AcT-2 A Novel Myotropic and Antimicrobial Type 2 Tryptophyllin fromthe Skin Secretion of the Central American Red-Eyed Leaf FrogAgalychnis callidryasGe L Lyu P Zhou M Zhang H Wan Y Li B Li R Wang L Chen T amp Shaw C (2014) AcT-2 ANovel Myotropic and Antimicrobial Type 2 Tryptophyllin from the Skin Secretion of the Central American Red-Eyed Leaf Frog Agalychnis callidryas The Scientific World Journal 2014 [158546]httpsdoiorg1011552014158546
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Research ArticleAcT-2 A Novel Myotropic and Antimicrobial Type 2Tryptophyllin from the Skin Secretion of the Central AmericanRed-Eyed Leaf Frog Agalychnis callidryas
Lilin Ge Peng Lyu Mei Zhou Huiling Zhang Yuantai WanBin Li Renjie Li Lei Wang Tianbao Chen and Chris Shaw
Natural Drug Discovery Group School of Pharmacy Queenrsquos University Belfast BT9 7BL UK
Correspondence should be addressed to Renjie Li rliqubacuk and Lei Wang lwangqubacuk
Received 13 December 2013 Accepted 8 January 2014 Published 13 February 2014
Academic Editors A Sacchetti and H-S Won
Copyright copy 2014 Lilin Ge et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Tryptophyllins are a diverse family of amphibian peptides originally found in extracts of phyllomedusine frog skin by chemicalmeansTheir biological activities remain obscure Here we describe the isolation and preliminary pharmacological characterizationof a novel type 2 tryptophyllin named AcT-2 from the skin secretion of the red-eyed leaf frog Agalychnis callidryas The peptidewas initially identified during smooth muscle pharmacological screening of skin secretion HPLC fractions and the unique primarystructuremdashGMRPPWF-NH
2mdashwas established by both Edman degradation and electrosprayMSMS fragmentation sequencing A
cDNA encoding the biosynthetic precursor of AcT-2 was successfully cloned from a skin secretion-derived cDNA library by meansof RACEPCRand this contained an open-reading frame consisting of 62 amino acid residueswith a singleAcT-2 encoding sequencelocated towards the C-terminus A synthetic replicate of AcT-2 was found to relax arterial smooth muscle (EC
50= 51 nM) and to
contract rat urinary bladder smooth muscle (EC50= 93 120583M) The peptide could also inhibit the growth of the microorganisms
Staphylococcus aureus (MIC = 256mgL) Escherichia coli (MIC = 512mgL) and Candida albicans (128mgL) AcT-2 is thus thefirst amphibian skin tryptophyllin found to possess both myotropic and antimicrobial activities
1 Introduction
Amphibians represent the most ancient group of terrestrialvertebrates and are widely distributed globally occurringon all continents with the exception of Antarctica [1 2]Their continuing persistence in the biosphere can in partbe attributed to their highly developed and potent skinsecretions that act as a front-line defense against potentialpredators and microbes [1 2] The biological potency of theirskins and associated secretions were recognized by manyancient peoples who used such in shamanic rituals and asmedicines for diverse ailments [1 2] Some applications ofthese secretions include those of bufonid toads as stimulantsin traditional Chinese medicine those of tropical poisonfrogs (dendrobatids) as arrow-tip poisons and those of giantmonkey frogs (Phyllomedusa bicolor) in ldquohunting magicrdquomdasha purging and sensory-enhancing ritual performed by someSouth-American natives prior to hunting [3]
The unlocking of the molecular secrets of amphibiandefensive skin secretions was predominantly initiated bytwo twentieth-century pioneer pharmacological chemistsmdashVittorio Erspamer (1909ndash1999) (endogenous peptides andbiogenic amines) and John Daly (1933ndash2008) (exogenousalkaloids) Their combined efforts resulted in the unravellingof the molecular structures and biological actions of severalthousands of such molecules representing many classes ofbiochemical studies on which have served to reveal manynovel regulatory systems and potential drug targets in mam-malian systems [1ndash4]
The application of modern analytical technologies suchas molecular cloning proteomics and mass spectrometryto the study of the molecular complexity of amphibian skinsecretions continues on where the pioneers left off withever-increasing numbers of unique molecules of diversefunctions being revealed [5] One of the major goals of suchcontemporary research is directed towards the identification
Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 158546 7 pageshttpdxdoiorg1011552014158546
2 The Scientific World Journal
of natural leads for therapeutic development or to identifynovel disease-related drug targets [6 7]
The bioactive peptides represent one of the largest andmost functionally diverse groups of biochemicals identifiedthus far from amphibian skin and unlike the diet-acquiredalkaloids these are of endogenous origin and are geneticallyencoded [1ndash4] While many individual peptides have beenisolated they fall into two broad functional groupingsmdashthosewith pharmacological activity and those with antimicrobialactivity [1ndash4]
Tryptophyllins (TPHs) are a large and heterogenousgroup of peptides that did not fall into either category whenfirst identified as they were originally isolated from theskin of the South-American leaf frog Phyllomedusa rohdeithrough chemical means by nature of their positive Ehrlichrsquosreactionmdasha color-producing chemical reaction for indoles(tryptophan residues) [2] All original TPHs possessed atryptophanyl residue at position 2 from the C-terminus andprolyl residues at position 2 andor 3 from the N-terminus[2] although some TPHs more recently identified are devoidof tryptophanyl residues [2] Due to the increasing numbersof TPHs being identified and their apparent structural het-erogeneity this peptide family has been divided into threetypesmdash(1) tryptophyllin-1 (T-1) peptides the N-terminaldoublet KP- a tryptophanyl residue at position 5 and a prolylresidue at position 7 from the N-terminus are all highlyconserved in this group of peptides that typically possess 7-8residues (2) tryptophyllin-2 (T-2) peptides these are variablein length contain 4 to 7 residues and all contain an internal-PW doublet (3) tryptophyllin-3 (T-3) peptides the mostconserved TPHs containing 13 residues with conservativesubstitutions at positions 2 5 6 and 13 from the N-terminusThe N-terminal pGlu- -KP- at positions 3 and 4 andthe 7ndash12 sequence -PPPIYP- are all completely conserved[2 8ndash10]
Although the structures of many TPHs have been knownfor more than twenty-five years their biological actions haveremained somewhat obscure until recently In early reportsTPHs were described as having some general metaboliceffects such as stimulating liver protein synthesis and anti-bodies raised to several of these peptides produced positiveimmunostaining in pituitary gonadotrophs [2] In morerecent times two tryptophyllins (PdT-1 and PdT-2) fromthe skin secretion of the Mexican leaf frog Pachymedusadacnicolor were found to have potent pharmacological effectson different rat smooth muscles [8 9]
Here we describe the identification structural andfunctional characterization of a novel Type-2 tryptophyllinnamed AcT-2 from the skin secretion of the Central Amer-ican red-eyed leaf frog Agalychnis callidryas This peptidewas first identified through pharmacological screening formyotropic activity and was subsequently in addition foundto have a broad spectrum of antimicrobial activity albeitrelatively low potency Unusually the peptide was found topossess a higher potency against the yeast Candida albicansthan against the model Gram-positive and Gram-negativebacteria employed
2 Materials and Methods
21 Acquisition of Skin Secretions Adult red-eyed tree frogsAgalychnis callidryas (119899 = 6) of the Costa Rican type werehoused in a purpose-designed terrarium under a 12 h12 hlightdark cycle and were fed multivitamin-loaded cricketsthree times per week Skin secretions were obtained by trans-dermal electrical stimulation (4ms pulse width 50Hz 6V)in accordance with the method of Tyler et al [11] followinga three-month settling-in period The skin secretions werewashed from the skin using deionized water snap frozenin liquid nitrogen lyophilized and stored at minus20∘C prior toanalyses
22 Reverse Phase HPLC Fractionation of Lyophilized SkinSecretion Five milligrams of lyophilized skin secretion weredissolved in 1mL of 0059995 (vv) trifluoroacetic acid(TFA)water and clarified by centrifugationThe supernatantwas directly injected onto a CECIL reverse phase HPLCsystem (Milton Technical Centre Cambridge UK) fittedwithan analytical column (Phenomenex Jupiter C5 300 A poresize 250 times 4mm) The linear elution gradient employed wasformed from 0059995 (vv) TFAwater to 0051995800(vvv) TFAwateracetonitrile in 80min at a flow rate of1mLmin The effluent absorbance was monitored at 120582 =214 nm and fractions of approximately 1mL were collectedautomatically atminute intervals Samples (100120583L) from eachchromatographic fraction were removed lyophilized andstored at minus20∘C prior to analysis for myoactivity using ratsmooth muscle bioassays
23 Rat Smooth Muscle Bioassays Male Wistar rats (250ndash300 g) were euthanized by carbon dioxide asphyxiation fol-lowed by cervical dislocation under appropriate UK animalresearch lisences and following local institutional guidelinesAfter removing the fur on the abdomen of rats the bodycavity was opened and any visible fat was removed Theexposed urinary bladder and the tail were carefully removedAll dissected tissues were placed in ice-cold Krebrsquos solu-tion (118mM NaCl 47mM KCl 25mM NaHCO
3 115mM
NaH2PO4 25mM CaCl
2 11 mM MgCl
2 and 56mM glu-
cose) which was aerated with 95 O25 CO
2gas mixture
Strips of urinary bladder and rings of dissected tail arterywere prepared and tied with fine silk ligatures (02mm) ateach end These preparations were attached to a fixed pin atone end and to a transducer at the other end Krebrsquos solution(at 37∘C) flowing through the organ baths at 2mLmin witha constant bubbling of 95 O
25 CO
2maintained the
tissues in a viable state for gt2 h An equilibration period of20min was used for each preparation after which 60mMKCl was added to test the responsiveness of urinary bladdermuscle strips and 10minus3M phenylephrine was added to testthe responsiveness of the tail artery rings and to causepreconstriction Following these tests viable preparationswere used to screen prepared samples of reverse phase HPLCfractions of Agalychnis callidryas skin secretion Changes intension of smooth muscle preparations were detected bypressure transducers connected to a PowerLab System (AD
The Scientific World Journal 3
Instruments Pty Ltd) Data were analyzed using GraphPadPrism software anddata pointswere expressed asmean valuesplusmn standard errors 119899 = 6 in each case
24 Structural Characterization of the Novel Peptide A singlereverse phase HPLC fraction was found to have a potentrelaxation effect on tail artery smooth muscle a less potentcontractile action on urinary bladder smooth muscle stripsand amoderately potent antimicrobial effect A sample of thisfraction was subjected to MALDI-TOF mass spectrometryusing a Perseptive BiosystemsVoyagerDEmass spectrometerin positive detectionmodewith120572-cyano-4-hydroxycinnamicacid as matrix Subsequent to this analysis the single majorpeptide resolved was subjected to MSMS fragmentationsequencing using an LCQ Fleet electrospray ion-trap massspectrometer (Thermo-Fisher San Jose CA USA)
