *******

NPY (frog) YPSKPDNPGEDAPAEDMAKYYSALRHYINLITRQRYa 72 32**.** . ***** .. *.* ** . ******** .******

PP (frog) APSEPHHPGDQATPDQLAQYYSDLYQYITFITRPRFa 36 31*.** * ** *.** * * * * ** *** * **

PYY (porcine) YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRYa 75 19**.****.********** .* ..***** **********

NPY (porcine) YPSKPDNPGEDAPAEDLARYYSALRHYINLITRQRYa 70 21** **.* *****..*.**.* .********.*******

Pairwise sequence alignments were performed with CLUSTAL v multiple sequence alignment software(40). A Dayhoff PAM 250 matrix was used in peptide comparisons (41). Similarity scores werecalculated by the method of Wilbur and Lipman (42). Identical (*) and similar (.) residues amongsequences are highlighted. a, Amide.

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SPYY B100-

80-

>a)

c 60-n

cox 240-

20-

0 JI

0 30 60 90Time, min

I..I I0 10-10 10-9 10-8 10-7 10-6

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FIG. 3. Effect of synthetic SPYY and SPYY-(14-36) on a-MSH secretion by perifused frog NILs. (A) Kinetics of the response of the NILsto graded doses of SPYY. After a 90-min equilibration period, the lobes were exposed for 20 min to various concentrations of SPYY: *, 5 x

10-10 M; o, 3 x 10-9 M; v, 10-8 M; E, 3 x 10-7 M. The spontaneous level of a-MSH release (A; 100% basal level) was calculated as the meana-MSH secretion rate during the 30-min period just preceding the administration of SPYY. (B) Semilogarithmic plot comparing the effects ofSPYY (e) and SPYY-(14-36) (v) on a-MSH release. All experimental values were calculated from data similar to those presented in A. Valuesare means (±SEM) of four to six independent experiments.

and 30 min after the onset of infusion of SPYY (Fig. 3A). Theeffect of SPYY was reversible whatever the dose of thepeptide administered, and the basal secretory level wasrecovered within 60 min after the end of infusion of thepeptide solution. Using a series of experiments similar tothose presented in Fig. 3A, dose-response curves wereestablished with graded concentrations of SPYY and SPYY-(14-36) (Fig. 3B). A significant inhibition of a-MSH releasewas observed after administration of 5 x 10-10 M SPYY.Half-maximal inhibition occurred at a dose of 3.5 x 10-9 MSPYY and 2 x 10-7 M SPYY-(14-36). At a dose of 10-7 M,SPYY caused 93% inhibition of a-MSH release.

DISCUSSIONThis study presents structural characterization of a memberof the PP/NPY/PYY peptide family in the skin ofa frog. Thesequences of P. bicolor SPYY and R. ridibunda PYY (33)differ by only two amino acid substitutions at positions 7 and18. The substitution at position 18 (Thr -* Asn) can be

accomplished by the exchange of a single nucleotide in thecorresponding cDNA sequence. Conversely, the degree ofsimilarity between the sequences of SPYY on the one handand frog NPY (32) or PP (31) on the other hand was muchlower. The strong similarity of the primary structures of P.bicolor SPYY and R. ridibunda PYY suggests that SPYYmay simply correspond to the expression product of the PYYgene in the frog skin. Alternatively, two closely related genesencoding PYY and SPYY, with differential tissue expression,may exist in the genome of P. bicolor as previously demon-strated for brain and skin prothyrotropin-releasing hormone(43, 44). Structural characterization of PYY from the gut ofP. bicolor should help to determine whether SPYY and PYYoriginate from expression of a single gene.We have previously shown that, in amphibians, NPY acts

as a physiologically important inhibitor of the activity of thepars intermedia of the pituitary (36, 37, 45). The present datademonstrate that SPYY is also a potent inhibitor of a-MSHsecretion in vitro. Since the C-terminal peptide SPYY-(14-36) was capable of inhibiting a-MSH release (although thisshort-chain analog was less potent than SPYY), it appearsthat the action of SPYY is likely mediated through Y2

receptors (46). Thyrotropin-releasing hormone, another pep-tide produced by frog skin (43), is also involved in the controlof a-MSH release from the pars intermedia (38, 47). The factthat dermal melanocytes are the main target cells for a-MSH,together with the occurrence of thyrotropin-releasing hor-mone and SPYY in frog skin, suggests the existence of aregulatory loop between the pars intermedia of the pituitaryand the skin in amphibians.The isolation in the skin ofP. bicolor of a peptide from the

PP/NPY/PYY family validates the concept of the potentialuse of frog skin for identification of specific regulatorypeptides (48-50). The skin ofamphibians contains both a richvariety and large amounts of biologically active peptides. Forinstance, in the present study, SPYY was purified from theskin of a single specimen (-1 g), while the related peptidesNPY and PYY have previously been isolated from 1200brains (=95 g) and 400 intestines (==270 g) ofR. ridibunda (32,33). The huge concentration of peptides stored in amphibianskin has already allowed the identification of 110 peptidesincluding hormones, neuropeptides, and defensive peptides(10, 11, 51). As a result, frog skin proves to be a very usefulorgan for characterization of peptides and for elucidation oftheir biosynthetic pathways.

The expert technical assistance of J. J. Montagne is gratefullyacknowledged. This work was supported in part by funds from theCentre National de la Recherche Scientifique, the Institut Nationalde la Sante et de la Recherche Mddicale (CRE 89-4015 and 92-0508),the European Economic Community (ERBCHRXCT920017), andthe Conseil Regional de Haute-Normandie.

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