25 Molecular Cloning of the Novel Peptide BiosyntheticPrecursor-Encoding cDNA Five milligrams of lyophilizedskin secretion were dissolved in 1mL of cell lysismRNAprotection buffer that was supplied by Dynal Biotech UKPolyadenylated mRNA was then isolated from this usingmagnetic oligo-dT Dynabeads (Dynal Biotech UK) asper manufacturerrsquos instructions The isolated polyadenylatedmRNA was then subjected to 51015840- and 31015840-rapid amplificationof cDNA ends (RACE) procedures to obtain full-length novelpeptide precursor nucleic acid sequence data using a SMART-RACE kit (Clontech UK) likewise as per manufacturerrsquosinstructions Briefly the 31015840-RACE reactions employed anested universal (NUP) primer (supplied with the kit) and adegenerate sense primer (SP 51015840-GGIATGMGICCICCITGG-31015840) (I = deoxyinosine M = AC) that was complementary tothe putative amino acid sequence GMRPPW- of the novelpeptide The 31015840-RACE reactions were purified and clonedusing a pGEM-T vector system (Promega Corporation)and sequenced using an ABI 3100 automated sequencerThe sequence data obtained from the 31015840-RACE productwere used to design a specific antisense primer (ASP 51015840-CGGCACTATTACTGATAATTGTGCT-31015840) to a defined sitewithin the 31015840 nontranslated region of the novel peptideprecursor-encoding transcripts 51015840-RACE was carried outusing these primers in conjunction with the NUP primer andresultant products were purified cloned and sequenced
26 Solid-Phase Peptide Synthesis Once the unequivocalprimary structure of the novel peptide had been establishedit was chemically synthesized by solid-phase Fmoc chem-istry using a PS3 automated solid-phase peptide synthesizer(Protein Technologies Inc AZ USA) Following cleavageof the synthetic peptide from the resin and deprotection ofthe side-chain protecting groups the resultant material waslyophilized and then purified by HPLC The major productwas subjected toMALDI-TOFmass spectrometry to establishboth degree of purity and authenticity of structure
27 Preliminary Pharmacological Characterization of the Syn-thetic Novel Peptide on Rat Tail Artery and Urinary BladderSmooth Muscle The synthetic peptide was initially prepared
as a stock in Krebrsquos solution (118mM NaCl 47mM KCl25mM NaHCO
3 115mM NaH
2PO4 25mM CaCl
2 11mM
MgCl2 and 56mM glucose) at a concentration of 10minus3M
Working concentrations of peptide ranging from 10minus3 to1011M were prepared prior to each experiment and wereapplied to tissues (119899 = 6) progressively from low to highconcentrations Data obtained from the PowerLab System(AD Instruments Pty Ltd) were analyzed by Studentrsquos t-testthrough GraphPad Prism software to obtain the mean andstandard error of responses and using these datasets dose-response curves were constructed using a best-fit algorithm
28 Determination of Minimal Inhibitory Concentrations(MICs) of the Synthetic Novel Peptide for Model Microor-ganisms Minimal inhibitory concentrations (MICs) of thesynthetic peptide were assessed against three standard modelmicroorganisms the Gram-positive bacterium Staphylococ-cus aureus (S aureus NCTC 10788) the Gram-negativebacterium Escherichia coli (E coli NCTC 10418) and thepathogenic yeast Candida albicans (C albicans NCPF 1467)The synthetic peptidewas initially dissolved in a small volumeof dimethylsulfoxide (DMSO) and made up to 1mL withsterile Muellar-Hinton broth (MHB) Doubling dilutions ofthis peptide stock solution peptide were made from 512ndash1mgL in sterile MHB and were incubated with microorgan-ism cultures (106 colony forming units (CFU)mL) in 96-wellmicrotiter cell culture plates for 18 h at 37∘C in a humidifiedatmosphere MHB alone was used as a negative control andeachmicroorganism inMHBwith no peptide addedwas usedas positive controls After incubation the growth of microor-ganisms was determined by means of measuring opticaldensity (OD) at 120582 = 550 nm using an ELISA plate reader(Biolise BioTek EL808) Minimal inhibitory concentrations(MICs) were defined as the lowest concentration at which nogrowth was detectable
29 Hemolysis Assay Peptide solutions in a range of con-centrations were prepared as described in Section 28 but in09 (wv) aqueous NaCl solution prior to performing thehemolysis assay A 2 suspension of washed horse red bloodcells in this solution was prepared from defibrinated horseblood (TCS Biosciences Ltd UK) and samples of this wereincubated at 37∘C for 120min with a range of peptide con-centrations similar to those employed for the antimicrobialassays Lysis of red cells was assessed by measurement ofthe optical density of supernatants following centrifugationat 120582 = 550 nm using an ELISA plate reader (Biolise BioTekEL808) Negative controls employed consisted of a 2 (vv)red cell suspension alone and positive controls consisted ofa 2 (vv) red cell suspension and an equal volume of salinecontaining 2 (vv) of the nonionic detergent Triton X-100(Sigma-Aldrich)
3 Results
31 Identification and Structural Characterization of the NovelPeptide Reverse phase HPLC fraction 33 of Agalychniscallidryas skin secretion (Figure 1(a)) was found to contain
4 The Scientific World Journal
Time (mmss)
36
218
400
583
765
2801 3048 3335 3622 3909 4156
AcT-2
Abso
rban
ce (120582
214
mA
)
(a)
1 Seq 21 5802 2952 G 72 18907 9504 M 83243 41672 63 34517 17309 R 70139 35120 54 44222 22162 P 54529 27315 45 53928 27014 P 44823 22462 36 72536 36318 W 35118 17609 27 F-
amidated16510 8305 1
b(1+) b(2+) y(1+) y(2+)
(b)
Figure 1 Region of reverse phase HPLC chromatogram of Agalychnis callidryas skin secretion indicating the elution positionretentiontime (arrow) of absorbance peak containing the peptide AcT-2 (a) Expected singly and doubly charged b-ion and y-ions arising fromfragmentation of AcT-2 as predicted using the MS-Product program available through Protein Prospector online Observed fragment ionsare indicated in bold type face and are underlined (b)
a myoactive peptide following preliminary smooth musclepharmacological screening and a sample of this fractionwas subjected to MALDI-TOF mass spectrometry whichindicated a relatively high degree of purity of a peptidewith an mz (M+H)+ of 88925 The doubly charged ion ofthis peptide was subsequently identified following electro-spray MS analysis and subjected to MSMS fragmentationsequencing (Figure 1(b)) This produced the tentative aminoacid sequence GMRPPWF based upon identification of b-and y-ion series The peptide was also deemed to be C-terminally amidated Bioinformatic analysis produced no hitswith any archived amphibian skin peptide but with twosynthetic antifungal peptides named PAF-26 and combi-1[12 13] (Figure 2(a)) Of note was the presence of residues3ndash8 of the novel myotropic peptide in a C-terminally-located domain of a prophenin-2-like protein from the killerwhale (Orcinus orca) that contains a cathelicidin sequence(accession no XP004284013) (Figure 2(a)) However theorigin and structural characteristics of the novel myotropicpeptide an internal -PPW- sequence and C-terminal amida-tion indicated that it was a member of the amphibian skintryptophyllin family subtype T-2 and thus it was namedAgalychnis callidryas tryptophyllin-2 (AcT-2) in accordance
32 Molecular Cloning of AcT-2 Biosynthetic Precursor-Encoding cDNA A cDNA encoding the AcT-2 precursorprotein was successfully and repeatedly cloned using theRACEPCR strategy employedThe sequencewas representedin at least 30 clones after employing repetitive PCR andcloning procedures The complete nucleotide and translatedopen-reading frame amino acid sequences of the clonedAcT-2 biosynthetic precursor-encoding cDNA are shown inFigure 2(b)The deduced open-reading frame consisted of 62amino acid residues and shared a similar architecture with
the previously reported precursors of the tryptophyllins PdT-1 and PdT-2 from Pachymedusa dacnicolor skin secretion(Chen et al 2004 Wang et al 2009) (Figure 2(c)) Thisconsisted of an N-terminal putative signal peptide an acidicamino acid residue-rich spacer peptide domain a single copyof a mature AcT-2 sequence and a C-terminal processingand amidation site (Figure 2(c)) As in the Pachymedusadacnicolor tryptophyllin precursors the mature peptide wasflanked N-terminally by a double basic amino acid residuepropeptide convertase processing site - -RR- cleavage ofwhich generated the N-terminus of mature AcT-2 The C-terminal region of themature AcT-2 peptide was flanked by atripeptide sequence -GKK in commonwith the precursor ofPdT-2 The double basic amino acid motif -KK is removedby propeptide convertase and the resulting C-terminal glycyl(G) residue serves as an amide donor through the action ofamidating enzyme complex
33 Smooth Muscle Activity of Synthetic AcT-2 SyntheticAcT-2 was found to be active in smooth muscle preparationsfrom both rat tail artery and urinary bladder but in differentways and at different potencies In rat tail artery smoothmus-cle preparations AcT-2 caused a dose-dependent relaxationwith an EC
50of 51 nM (Figure 3(a)) In contrast the peptide
induced a dose-dependent contraction of urinary bladdersmooth muscle with an EC
50of 93 120583M (Figure 3(b))
34 Antimicrobial and Hemolytic Activity of AcT-2 AcT-2was somewhat unexpectedly found to possess a broad spec-trum of antimicrobial activity MICs obtained with the threemodel test organisms were as follows S aureus (256mgL)E coli (512mgL) and C albicans (128mgL) (Figures 4(a)ndash4(c)) The order of sensitivity of the test organisms employedwas thus C albicans gt S aureus gt E coli which is a most
---------1--------- ---- --------2 ---------- - - - - - --3 4--AcT-2 M ASV KKS VFLVLFLGF ISISFC DEEKR EDDEEGNEREEKREIHEEG N Q E E R R GMRPPW F GK K
PdT-2 M DFL RKS LFLVLFLGF VSISFC DEEKR EDDDENHGSEEKREIHEEG N Q E E R R DMSPPW H GK K
PdT-1 M NFL KKS LFLVLFLGF VSISFC DEEKR QDDDEGNEREEKKEIQEDG N Q E E R R DKPPAW VP GK -
larr larr larr larrrarr rarr rarr rarr
(c)
Figure 2 Comparison of the primary structure of AcT-2 with those of similar peptides PAF26 and combi-1 are synthetic antifungal peptidesisolated from combinatorial peptide libraries [12 13] Prophenin-2-like is a region of an antimicrobial polypeptide cathelicidin sequencedfrom a genomic DNA template of the killer whale (Orcinus orca) (accession no XP004284013) Conserved amino acid residues are shadedblack and consensus residues are shaded grey (a) Nucleotide and translated open-reading frame amino acid sequence of a cloned cDNAencoding the biosynthetic precursor of AcT-2 The putative signal peptide is double-underlined the mature peptide is single-underlinedand the stop codon is indicated by an asterisk (b) Alignments of complete open-reading frame amino acid sequences of AcT-2 precursor-encoding cDNA with the two top hits obtained following BLAST analysis using the NCBI portal PdT-1 and PdT-2 represent Pachymedusadacnicolor tryptophyllin-1 and -2 respectively Conserved amino acids are shaded black and consensus amino acids are shaded grey Gapshave been inserted tomaximize alignments (1) Putative signal peptide domain (2) Acidic spacer peptide domain (3)Mature peptide domain(4) C-terminal processing site (c)
Figure 3 Dose-response curves obtained using synthetic AcT-2 on rat smooth muscle preparations (a) Effect of the peptide on tail arterysmoothmuscle preparations expressed asmeans and standard errors (119899 = 6) EC
50was found to be 51 nM (b) Effect of the peptide on urinary
bladder smooth muscle preparations expressed as means and standard errors (119899 = 6) EC50was found to be 93 120583M
unusual profile for previously reported typical amphibianskin cationic amphipathic 120572-helical antimicrobial peptidesAlso worthy of note was the virtual lack of hemolytic activityof AcT-2 even up to the highest concentration (512mgL)employed (Figure 4(d))
4 Discussion
Central-American red-eyed leaf frogs (Agalychnis callidryas)are probably the most universally recognized frogs in theworld having been used extensively by advertising agencies
Figure 4Minimal inhibitory concentration (MIC) curves obtained following incubation of synthetic AcT-2 with S aureus (a) E coli (b) andC albicans (c) The MIC value for each microorganism is indicated in respective panels by asterisks (d) The hemolytic activity of syntheticAcT-2 The arrow shows the highest concentration of peptide (512mgL) which was the only concentration at which hemolysis was observed(85)
due to their strikingly beautiful colors Although they areone of the most widely available of the phyllomedusine leaffrogs studies on their defensive skin secretion peptides havenot been carried out with the same degree of focus that hasbeen given to many of their more obscure relatives [2 14]Red-eyed leaf frogs are most vividly colored with vibrantlime-green dorsal surfaces white ventral surfaces cream andcobalt-blue striped sides and bright orange feet Despite thisfrogs are difficult to see as they press themselves againstleaves and pull their limbs tightly towards their bodies thusbecoming a green blob on the leaf surface Their brightcoloration however is only present during the day when thefrogs are largely inactive while during the hours of darknesswhenever the frogs are most active their colors are muchdrabber favouring shades of grey and brown [15]
The potent cocktails of defensive molecules presentin skin secretions effectively constitutes their front-line ofdefense against predators [1 2] To date several peptideswith a range of biological effects have been reported fromthese skin secretions and these include both antimicrobialand pharmacologically active peptides [1 2 16] HoweverAcT-2 described here represents the first skin secretiontryptophyllin described from this species despite severalhaving been reported from Phyllomedusa species over thepast decades [2] A recent report byWang et al [9] describeda simple rational scheme to classify the rather structurally-heterogenous group of amphibian skin peptides collectivelynamed tryptophyllins [9] In this peptideswere classified intoone of three groups named T-1 T-2 and T-3 tryptophyllinsbased on some common structural features On this basisAcT-2 shows the features of a type-2 (T-2) tryptophyllin incommon with PdT-2 [9] as both possess an internal -PW-sequence motif (Figure 2(c)) However despite the presence
of this common internal structural feature AcT-2 and PdT-2 display radically different potencies in stimulating thecontraction of rat uterus smooth muscle (EC
50values AcT-2
= 93 120583M PdT-2 = 4 nM) indicating that other features of theprimary structure influence their receptor interactions [9]However AcT-2 was found to possess a most potent arterialsmooth muscle relaxant effect (EC
50= 51 nM)mdashan effect not
observed with PdT-2 [9] The smooth muscle pharmacol-ogy and molecular targets for these Type-2 tryptophyllinsin mammalian tissues obviously warrant further in-depthinvestigation
As demonstrated in this study AcT-2 was unexpectedlyfound to possess antimicrobial activity albeit relatively lowpotency using three model test microorganisms with theorder of sensitivity being C albicans gt S aureus gt E coli orin other words yeast gt Gram-positive bacterium gt Gram-negative bacterium Related to this observation was thatfollowing database interrogation with the primary structureof AcT-2 three peptides were found which displayed somedegree of structural identity (Figure 2(a)) Two of thesenamed PAF26 and combi-1 were synthetic in nature andwere found through the screening of combinatorial peptidelibraries for antifungal activity [12 13] The third representeda cathelicidin domain of a prophenin-2-like protein deducedfrom a cloned cDNA of the killer whale (Orcinus orca)(accession no XP004284013) Interestingly all three AcT-2-like peptides were associated with antimicrobial functionswith the former two being specifically against fungi
While AcT-2 is of relatively low potency when comparedwith many other amphibian skin antimicrobial peptidesmost if not all of the latter act through a mechanism ofmicrobial target cell membrane lysis and some are stronglyhemolytic [17ndash19] To achieve this form of nonspecific
The Scientific World Journal 7
antimicrobial action such peptides are generallymuch longerin amino acid chain length than AcT-2 [17 19] ThusAcT-2 may act upon a different and possibly intracellulartarget rather than by direct lytic action on the cytoplasmicmembranemdasha mode of action that has actually been pro-posed for some ldquoclassicalrdquo antimicrobial peptides as well [1719] In support of this proposal to some degree is the lack ofhemolytic activity of AcT-2 observed following its incubationwith horse erythrocytes (Figure 4(d))
In conclusion the novel amphibian skin tryptophyllinnamed AcT-2 described here has been demonstrated tohave both selective mammalian smooth muscle myotropiceffects and antimicrobial activitymdashfindings which add toour increasing knowledge of the biological effects of thisenigmatic family of amphibian skin peptides
Thenucleotide sequence ofAcT-2 from the skin secretionof Agalychnis callidryas has been deposited in the EMBLNucleotide Sequence Database under the accession codeHG710094
Conflict of Interests
The authors declare that they have no conflict of interests
Authorsrsquo Contribution
Lilin Ge and Peng Lyu contributed equally to this work
References
[1] B T Clarke ldquoThe natural history of amphibian skin secretionstheir normal functioning and potential medical applicationsrdquoBiological Reviews of the Cambridge Philosophical Society vol72 no 3 pp 365ndash379 1997
[2] V Erspamer ldquoBioactive secretions of the integumentrdquo inAmphibian Biologymdashvol 1 the Integument H Heatwole andG T Barthalmus Eds pp 179ndash350 Surrey Beatty amp SonsChipping Norton 1994
[3] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[4] J W Daly T F Spande and H M Garraffo ldquoAlkaloidsfrom amphibian skin a tabulation of over eight-hundredcompoundsrdquo Journal of Natural Products vol 68 no 10 pp1556ndash1575 2005
[5] F E Koehn ldquoHigh impact technologies for natural productsscreeningrdquo Progress in Drug Research vol 65 pp 176ndash210 2008
[6] R P Borris ldquoNatural products research perspectives from amajor pharmaceutical companyrdquo Journal of Ethnopharmacol-ogy vol 51 no 1ndash3 pp 29ndash38 1996
[7] GM Cragg andD J Newman ldquoNatural products a continuingsource of novel drug leadsrdquo Biochimica et Biophysica Acta vol1830 pp 3670ndash3695 2013
[8] T ChenD F OrrMOrsquoRourke et al ldquoPachymedusa dacnicolortryptophyllin-1 structural characterization pharmacologicalactivity and cloning of precursor cDNArdquo Regulatory Peptidesvol 117 no 1 pp 25ndash32 2004
[9] L Wang M Zhou T Chen B Walker and C Shaw ldquoPdT-2 anovelmyotropic Type-2 tryptophyllin from the skin secretion of
the Mexican giant leaf frog Pachymedusa dacnicolorrdquo Peptidesvol 30 no 8 pp 1557ndash1561 2009
[10] RWang T ChenM Zhou LWang andC Shaw ldquoPsT-1 a newtryptophyllin peptide from the skin secretion of Waxy MonkeyLeaf Frog Phyllomedusa sauvageirdquoRegulatory Peptides vol 184pp 14ndash21 2013
[11] M J Tyler D J M Stone and J H Bowie ldquoA novel method forthe release and collection of dermal glandular secretions fromthe skin of frogsrdquo Journal of Pharmacological and ToxicologicalMethods vol 28 no 4 pp 199ndash200 1992
[12] D I Chan E J Prenner and H J Vogel ldquoTryptophan- andarginine-rich antimicrobial peptides structures and mecha-nisms of actionrdquo Biochimica et Biophysica Acta vol 1758 no9 pp 1184ndash1202 2006
[13] A Munoz B Lopez-Garcıa and J F Marcos ldquoStudies onthe mode of action of the antifungal hexapeptide PAF26rdquoAntimicrobial Agents and Chemotherapy vol 50 no 11 pp3847ndash3855 2006
[14] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[15] J G Walls Red-Eyes and Other Leaf-Frogs TFH Publications1996
[16] J M Conlon J Kolodziejek and N Nowotny ldquoAntimicrobialpeptides from ranid frogs taxonomic and phylogeneticmarkersand a potential source of new therapeutic agentsrdquo Biochimica etBiophysica Acta vol 1696 no 1 pp 1ndash14 2004
[17] J M Conlon ldquoThe contribution of skin antimicrobial peptidesto the system of innate immunity in anuransrdquo Cell and TissueResearch vol 343 no 1 pp 201ndash212 2011
[18] R M Epand and H J Vogel ldquoDiversity of antimicrobial pep-tides and their mechanisms of actionrdquo Biochimica et BiophysicaActa vol 1462 no 1-2 pp 11ndash28 1999
[19] M Simmaco G Mignogna and D Barra ldquoAntimicrobial pep-tides from amphibian skin what do they tell usrdquo Biopolymersvol 47 no 6 pp 435ndash450 1998
Research ArticleAcT-2 A Novel Myotropic and Antimicrobial Type 2Tryptophyllin from the Skin Secretion of the Central AmericanRed-Eyed Leaf Frog Agalychnis callidryas
Lilin Ge Peng Lyu Mei Zhou Huiling Zhang Yuantai WanBin Li Renjie Li Lei Wang Tianbao Chen and Chris Shaw
Natural Drug Discovery Group School of Pharmacy Queenrsquos University Belfast BT9 7BL UK
Correspondence should be addressed to Renjie Li rliqubacuk and Lei Wang lwangqubacuk
Received 13 December 2013 Accepted 8 January 2014 Published 13 February 2014
Academic Editors A Sacchetti and H-S Won
Copyright copy 2014 Lilin Ge et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Tryptophyllins are a diverse family of amphibian peptides originally found in extracts of phyllomedusine frog skin by chemicalmeansTheir biological activities remain obscure Here we describe the isolation and preliminary pharmacological characterizationof a novel type 2 tryptophyllin named AcT-2 from the skin secretion of the red-eyed leaf frog Agalychnis callidryas The peptidewas initially identified during smooth muscle pharmacological screening of skin secretion HPLC fractions and the unique primarystructuremdashGMRPPWF-NH
2mdashwas established by both Edman degradation and electrosprayMSMS fragmentation sequencing A
cDNA encoding the biosynthetic precursor of AcT-2 was successfully cloned from a skin secretion-derived cDNA library by meansof RACEPCRand this contained an open-reading frame consisting of 62 amino acid residueswith a singleAcT-2 encoding sequencelocated towards the C-terminus A synthetic replicate of AcT-2 was found to relax arterial smooth muscle (EC
50= 51 nM) and to
contract rat urinary bladder smooth muscle (EC50= 93 120583M) The peptide could also inhibit the growth of the microorganisms
Staphylococcus aureus (MIC = 256mgL) Escherichia coli (MIC = 512mgL) and Candida albicans (128mgL) AcT-2 is thus thefirst amphibian skin tryptophyllin found to possess both myotropic and antimicrobial activities
1 Introduction
Amphibians represent the most ancient group of terrestrialvertebrates and are widely distributed globally occurringon all continents with the exception of Antarctica [1 2]Their continuing persistence in the biosphere can in partbe attributed to their highly developed and potent skinsecretions that act as a front-line defense against potentialpredators and microbes [1 2] The biological potency of theirskins and associated secretions were recognized by manyancient peoples who used such in shamanic rituals and asmedicines for diverse ailments [1 2] Some applications ofthese secretions include those of bufonid toads as stimulantsin traditional Chinese medicine those of tropical poisonfrogs (dendrobatids) as arrow-tip poisons and those of giantmonkey frogs (Phyllomedusa bicolor) in ldquohunting magicrdquomdasha purging and sensory-enhancing ritual performed by someSouth-American natives prior to hunting [3]
The unlocking of the molecular secrets of amphibiandefensive skin secretions was predominantly initiated bytwo twentieth-century pioneer pharmacological chemistsmdashVittorio Erspamer (1909ndash1999) (endogenous peptides andbiogenic amines) and John Daly (1933ndash2008) (exogenousalkaloids) Their combined efforts resulted in the unravellingof the molecular structures and biological actions of severalthousands of such molecules representing many classes ofbiochemical studies on which have served to reveal manynovel regulatory systems and potential drug targets in mam-malian systems [1ndash4]
The application of modern analytical technologies suchas molecular cloning proteomics and mass spectrometryto the study of the molecular complexity of amphibian skinsecretions continues on where the pioneers left off withever-increasing numbers of unique molecules of diversefunctions being revealed [5] One of the major goals of suchcontemporary research is directed towards the identification
Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 158546 7 pageshttpdxdoiorg1011552014158546
2 The Scientific World Journal
of natural leads for therapeutic development or to identifynovel disease-related drug targets [6 7]
The bioactive peptides represent one of the largest andmost functionally diverse groups of biochemicals identifiedthus far from amphibian skin and unlike the diet-acquiredalkaloids these are of endogenous origin and are geneticallyencoded [1ndash4] While many individual peptides have beenisolated they fall into two broad functional groupingsmdashthosewith pharmacological activity and those with antimicrobialactivity [1ndash4]
Tryptophyllins (TPHs) are a large and heterogenousgroup of peptides that did not fall into either category whenfirst identified as they were originally isolated from theskin of the South-American leaf frog Phyllomedusa rohdeithrough chemical means by nature of their positive Ehrlichrsquosreactionmdasha color-producing chemical reaction for indoles(tryptophan residues) [2] All original TPHs possessed atryptophanyl residue at position 2 from the C-terminus andprolyl residues at position 2 andor 3 from the N-terminus[2] although some TPHs more recently identified are devoidof tryptophanyl residues [2] Due to the increasing numbersof TPHs being identified and their apparent structural het-erogeneity this peptide family has been divided into threetypesmdash(1) tryptophyllin-1 (T-1) peptides the N-terminaldoublet KP- a tryptophanyl residue at position 5 and a prolylresidue at position 7 from the N-terminus are all highlyconserved in this group of peptides that typically possess 7-8residues (2) tryptophyllin-2 (T-2) peptides these are variablein length contain 4 to 7 residues and all contain an internal-PW doublet (3) tryptophyllin-3 (T-3) peptides the mostconserved TPHs containing 13 residues with conservativesubstitutions at positions 2 5 6 and 13 from the N-terminusThe N-terminal pGlu- -KP- at positions 3 and 4 andthe 7ndash12 sequence -PPPIYP- are all completely conserved[2 8ndash10]
Although the structures of many TPHs have been knownfor more than twenty-five years their biological actions haveremained somewhat obscure until recently In early reportsTPHs were described as having some general metaboliceffects such as stimulating liver protein synthesis and anti-bodies raised to several of these peptides produced positiveimmunostaining in pituitary gonadotrophs [2] In morerecent times two tryptophyllins (PdT-1 and PdT-2) fromthe skin secretion of the Mexican leaf frog Pachymedusadacnicolor were found to have potent pharmacological effectson different rat smooth muscles [8 9]
Here we describe the identification structural andfunctional characterization of a novel Type-2 tryptophyllinnamed AcT-2 from the skin secretion of the Central Amer-ican red-eyed leaf frog Agalychnis callidryas This peptidewas first identified through pharmacological screening formyotropic activity and was subsequently in addition foundto have a broad spectrum of antimicrobial activity albeitrelatively low potency Unusually the peptide was found topossess a higher potency against the yeast Candida albicansthan against the model Gram-positive and Gram-negativebacteria employed
2 Materials and Methods
21 Acquisition of Skin Secretions Adult red-eyed tree frogsAgalychnis callidryas (119899 = 6) of the Costa Rican type werehoused in a purpose-designed terrarium under a 12 h12 hlightdark cycle and were fed multivitamin-loaded cricketsthree times per week Skin secretions were obtained by trans-dermal electrical stimulation (4ms pulse width 50Hz 6V)in accordance with the method of Tyler et al [11] followinga three-month settling-in period The skin secretions werewashed from the skin using deionized water snap frozenin liquid nitrogen lyophilized and stored at minus20∘C prior toanalyses
22 Reverse Phase HPLC Fractionation of Lyophilized SkinSecretion Five milligrams of lyophilized skin secretion weredissolved in 1mL of 0059995 (vv) trifluoroacetic acid(TFA)water and clarified by centrifugationThe supernatantwas directly injected onto a CECIL reverse phase HPLCsystem (Milton Technical Centre Cambridge UK) fittedwithan analytical column (Phenomenex Jupiter C5 300 A poresize 250 times 4mm) The linear elution gradient employed wasformed from 0059995 (vv) TFAwater to 0051995800(vvv) TFAwateracetonitrile in 80min at a flow rate of1mLmin The effluent absorbance was monitored at 120582 =214 nm and fractions of approximately 1mL were collectedautomatically atminute intervals Samples (100120583L) from eachchromatographic fraction were removed lyophilized andstored at minus20∘C prior to analysis for myoactivity using ratsmooth muscle bioassays
23 Rat Smooth Muscle Bioassays Male Wistar rats (250ndash300 g) were euthanized by carbon dioxide asphyxiation fol-lowed by cervical dislocation under appropriate UK animalresearch lisences and following local institutional guidelinesAfter removing the fur on the abdomen of rats the bodycavity was opened and any visible fat was removed Theexposed urinary bladder and the tail were carefully removedAll dissected tissues were placed in ice-cold Krebrsquos solu-tion (118mM NaCl 47mM KCl 25mM NaHCO
3 115mM
NaH2PO4 25mM CaCl
2 11 mM MgCl
2 and 56mM glu-
cose) which was aerated with 95 O25 CO
2gas mixture
Strips of urinary bladder and rings of dissected tail arterywere prepared and tied with fine silk ligatures (02mm) ateach end These preparations were attached to a fixed pin atone end and to a transducer at the other end Krebrsquos solution(at 37∘C) flowing through the organ baths at 2mLmin witha constant bubbling of 95 O
25 CO
2maintained the
tissues in a viable state for gt2 h An equilibration period of20min was used for each preparation after which 60mMKCl was added to test the responsiveness of urinary bladdermuscle strips and 10minus3M phenylephrine was added to testthe responsiveness of the tail artery rings and to causepreconstriction Following these tests viable preparationswere used to screen prepared samples of reverse phase HPLCfractions of Agalychnis callidryas skin secretion Changes intension of smooth muscle preparations were detected bypressure transducers connected to a PowerLab System (AD
The Scientific World Journal 3
Instruments Pty Ltd) Data were analyzed using GraphPadPrism software anddata pointswere expressed asmean valuesplusmn standard errors 119899 = 6 in each case
24 Structural Characterization of the Novel Peptide A singlereverse phase HPLC fraction was found to have a potentrelaxation effect on tail artery smooth muscle a less potentcontractile action on urinary bladder smooth muscle stripsand amoderately potent antimicrobial effect A sample of thisfraction was subjected to MALDI-TOF mass spectrometryusing a Perseptive BiosystemsVoyagerDEmass spectrometerin positive detectionmodewith120572-cyano-4-hydroxycinnamicacid as matrix Subsequent to this analysis the single majorpeptide resolved was subjected to MSMS fragmentationsequencing using an LCQ Fleet electrospray ion-trap massspectrometer (Thermo-Fisher San Jose CA USA)
25 Molecular Cloning of the Novel Peptide BiosyntheticPrecursor-Encoding cDNA Five milligrams of lyophilizedskin secretion were dissolved in 1mL of cell lysismRNAprotection buffer that was supplied by Dynal Biotech UKPolyadenylated mRNA was then isolated from this usingmagnetic oligo-dT Dynabeads (Dynal Biotech UK) asper manufacturerrsquos instructions The isolated polyadenylatedmRNA was then subjected to 51015840- and 31015840-rapid amplificationof cDNA ends (RACE) procedures to obtain full-length novelpeptide precursor nucleic acid sequence data using a SMART-RACE kit (Clontech UK) likewise as per manufacturerrsquosinstructions Briefly the 31015840-RACE reactions employed anested universal (NUP) primer (supplied with the kit) and adegenerate sense primer (SP 51015840-GGIATGMGICCICCITGG-31015840) (I = deoxyinosine M = AC) that was complementary tothe putative amino acid sequence GMRPPW- of the novelpeptide The 31015840-RACE reactions were purified and clonedusing a pGEM-T vector system (Promega Corporation)and sequenced using an ABI 3100 automated sequencerThe sequence data obtained from the 31015840-RACE productwere used to design a specific antisense primer (ASP 51015840-CGGCACTATTACTGATAATTGTGCT-31015840) to a defined sitewithin the 31015840 nontranslated region of the novel peptideprecursor-encoding transcripts 51015840-RACE was carried outusing these primers in conjunction with the NUP primer andresultant products were purified cloned and sequenced
26 Solid-Phase Peptide Synthesis Once the unequivocalprimary structure of the novel peptide had been establishedit was chemically synthesized by solid-phase Fmoc chem-istry using a PS3 automated solid-phase peptide synthesizer(Protein Technologies Inc AZ USA) Following cleavageof the synthetic peptide from the resin and deprotection ofthe side-chain protecting groups the resultant material waslyophilized and then purified by HPLC The major productwas subjected toMALDI-TOFmass spectrometry to establishboth degree of purity and authenticity of structure
27 Preliminary Pharmacological Characterization of the Syn-thetic Novel Peptide on Rat Tail Artery and Urinary BladderSmooth Muscle The synthetic peptide was initially prepared
as a stock in Krebrsquos solution (118mM NaCl 47mM KCl25mM NaHCO
3 115mM NaH
2PO4 25mM CaCl
2 11mM
MgCl2 and 56mM glucose) at a concentration of 10minus3M
Working concentrations of peptide ranging from 10minus3 to1011M were prepared prior to each experiment and wereapplied to tissues (119899 = 6) progressively from low to highconcentrations Data obtained from the PowerLab System(AD Instruments Pty Ltd) were analyzed by Studentrsquos t-testthrough GraphPad Prism software to obtain the mean andstandard error of responses and using these datasets dose-response curves were constructed using a best-fit algorithm
28 Determination of Minimal Inhibitory Concentrations(MICs) of the Synthetic Novel Peptide for Model Microor-ganisms Minimal inhibitory concentrations (MICs) of thesynthetic peptide were assessed against three standard modelmicroorganisms the Gram-positive bacterium Staphylococ-cus aureus (S aureus NCTC 10788) the Gram-negativebacterium Escherichia coli (E coli NCTC 10418) and thepathogenic yeast Candida albicans (C albicans NCPF 1467)The synthetic peptidewas initially dissolved in a small volumeof dimethylsulfoxide (DMSO) and made up to 1mL withsterile Muellar-Hinton broth (MHB) Doubling dilutions ofthis peptide stock solution peptide were made from 512ndash1mgL in sterile MHB and were incubated with microorgan-ism cultures (106 colony forming units (CFU)mL) in 96-wellmicrotiter cell culture plates for 18 h at 37∘C in a humidifiedatmosphere MHB alone was used as a negative control andeachmicroorganism inMHBwith no peptide addedwas usedas positive controls After incubation the growth of microor-ganisms was determined by means of measuring opticaldensity (OD) at 120582 = 550 nm using an ELISA plate reader(Biolise BioTek EL808) Minimal inhibitory concentrations(MICs) were defined as the lowest concentration at which nogrowth was detectable
29 Hemolysis Assay Peptide solutions in a range of con-centrations were prepared as described in Section 28 but in09 (wv) aqueous NaCl solution prior to performing thehemolysis assay A 2 suspension of washed horse red bloodcells in this solution was prepared from defibrinated horseblood (TCS Biosciences Ltd UK) and samples of this wereincubated at 37∘C for 120min with a range of peptide con-centrations similar to those employed for the antimicrobialassays Lysis of red cells was assessed by measurement ofthe optical density of supernatants following centrifugationat 120582 = 550 nm using an ELISA plate reader (Biolise BioTekEL808) Negative controls employed consisted of a 2 (vv)red cell suspension alone and positive controls consisted ofa 2 (vv) red cell suspension and an equal volume of salinecontaining 2 (vv) of the nonionic detergent Triton X-100(Sigma-Aldrich)
3 Results
31 Identification and Structural Characterization of the NovelPeptide Reverse phase HPLC fraction 33 of Agalychniscallidryas skin secretion (Figure 1(a)) was found to contain
4 The Scientific World Journal
Time (mmss)
36
218
400
583
765
2801 3048 3335 3622 3909 4156
AcT-2
Abso
rban
ce (120582
214
mA
)
(a)
1 Seq 21 5802 2952 G 72 18907 9504 M 83243 41672 63 34517 17309 R 70139 35120 54 44222 22162 P 54529 27315 45 53928 27014 P 44823 22462 36 72536 36318 W 35118 17609 27 F-
amidated16510 8305 1
b(1+) b(2+) y(1+) y(2+)
(b)
Figure 1 Region of reverse phase HPLC chromatogram of Agalychnis callidryas skin secretion indicating the elution positionretentiontime (arrow) of absorbance peak containing the peptide AcT-2 (a) Expected singly and doubly charged b-ion and y-ions arising fromfragmentation of AcT-2 as predicted using the MS-Product program available through Protein Prospector online Observed fragment ionsare indicated in bold type face and are underlined (b)
a myoactive peptide following preliminary smooth musclepharmacological screening and a sample of this fractionwas subjected to MALDI-TOF mass spectrometry whichindicated a relatively high degree of purity of a peptidewith an mz (M+H)+ of 88925 The doubly charged ion ofthis peptide was subsequently identified following electro-spray MS analysis and subjected to MSMS fragmentationsequencing (Figure 1(b)) This produced the tentative aminoacid sequence GMRPPWF based upon identification of b-and y-ion series The peptide was also deemed to be C-terminally amidated Bioinformatic analysis produced no hitswith any archived amphibian skin peptide but with twosynthetic antifungal peptides named PAF-26 and combi-1[12 13] (Figure 2(a)) Of note was the presence of residues3ndash8 of the novel myotropic peptide in a C-terminally-located domain of a prophenin-2-like protein from the killerwhale (Orcinus orca) that contains a cathelicidin sequence(accession no XP004284013) (Figure 2(a)) However theorigin and structural characteristics of the novel myotropicpeptide an internal -PPW- sequence and C-terminal amida-tion indicated that it was a member of the amphibian skintryptophyllin family subtype T-2 and thus it was namedAgalychnis callidryas tryptophyllin-2 (AcT-2) in accordance
32 Molecular Cloning of AcT-2 Biosynthetic Precursor-Encoding cDNA A cDNA encoding the AcT-2 precursorprotein was successfully and repeatedly cloned using theRACEPCR strategy employedThe sequencewas representedin at least 30 clones after employing repetitive PCR andcloning procedures The complete nucleotide and translatedopen-reading frame amino acid sequences of the clonedAcT-2 biosynthetic precursor-encoding cDNA are shown inFigure 2(b)The deduced open-reading frame consisted of 62amino acid residues and shared a similar architecture with
the previously reported precursors of the tryptophyllins PdT-1 and PdT-2 from Pachymedusa dacnicolor skin secretion(Chen et al 2004 Wang et al 2009) (Figure 2(c)) Thisconsisted of an N-terminal putative signal peptide an acidicamino acid residue-rich spacer peptide domain a single copyof a mature AcT-2 sequence and a C-terminal processingand amidation site (Figure 2(c)) As in the Pachymedusadacnicolor tryptophyllin precursors the mature peptide wasflanked N-terminally by a double basic amino acid residuepropeptide convertase processing site - -RR- cleavage ofwhich generated the N-terminus of mature AcT-2 The C-terminal region of themature AcT-2 peptide was flanked by atripeptide sequence -GKK in commonwith the precursor ofPdT-2 The double basic amino acid motif -KK is removedby propeptide convertase and the resulting C-terminal glycyl(G) residue serves as an amide donor through the action ofamidating enzyme complex
33 Smooth Muscle Activity of Synthetic AcT-2 SyntheticAcT-2 was found to be active in smooth muscle preparationsfrom both rat tail artery and urinary bladder but in differentways and at different potencies In rat tail artery smoothmus-cle preparations AcT-2 caused a dose-dependent relaxationwith an EC
50of 51 nM (Figure 3(a)) In contrast the peptide
induced a dose-dependent contraction of urinary bladdersmooth muscle with an EC
50of 93 120583M (Figure 3(b))
34 Antimicrobial and Hemolytic Activity of AcT-2 AcT-2was somewhat unexpectedly found to possess a broad spec-trum of antimicrobial activity MICs obtained with the threemodel test organisms were as follows S aureus (256mgL)E coli (512mgL) and C albicans (128mgL) (Figures 4(a)ndash4(c)) The order of sensitivity of the test organisms employedwas thus C albicans gt S aureus gt E coli which is a most
---------1--------- ---- --------2 ---------- - - - - - --3 4--AcT-2 M ASV KKS VFLVLFLGF ISISFC DEEKR EDDEEGNEREEKREIHEEG N Q E E R R GMRPPW F GK K
PdT-2 M DFL RKS LFLVLFLGF VSISFC DEEKR EDDDENHGSEEKREIHEEG N Q E E R R DMSPPW H GK K
PdT-1 M NFL KKS LFLVLFLGF VSISFC DEEKR QDDDEGNEREEKKEIQEDG N Q E E R R DKPPAW VP GK -
larr larr larr larrrarr rarr rarr rarr
(c)
Figure 2 Comparison of the primary structure of AcT-2 with those of similar peptides PAF26 and combi-1 are synthetic antifungal peptidesisolated from combinatorial peptide libraries [12 13] Prophenin-2-like is a region of an antimicrobial polypeptide cathelicidin sequencedfrom a genomic DNA template of the killer whale (Orcinus orca) (accession no XP004284013) Conserved amino acid residues are shadedblack and consensus residues are shaded grey (a) Nucleotide and translated open-reading frame amino acid sequence of a cloned cDNAencoding the biosynthetic precursor of AcT-2 The putative signal peptide is double-underlined the mature peptide is single-underlinedand the stop codon is indicated by an asterisk (b) Alignments of complete open-reading frame amino acid sequences of AcT-2 precursor-encoding cDNA with the two top hits obtained following BLAST analysis using the NCBI portal PdT-1 and PdT-2 represent Pachymedusadacnicolor tryptophyllin-1 and -2 respectively Conserved amino acids are shaded black and consensus amino acids are shaded grey Gapshave been inserted tomaximize alignments (1) Putative signal peptide domain (2) Acidic spacer peptide domain (3)Mature peptide domain(4) C-terminal processing site (c)
Figure 3 Dose-response curves obtained using synthetic AcT-2 on rat smooth muscle preparations (a) Effect of the peptide on tail arterysmoothmuscle preparations expressed asmeans and standard errors (119899 = 6) EC
50was found to be 51 nM (b) Effect of the peptide on urinary
bladder smooth muscle preparations expressed as means and standard errors (119899 = 6) EC50was found to be 93 120583M
unusual profile for previously reported typical amphibianskin cationic amphipathic 120572-helical antimicrobial peptidesAlso worthy of note was the virtual lack of hemolytic activityof AcT-2 even up to the highest concentration (512mgL)employed (Figure 4(d))
4 Discussion
Central-American red-eyed leaf frogs (Agalychnis callidryas)are probably the most universally recognized frogs in theworld having been used extensively by advertising agencies
Figure 4Minimal inhibitory concentration (MIC) curves obtained following incubation of synthetic AcT-2 with S aureus (a) E coli (b) andC albicans (c) The MIC value for each microorganism is indicated in respective panels by asterisks (d) The hemolytic activity of syntheticAcT-2 The arrow shows the highest concentration of peptide (512mgL) which was the only concentration at which hemolysis was observed(85)
due to their strikingly beautiful colors Although they areone of the most widely available of the phyllomedusine leaffrogs studies on their defensive skin secretion peptides havenot been carried out with the same degree of focus that hasbeen given to many of their more obscure relatives [2 14]Red-eyed leaf frogs are most vividly colored with vibrantlime-green dorsal surfaces white ventral surfaces cream andcobalt-blue striped sides and bright orange feet Despite thisfrogs are difficult to see as they press themselves againstleaves and pull their limbs tightly towards their bodies thusbecoming a green blob on the leaf surface Their brightcoloration however is only present during the day when thefrogs are largely inactive while during the hours of darknesswhenever the frogs are most active their colors are muchdrabber favouring shades of grey and brown [15]
The potent cocktails of defensive molecules presentin skin secretions effectively constitutes their front-line ofdefense against predators [1 2] To date several peptideswith a range of biological effects have been reported fromthese skin secretions and these include both antimicrobialand pharmacologically active peptides [1 2 16] HoweverAcT-2 described here represents the first skin secretiontryptophyllin described from this species despite severalhaving been reported from Phyllomedusa species over thepast decades [2] A recent report byWang et al [9] describeda simple rational scheme to classify the rather structurally-heterogenous group of amphibian skin peptides collectivelynamed tryptophyllins [9] In this peptideswere classified intoone of three groups named T-1 T-2 and T-3 tryptophyllinsbased on some common structural features On this basisAcT-2 shows the features of a type-2 (T-2) tryptophyllin incommon with PdT-2 [9] as both possess an internal -PW-sequence motif (Figure 2(c)) However despite the presence
of this common internal structural feature AcT-2 and PdT-2 display radically different potencies in stimulating thecontraction of rat uterus smooth muscle (EC
50values AcT-2
= 93 120583M PdT-2 = 4 nM) indicating that other features of theprimary structure influence their receptor interactions [9]However AcT-2 was found to possess a most potent arterialsmooth muscle relaxant effect (EC
50= 51 nM)mdashan effect not
observed with PdT-2 [9] The smooth muscle pharmacol-ogy and molecular targets for these Type-2 tryptophyllinsin mammalian tissues obviously warrant further in-depthinvestigation
As demonstrated in this study AcT-2 was unexpectedlyfound to possess antimicrobial activity albeit relatively lowpotency using three model test microorganisms with theorder of sensitivity being C albicans gt S aureus gt E coli orin other words yeast gt Gram-positive bacterium gt Gram-negative bacterium Related to this observation was thatfollowing database interrogation with the primary structureof AcT-2 three peptides were found which displayed somedegree of structural identity (Figure 2(a)) Two of thesenamed PAF26 and combi-1 were synthetic in nature andwere found through the screening of combinatorial peptidelibraries for antifungal activity [12 13] The third representeda cathelicidin domain of a prophenin-2-like protein deducedfrom a cloned cDNA of the killer whale (Orcinus orca)(accession no XP004284013) Interestingly all three AcT-2-like peptides were associated with antimicrobial functionswith the former two being specifically against fungi
While AcT-2 is of relatively low potency when comparedwith many other amphibian skin antimicrobial peptidesmost if not all of the latter act through a mechanism ofmicrobial target cell membrane lysis and some are stronglyhemolytic [17ndash19] To achieve this form of nonspecific
The Scientific World Journal 7
antimicrobial action such peptides are generallymuch longerin amino acid chain length than AcT-2 [17 19] ThusAcT-2 may act upon a different and possibly intracellulartarget rather than by direct lytic action on the cytoplasmicmembranemdasha mode of action that has actually been pro-posed for some ldquoclassicalrdquo antimicrobial peptides as well [1719] In support of this proposal to some degree is the lack ofhemolytic activity of AcT-2 observed following its incubationwith horse erythrocytes (Figure 4(d))
In conclusion the novel amphibian skin tryptophyllinnamed AcT-2 described here has been demonstrated tohave both selective mammalian smooth muscle myotropiceffects and antimicrobial activitymdashfindings which add toour increasing knowledge of the biological effects of thisenigmatic family of amphibian skin peptides
Thenucleotide sequence ofAcT-2 from the skin secretionof Agalychnis callidryas has been deposited in the EMBLNucleotide Sequence Database under the accession codeHG710094
Conflict of Interests
The authors declare that they have no conflict of interests
Authorsrsquo Contribution
Lilin Ge and Peng Lyu contributed equally to this work
References
[1] B T Clarke ldquoThe natural history of amphibian skin secretionstheir normal functioning and potential medical applicationsrdquoBiological Reviews of the Cambridge Philosophical Society vol72 no 3 pp 365ndash379 1997
[2] V Erspamer ldquoBioactive secretions of the integumentrdquo inAmphibian Biologymdashvol 1 the Integument H Heatwole andG T Barthalmus Eds pp 179ndash350 Surrey Beatty amp SonsChipping Norton 1994
[3] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[4] J W Daly T F Spande and H M Garraffo ldquoAlkaloidsfrom amphibian skin a tabulation of over eight-hundredcompoundsrdquo Journal of Natural Products vol 68 no 10 pp1556ndash1575 2005
[5] F E Koehn ldquoHigh impact technologies for natural productsscreeningrdquo Progress in Drug Research vol 65 pp 176ndash210 2008
[6] R P Borris ldquoNatural products research perspectives from amajor pharmaceutical companyrdquo Journal of Ethnopharmacol-ogy vol 51 no 1ndash3 pp 29ndash38 1996
[7] GM Cragg andD J Newman ldquoNatural products a continuingsource of novel drug leadsrdquo Biochimica et Biophysica Acta vol1830 pp 3670ndash3695 2013
[8] T ChenD F OrrMOrsquoRourke et al ldquoPachymedusa dacnicolortryptophyllin-1 structural characterization pharmacologicalactivity and cloning of precursor cDNArdquo Regulatory Peptidesvol 117 no 1 pp 25ndash32 2004
[9] L Wang M Zhou T Chen B Walker and C Shaw ldquoPdT-2 anovelmyotropic Type-2 tryptophyllin from the skin secretion of
the Mexican giant leaf frog Pachymedusa dacnicolorrdquo Peptidesvol 30 no 8 pp 1557ndash1561 2009
[10] RWang T ChenM Zhou LWang andC Shaw ldquoPsT-1 a newtryptophyllin peptide from the skin secretion of Waxy MonkeyLeaf Frog Phyllomedusa sauvageirdquoRegulatory Peptides vol 184pp 14ndash21 2013
[11] M J Tyler D J M Stone and J H Bowie ldquoA novel method forthe release and collection of dermal glandular secretions fromthe skin of frogsrdquo Journal of Pharmacological and ToxicologicalMethods vol 28 no 4 pp 199ndash200 1992
[12] D I Chan E J Prenner and H J Vogel ldquoTryptophan- andarginine-rich antimicrobial peptides structures and mecha-nisms of actionrdquo Biochimica et Biophysica Acta vol 1758 no9 pp 1184ndash1202 2006
[13] A Munoz B Lopez-Garcıa and J F Marcos ldquoStudies onthe mode of action of the antifungal hexapeptide PAF26rdquoAntimicrobial Agents and Chemotherapy vol 50 no 11 pp3847ndash3855 2006
[14] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[15] J G Walls Red-Eyes and Other Leaf-Frogs TFH Publications1996
[16] J M Conlon J Kolodziejek and N Nowotny ldquoAntimicrobialpeptides from ranid frogs taxonomic and phylogeneticmarkersand a potential source of new therapeutic agentsrdquo Biochimica etBiophysica Acta vol 1696 no 1 pp 1ndash14 2004
[17] J M Conlon ldquoThe contribution of skin antimicrobial peptidesto the system of innate immunity in anuransrdquo Cell and TissueResearch vol 343 no 1 pp 201ndash212 2011
[18] R M Epand and H J Vogel ldquoDiversity of antimicrobial pep-tides and their mechanisms of actionrdquo Biochimica et BiophysicaActa vol 1462 no 1-2 pp 11ndash28 1999
[19] M Simmaco G Mignogna and D Barra ldquoAntimicrobial pep-tides from amphibian skin what do they tell usrdquo Biopolymersvol 47 no 6 pp 435ndash450 1998
of natural leads for therapeutic development or to identifynovel disease-related drug targets [6 7]
The bioactive peptides represent one of the largest andmost functionally diverse groups of biochemicals identifiedthus far from amphibian skin and unlike the diet-acquiredalkaloids these are of endogenous origin and are geneticallyencoded [1ndash4] While many individual peptides have beenisolated they fall into two broad functional groupingsmdashthosewith pharmacological activity and those with antimicrobialactivity [1ndash4]
Tryptophyllins (TPHs) are a large and heterogenousgroup of peptides that did not fall into either category whenfirst identified as they were originally isolated from theskin of the South-American leaf frog Phyllomedusa rohdeithrough chemical means by nature of their positive Ehrlichrsquosreactionmdasha color-producing chemical reaction for indoles(tryptophan residues) [2] All original TPHs possessed atryptophanyl residue at position 2 from the C-terminus andprolyl residues at position 2 andor 3 from the N-terminus[2] although some TPHs more recently identified are devoidof tryptophanyl residues [2] Due to the increasing numbersof TPHs being identified and their apparent structural het-erogeneity this peptide family has been divided into threetypesmdash(1) tryptophyllin-1 (T-1) peptides the N-terminaldoublet KP- a tryptophanyl residue at position 5 and a prolylresidue at position 7 from the N-terminus are all highlyconserved in this group of peptides that typically possess 7-8residues (2) tryptophyllin-2 (T-2) peptides these are variablein length contain 4 to 7 residues and all contain an internal-PW doublet (3) tryptophyllin-3 (T-3) peptides the mostconserved TPHs containing 13 residues with conservativesubstitutions at positions 2 5 6 and 13 from the N-terminusThe N-terminal pGlu- -KP- at positions 3 and 4 andthe 7ndash12 sequence -PPPIYP- are all completely conserved[2 8ndash10]
Although the structures of many TPHs have been knownfor more than twenty-five years their biological actions haveremained somewhat obscure until recently In early reportsTPHs were described as having some general metaboliceffects such as stimulating liver protein synthesis and anti-bodies raised to several of these peptides produced positiveimmunostaining in pituitary gonadotrophs [2] In morerecent times two tryptophyllins (PdT-1 and PdT-2) fromthe skin secretion of the Mexican leaf frog Pachymedusadacnicolor were found to have potent pharmacological effectson different rat smooth muscles [8 9]
Here we describe the identification structural andfunctional characterization of a novel Type-2 tryptophyllinnamed AcT-2 from the skin secretion of the Central Amer-ican red-eyed leaf frog Agalychnis callidryas This peptidewas first identified through pharmacological screening formyotropic activity and was subsequently in addition foundto have a broad spectrum of antimicrobial activity albeitrelatively low potency Unusually the peptide was found topossess a higher potency against the yeast Candida albicansthan against the model Gram-positive and Gram-negativebacteria employed
2 Materials and Methods
21 Acquisition of Skin Secretions Adult red-eyed tree frogsAgalychnis callidryas (119899 = 6) of the Costa Rican type werehoused in a purpose-designed terrarium under a 12 h12 hlightdark cycle and were fed multivitamin-loaded cricketsthree times per week Skin secretions were obtained by trans-dermal electrical stimulation (4ms pulse width 50Hz 6V)in accordance with the method of Tyler et al [11] followinga three-month settling-in period The skin secretions werewashed from the skin using deionized water snap frozenin liquid nitrogen lyophilized and stored at minus20∘C prior toanalyses
22 Reverse Phase HPLC Fractionation of Lyophilized SkinSecretion Five milligrams of lyophilized skin secretion weredissolved in 1mL of 0059995 (vv) trifluoroacetic acid(TFA)water and clarified by centrifugationThe supernatantwas directly injected onto a CECIL reverse phase HPLCsystem (Milton Technical Centre Cambridge UK) fittedwithan analytical column (Phenomenex Jupiter C5 300 A poresize 250 times 4mm) The linear elution gradient employed wasformed from 0059995 (vv) TFAwater to 0051995800(vvv) TFAwateracetonitrile in 80min at a flow rate of1mLmin The effluent absorbance was monitored at 120582 =214 nm and fractions of approximately 1mL were collectedautomatically atminute intervals Samples (100120583L) from eachchromatographic fraction were removed lyophilized andstored at minus20∘C prior to analysis for myoactivity using ratsmooth muscle bioassays
23 Rat Smooth Muscle Bioassays Male Wistar rats (250ndash300 g) were euthanized by carbon dioxide asphyxiation fol-lowed by cervical dislocation under appropriate UK animalresearch lisences and following local institutional guidelinesAfter removing the fur on the abdomen of rats the bodycavity was opened and any visible fat was removed Theexposed urinary bladder and the tail were carefully removedAll dissected tissues were placed in ice-cold Krebrsquos solu-tion (118mM NaCl 47mM KCl 25mM NaHCO
3 115mM
NaH2PO4 25mM CaCl
2 11 mM MgCl
2 and 56mM glu-
cose) which was aerated with 95 O25 CO
2gas mixture
Strips of urinary bladder and rings of dissected tail arterywere prepared and tied with fine silk ligatures (02mm) ateach end These preparations were attached to a fixed pin atone end and to a transducer at the other end Krebrsquos solution(at 37∘C) flowing through the organ baths at 2mLmin witha constant bubbling of 95 O
25 CO
2maintained the
tissues in a viable state for gt2 h An equilibration period of20min was used for each preparation after which 60mMKCl was added to test the responsiveness of urinary bladdermuscle strips and 10minus3M phenylephrine was added to testthe responsiveness of the tail artery rings and to causepreconstriction Following these tests viable preparationswere used to screen prepared samples of reverse phase HPLCfractions of Agalychnis callidryas skin secretion Changes intension of smooth muscle preparations were detected bypressure transducers connected to a PowerLab System (AD
The Scientific World Journal 3
Instruments Pty Ltd) Data were analyzed using GraphPadPrism software anddata pointswere expressed asmean valuesplusmn standard errors 119899 = 6 in each case
24 Structural Characterization of the Novel Peptide A singlereverse phase HPLC fraction was found to have a potentrelaxation effect on tail artery smooth muscle a less potentcontractile action on urinary bladder smooth muscle stripsand amoderately potent antimicrobial effect A sample of thisfraction was subjected to MALDI-TOF mass spectrometryusing a Perseptive BiosystemsVoyagerDEmass spectrometerin positive detectionmodewith120572-cyano-4-hydroxycinnamicacid as matrix Subsequent to this analysis the single majorpeptide resolved was subjected to MSMS fragmentationsequencing using an LCQ Fleet electrospray ion-trap massspectrometer (Thermo-Fisher San Jose CA USA)
25 Molecular Cloning of the Novel Peptide BiosyntheticPrecursor-Encoding cDNA Five milligrams of lyophilizedskin secretion were dissolved in 1mL of cell lysismRNAprotection buffer that was supplied by Dynal Biotech UKPolyadenylated mRNA was then isolated from this usingmagnetic oligo-dT Dynabeads (Dynal Biotech UK) asper manufacturerrsquos instructions The isolated polyadenylatedmRNA was then subjected to 51015840- and 31015840-rapid amplificationof cDNA ends (RACE) procedures to obtain full-length novelpeptide precursor nucleic acid sequence data using a SMART-RACE kit (Clontech UK) likewise as per manufacturerrsquosinstructions Briefly the 31015840-RACE reactions employed anested universal (NUP) primer (supplied with the kit) and adegenerate sense primer (SP 51015840-GGIATGMGICCICCITGG-31015840) (I = deoxyinosine M = AC) that was complementary tothe putative amino acid sequence GMRPPW- of the novelpeptide The 31015840-RACE reactions were purified and clonedusing a pGEM-T vector system (Promega Corporation)and sequenced using an ABI 3100 automated sequencerThe sequence data obtained from the 31015840-RACE productwere used to design a specific antisense primer (ASP 51015840-CGGCACTATTACTGATAATTGTGCT-31015840) to a defined sitewithin the 31015840 nontranslated region of the novel peptideprecursor-encoding transcripts 51015840-RACE was carried outusing these primers in conjunction with the NUP primer andresultant products were purified cloned and sequenced
26 Solid-Phase Peptide Synthesis Once the unequivocalprimary structure of the novel peptide had been establishedit was chemically synthesized by solid-phase Fmoc chem-istry using a PS3 automated solid-phase peptide synthesizer(Protein Technologies Inc AZ USA) Following cleavageof the synthetic peptide from the resin and deprotection ofthe side-chain protecting groups the resultant material waslyophilized and then purified by HPLC The major productwas subjected toMALDI-TOFmass spectrometry to establishboth degree of purity and authenticity of structure
27 Preliminary Pharmacological Characterization of the Syn-thetic Novel Peptide on Rat Tail Artery and Urinary BladderSmooth Muscle The synthetic peptide was initially prepared
as a stock in Krebrsquos solution (118mM NaCl 47mM KCl25mM NaHCO
3 115mM NaH
2PO4 25mM CaCl
2 11mM
MgCl2 and 56mM glucose) at a concentration of 10minus3M
Working concentrations of peptide ranging from 10minus3 to1011M were prepared prior to each experiment and wereapplied to tissues (119899 = 6) progressively from low to highconcentrations Data obtained from the PowerLab System(AD Instruments Pty Ltd) were analyzed by Studentrsquos t-testthrough GraphPad Prism software to obtain the mean andstandard error of responses and using these datasets dose-response curves were constructed using a best-fit algorithm
28 Determination of Minimal Inhibitory Concentrations(MICs) of the Synthetic Novel Peptide for Model Microor-ganisms Minimal inhibitory concentrations (MICs) of thesynthetic peptide were assessed against three standard modelmicroorganisms the Gram-positive bacterium Staphylococ-cus aureus (S aureus NCTC 10788) the Gram-negativebacterium Escherichia coli (E coli NCTC 10418) and thepathogenic yeast Candida albicans (C albicans NCPF 1467)The synthetic peptidewas initially dissolved in a small volumeof dimethylsulfoxide (DMSO) and made up to 1mL withsterile Muellar-Hinton broth (MHB) Doubling dilutions ofthis peptide stock solution peptide were made from 512ndash1mgL in sterile MHB and were incubated with microorgan-ism cultures (106 colony forming units (CFU)mL) in 96-wellmicrotiter cell culture plates for 18 h at 37∘C in a humidifiedatmosphere MHB alone was used as a negative control andeachmicroorganism inMHBwith no peptide addedwas usedas positive controls After incubation the growth of microor-ganisms was determined by means of measuring opticaldensity (OD) at 120582 = 550 nm using an ELISA plate reader(Biolise BioTek EL808) Minimal inhibitory concentrations(MICs) were defined as the lowest concentration at which nogrowth was detectable
29 Hemolysis Assay Peptide solutions in a range of con-centrations were prepared as described in Section 28 but in09 (wv) aqueous NaCl solution prior to performing thehemolysis assay A 2 suspension of washed horse red bloodcells in this solution was prepared from defibrinated horseblood (TCS Biosciences Ltd UK) and samples of this wereincubated at 37∘C for 120min with a range of peptide con-centrations similar to those employed for the antimicrobialassays Lysis of red cells was assessed by measurement ofthe optical density of supernatants following centrifugationat 120582 = 550 nm using an ELISA plate reader (Biolise BioTekEL808) Negative controls employed consisted of a 2 (vv)red cell suspension alone and positive controls consisted ofa 2 (vv) red cell suspension and an equal volume of salinecontaining 2 (vv) of the nonionic detergent Triton X-100(Sigma-Aldrich)
3 Results
31 Identification and Structural Characterization of the NovelPeptide Reverse phase HPLC fraction 33 of Agalychniscallidryas skin secretion (Figure 1(a)) was found to contain
4 The Scientific World Journal
Time (mmss)
36
218
400
583
765
2801 3048 3335 3622 3909 4156
AcT-2
Abso
rban
ce (120582
214
mA
)
(a)
1 Seq 21 5802 2952 G 72 18907 9504 M 83243 41672 63 34517 17309 R 70139 35120 54 44222 22162 P 54529 27315 45 53928 27014 P 44823 22462 36 72536 36318 W 35118 17609 27 F-
amidated16510 8305 1
b(1+) b(2+) y(1+) y(2+)
(b)
Figure 1 Region of reverse phase HPLC chromatogram of Agalychnis callidryas skin secretion indicating the elution positionretentiontime (arrow) of absorbance peak containing the peptide AcT-2 (a) Expected singly and doubly charged b-ion and y-ions arising fromfragmentation of AcT-2 as predicted using the MS-Product program available through Protein Prospector online Observed fragment ionsare indicated in bold type face and are underlined (b)
a myoactive peptide following preliminary smooth musclepharmacological screening and a sample of this fractionwas subjected to MALDI-TOF mass spectrometry whichindicated a relatively high degree of purity of a peptidewith an mz (M+H)+ of 88925 The doubly charged ion ofthis peptide was subsequently identified following electro-spray MS analysis and subjected to MSMS fragmentationsequencing (Figure 1(b)) This produced the tentative aminoacid sequence GMRPPWF based upon identification of b-and y-ion series The peptide was also deemed to be C-terminally amidated Bioinformatic analysis produced no hitswith any archived amphibian skin peptide but with twosynthetic antifungal peptides named PAF-26 and combi-1[12 13] (Figure 2(a)) Of note was the presence of residues3ndash8 of the novel myotropic peptide in a C-terminally-located domain of a prophenin-2-like protein from the killerwhale (Orcinus orca) that contains a cathelicidin sequence(accession no XP004284013) (Figure 2(a)) However theorigin and structural characteristics of the novel myotropicpeptide an internal -PPW- sequence and C-terminal amida-tion indicated that it was a member of the amphibian skintryptophyllin family subtype T-2 and thus it was namedAgalychnis callidryas tryptophyllin-2 (AcT-2) in accordance
32 Molecular Cloning of AcT-2 Biosynthetic Precursor-Encoding cDNA A cDNA encoding the AcT-2 precursorprotein was successfully and repeatedly cloned using theRACEPCR strategy employedThe sequencewas representedin at least 30 clones after employing repetitive PCR andcloning procedures The complete nucleotide and translatedopen-reading frame amino acid sequences of the clonedAcT-2 biosynthetic precursor-encoding cDNA are shown inFigure 2(b)The deduced open-reading frame consisted of 62amino acid residues and shared a similar architecture with
the previously reported precursors of the tryptophyllins PdT-1 and PdT-2 from Pachymedusa dacnicolor skin secretion(Chen et al 2004 Wang et al 2009) (Figure 2(c)) Thisconsisted of an N-terminal putative signal peptide an acidicamino acid residue-rich spacer peptide domain a single copyof a mature AcT-2 sequence and a C-terminal processingand amidation site (Figure 2(c)) As in the Pachymedusadacnicolor tryptophyllin precursors the mature peptide wasflanked N-terminally by a double basic amino acid residuepropeptide convertase processing site - -RR- cleavage ofwhich generated the N-terminus of mature AcT-2 The C-terminal region of themature AcT-2 peptide was flanked by atripeptide sequence -GKK in commonwith the precursor ofPdT-2 The double basic amino acid motif -KK is removedby propeptide convertase and the resulting C-terminal glycyl(G) residue serves as an amide donor through the action ofamidating enzyme complex
33 Smooth Muscle Activity of Synthetic AcT-2 SyntheticAcT-2 was found to be active in smooth muscle preparationsfrom both rat tail artery and urinary bladder but in differentways and at different potencies In rat tail artery smoothmus-cle preparations AcT-2 caused a dose-dependent relaxationwith an EC
50of 51 nM (Figure 3(a)) In contrast the peptide
induced a dose-dependent contraction of urinary bladdersmooth muscle with an EC
50of 93 120583M (Figure 3(b))
34 Antimicrobial and Hemolytic Activity of AcT-2 AcT-2was somewhat unexpectedly found to possess a broad spec-trum of antimicrobial activity MICs obtained with the threemodel test organisms were as follows S aureus (256mgL)E coli (512mgL) and C albicans (128mgL) (Figures 4(a)ndash4(c)) The order of sensitivity of the test organisms employedwas thus C albicans gt S aureus gt E coli which is a most
---------1--------- ---- --------2 ---------- - - - - - --3 4--AcT-2 M ASV KKS VFLVLFLGF ISISFC DEEKR EDDEEGNEREEKREIHEEG N Q E E R R GMRPPW F GK K
PdT-2 M DFL RKS LFLVLFLGF VSISFC DEEKR EDDDENHGSEEKREIHEEG N Q E E R R DMSPPW H GK K
PdT-1 M NFL KKS LFLVLFLGF VSISFC DEEKR QDDDEGNEREEKKEIQEDG N Q E E R R DKPPAW VP GK -
larr larr larr larrrarr rarr rarr rarr
(c)
Figure 2 Comparison of the primary structure of AcT-2 with those of similar peptides PAF26 and combi-1 are synthetic antifungal peptidesisolated from combinatorial peptide libraries [12 13] Prophenin-2-like is a region of an antimicrobial polypeptide cathelicidin sequencedfrom a genomic DNA template of the killer whale (Orcinus orca) (accession no XP004284013) Conserved amino acid residues are shadedblack and consensus residues are shaded grey (a) Nucleotide and translated open-reading frame amino acid sequence of a cloned cDNAencoding the biosynthetic precursor of AcT-2 The putative signal peptide is double-underlined the mature peptide is single-underlinedand the stop codon is indicated by an asterisk (b) Alignments of complete open-reading frame amino acid sequences of AcT-2 precursor-encoding cDNA with the two top hits obtained following BLAST analysis using the NCBI portal PdT-1 and PdT-2 represent Pachymedusadacnicolor tryptophyllin-1 and -2 respectively Conserved amino acids are shaded black and consensus amino acids are shaded grey Gapshave been inserted tomaximize alignments (1) Putative signal peptide domain (2) Acidic spacer peptide domain (3)Mature peptide domain(4) C-terminal processing site (c)
Figure 3 Dose-response curves obtained using synthetic AcT-2 on rat smooth muscle preparations (a) Effect of the peptide on tail arterysmoothmuscle preparations expressed asmeans and standard errors (119899 = 6) EC
50was found to be 51 nM (b) Effect of the peptide on urinary
bladder smooth muscle preparations expressed as means and standard errors (119899 = 6) EC50was found to be 93 120583M
unusual profile for previously reported typical amphibianskin cationic amphipathic 120572-helical antimicrobial peptidesAlso worthy of note was the virtual lack of hemolytic activityof AcT-2 even up to the highest concentration (512mgL)employed (Figure 4(d))
4 Discussion
Central-American red-eyed leaf frogs (Agalychnis callidryas)are probably the most universally recognized frogs in theworld having been used extensively by advertising agencies
Figure 4Minimal inhibitory concentration (MIC) curves obtained following incubation of synthetic AcT-2 with S aureus (a) E coli (b) andC albicans (c) The MIC value for each microorganism is indicated in respective panels by asterisks (d) The hemolytic activity of syntheticAcT-2 The arrow shows the highest concentration of peptide (512mgL) which was the only concentration at which hemolysis was observed(85)
due to their strikingly beautiful colors Although they areone of the most widely available of the phyllomedusine leaffrogs studies on their defensive skin secretion peptides havenot been carried out with the same degree of focus that hasbeen given to many of their more obscure relatives [2 14]Red-eyed leaf frogs are most vividly colored with vibrantlime-green dorsal surfaces white ventral surfaces cream andcobalt-blue striped sides and bright orange feet Despite thisfrogs are difficult to see as they press themselves againstleaves and pull their limbs tightly towards their bodies thusbecoming a green blob on the leaf surface Their brightcoloration however is only present during the day when thefrogs are largely inactive while during the hours of darknesswhenever the frogs are most active their colors are muchdrabber favouring shades of grey and brown [15]
The potent cocktails of defensive molecules presentin skin secretions effectively constitutes their front-line ofdefense against predators [1 2] To date several peptideswith a range of biological effects have been reported fromthese skin secretions and these include both antimicrobialand pharmacologically active peptides [1 2 16] HoweverAcT-2 described here represents the first skin secretiontryptophyllin described from this species despite severalhaving been reported from Phyllomedusa species over thepast decades [2] A recent report byWang et al [9] describeda simple rational scheme to classify the rather structurally-heterogenous group of amphibian skin peptides collectivelynamed tryptophyllins [9] In this peptideswere classified intoone of three groups named T-1 T-2 and T-3 tryptophyllinsbased on some common structural features On this basisAcT-2 shows the features of a type-2 (T-2) tryptophyllin incommon with PdT-2 [9] as both possess an internal -PW-sequence motif (Figure 2(c)) However despite the presence
of this common internal structural feature AcT-2 and PdT-2 display radically different potencies in stimulating thecontraction of rat uterus smooth muscle (EC
50values AcT-2
= 93 120583M PdT-2 = 4 nM) indicating that other features of theprimary structure influence their receptor interactions [9]However AcT-2 was found to possess a most potent arterialsmooth muscle relaxant effect (EC
50= 51 nM)mdashan effect not
observed with PdT-2 [9] The smooth muscle pharmacol-ogy and molecular targets for these Type-2 tryptophyllinsin mammalian tissues obviously warrant further in-depthinvestigation
As demonstrated in this study AcT-2 was unexpectedlyfound to possess antimicrobial activity albeit relatively lowpotency using three model test microorganisms with theorder of sensitivity being C albicans gt S aureus gt E coli orin other words yeast gt Gram-positive bacterium gt Gram-negative bacterium Related to this observation was thatfollowing database interrogation with the primary structureof AcT-2 three peptides were found which displayed somedegree of structural identity (Figure 2(a)) Two of thesenamed PAF26 and combi-1 were synthetic in nature andwere found through the screening of combinatorial peptidelibraries for antifungal activity [12 13] The third representeda cathelicidin domain of a prophenin-2-like protein deducedfrom a cloned cDNA of the killer whale (Orcinus orca)(accession no XP004284013) Interestingly all three AcT-2-like peptides were associated with antimicrobial functionswith the former two being specifically against fungi
While AcT-2 is of relatively low potency when comparedwith many other amphibian skin antimicrobial peptidesmost if not all of the latter act through a mechanism ofmicrobial target cell membrane lysis and some are stronglyhemolytic [17ndash19] To achieve this form of nonspecific
The Scientific World Journal 7
antimicrobial action such peptides are generallymuch longerin amino acid chain length than AcT-2 [17 19] ThusAcT-2 may act upon a different and possibly intracellulartarget rather than by direct lytic action on the cytoplasmicmembranemdasha mode of action that has actually been pro-posed for some ldquoclassicalrdquo antimicrobial peptides as well [1719] In support of this proposal to some degree is the lack ofhemolytic activity of AcT-2 observed following its incubationwith horse erythrocytes (Figure 4(d))
In conclusion the novel amphibian skin tryptophyllinnamed AcT-2 described here has been demonstrated tohave both selective mammalian smooth muscle myotropiceffects and antimicrobial activitymdashfindings which add toour increasing knowledge of the biological effects of thisenigmatic family of amphibian skin peptides
Thenucleotide sequence ofAcT-2 from the skin secretionof Agalychnis callidryas has been deposited in the EMBLNucleotide Sequence Database under the accession codeHG710094
Conflict of Interests
The authors declare that they have no conflict of interests
Authorsrsquo Contribution
Lilin Ge and Peng Lyu contributed equally to this work
References
[1] B T Clarke ldquoThe natural history of amphibian skin secretionstheir normal functioning and potential medical applicationsrdquoBiological Reviews of the Cambridge Philosophical Society vol72 no 3 pp 365ndash379 1997
[2] V Erspamer ldquoBioactive secretions of the integumentrdquo inAmphibian Biologymdashvol 1 the Integument H Heatwole andG T Barthalmus Eds pp 179ndash350 Surrey Beatty amp SonsChipping Norton 1994
[3] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[4] J W Daly T F Spande and H M Garraffo ldquoAlkaloidsfrom amphibian skin a tabulation of over eight-hundredcompoundsrdquo Journal of Natural Products vol 68 no 10 pp1556ndash1575 2005
[5] F E Koehn ldquoHigh impact technologies for natural productsscreeningrdquo Progress in Drug Research vol 65 pp 176ndash210 2008
[6] R P Borris ldquoNatural products research perspectives from amajor pharmaceutical companyrdquo Journal of Ethnopharmacol-ogy vol 51 no 1ndash3 pp 29ndash38 1996
[7] GM Cragg andD J Newman ldquoNatural products a continuingsource of novel drug leadsrdquo Biochimica et Biophysica Acta vol1830 pp 3670ndash3695 2013
[8] T ChenD F OrrMOrsquoRourke et al ldquoPachymedusa dacnicolortryptophyllin-1 structural characterization pharmacologicalactivity and cloning of precursor cDNArdquo Regulatory Peptidesvol 117 no 1 pp 25ndash32 2004
[9] L Wang M Zhou T Chen B Walker and C Shaw ldquoPdT-2 anovelmyotropic Type-2 tryptophyllin from the skin secretion of
the Mexican giant leaf frog Pachymedusa dacnicolorrdquo Peptidesvol 30 no 8 pp 1557ndash1561 2009
[10] RWang T ChenM Zhou LWang andC Shaw ldquoPsT-1 a newtryptophyllin peptide from the skin secretion of Waxy MonkeyLeaf Frog Phyllomedusa sauvageirdquoRegulatory Peptides vol 184pp 14ndash21 2013
[11] M J Tyler D J M Stone and J H Bowie ldquoA novel method forthe release and collection of dermal glandular secretions fromthe skin of frogsrdquo Journal of Pharmacological and ToxicologicalMethods vol 28 no 4 pp 199ndash200 1992
[12] D I Chan E J Prenner and H J Vogel ldquoTryptophan- andarginine-rich antimicrobial peptides structures and mecha-nisms of actionrdquo Biochimica et Biophysica Acta vol 1758 no9 pp 1184ndash1202 2006
[13] A Munoz B Lopez-Garcıa and J F Marcos ldquoStudies onthe mode of action of the antifungal hexapeptide PAF26rdquoAntimicrobial Agents and Chemotherapy vol 50 no 11 pp3847ndash3855 2006
[14] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[15] J G Walls Red-Eyes and Other Leaf-Frogs TFH Publications1996
[16] J M Conlon J Kolodziejek and N Nowotny ldquoAntimicrobialpeptides from ranid frogs taxonomic and phylogeneticmarkersand a potential source of new therapeutic agentsrdquo Biochimica etBiophysica Acta vol 1696 no 1 pp 1ndash14 2004
[17] J M Conlon ldquoThe contribution of skin antimicrobial peptidesto the system of innate immunity in anuransrdquo Cell and TissueResearch vol 343 no 1 pp 201ndash212 2011
[18] R M Epand and H J Vogel ldquoDiversity of antimicrobial pep-tides and their mechanisms of actionrdquo Biochimica et BiophysicaActa vol 1462 no 1-2 pp 11ndash28 1999
[19] M Simmaco G Mignogna and D Barra ldquoAntimicrobial pep-tides from amphibian skin what do they tell usrdquo Biopolymersvol 47 no 6 pp 435ndash450 1998
Instruments Pty Ltd) Data were analyzed using GraphPadPrism software anddata pointswere expressed asmean valuesplusmn standard errors 119899 = 6 in each case
24 Structural Characterization of the Novel Peptide A singlereverse phase HPLC fraction was found to have a potentrelaxation effect on tail artery smooth muscle a less potentcontractile action on urinary bladder smooth muscle stripsand amoderately potent antimicrobial effect A sample of thisfraction was subjected to MALDI-TOF mass spectrometryusing a Perseptive BiosystemsVoyagerDEmass spectrometerin positive detectionmodewith120572-cyano-4-hydroxycinnamicacid as matrix Subsequent to this analysis the single majorpeptide resolved was subjected to MSMS fragmentationsequencing using an LCQ Fleet electrospray ion-trap massspectrometer (Thermo-Fisher San Jose CA USA)
25 Molecular Cloning of the Novel Peptide BiosyntheticPrecursor-Encoding cDNA Five milligrams of lyophilizedskin secretion were dissolved in 1mL of cell lysismRNAprotection buffer that was supplied by Dynal Biotech UKPolyadenylated mRNA was then isolated from this usingmagnetic oligo-dT Dynabeads (Dynal Biotech UK) asper manufacturerrsquos instructions The isolated polyadenylatedmRNA was then subjected to 51015840- and 31015840-rapid amplificationof cDNA ends (RACE) procedures to obtain full-length novelpeptide precursor nucleic acid sequence data using a SMART-RACE kit (Clontech UK) likewise as per manufacturerrsquosinstructions Briefly the 31015840-RACE reactions employed anested universal (NUP) primer (supplied with the kit) and adegenerate sense primer (SP 51015840-GGIATGMGICCICCITGG-31015840) (I = deoxyinosine M = AC) that was complementary tothe putative amino acid sequence GMRPPW- of the novelpeptide The 31015840-RACE reactions were purified and clonedusing a pGEM-T vector system (Promega Corporation)and sequenced using an ABI 3100 automated sequencerThe sequence data obtained from the 31015840-RACE productwere used to design a specific antisense primer (ASP 51015840-CGGCACTATTACTGATAATTGTGCT-31015840) to a defined sitewithin the 31015840 nontranslated region of the novel peptideprecursor-encoding transcripts 51015840-RACE was carried outusing these primers in conjunction with the NUP primer andresultant products were purified cloned and sequenced
26 Solid-Phase Peptide Synthesis Once the unequivocalprimary structure of the novel peptide had been establishedit was chemically synthesized by solid-phase Fmoc chem-istry using a PS3 automated solid-phase peptide synthesizer(Protein Technologies Inc AZ USA) Following cleavageof the synthetic peptide from the resin and deprotection ofthe side-chain protecting groups the resultant material waslyophilized and then purified by HPLC The major productwas subjected toMALDI-TOFmass spectrometry to establishboth degree of purity and authenticity of structure
27 Preliminary Pharmacological Characterization of the Syn-thetic Novel Peptide on Rat Tail Artery and Urinary BladderSmooth Muscle The synthetic peptide was initially prepared
as a stock in Krebrsquos solution (118mM NaCl 47mM KCl25mM NaHCO
3 115mM NaH
2PO4 25mM CaCl
2 11mM
MgCl2 and 56mM glucose) at a concentration of 10minus3M
Working concentrations of peptide ranging from 10minus3 to1011M were prepared prior to each experiment and wereapplied to tissues (119899 = 6) progressively from low to highconcentrations Data obtained from the PowerLab System(AD Instruments Pty Ltd) were analyzed by Studentrsquos t-testthrough GraphPad Prism software to obtain the mean andstandard error of responses and using these datasets dose-response curves were constructed using a best-fit algorithm
28 Determination of Minimal Inhibitory Concentrations(MICs) of the Synthetic Novel Peptide for Model Microor-ganisms Minimal inhibitory concentrations (MICs) of thesynthetic peptide were assessed against three standard modelmicroorganisms the Gram-positive bacterium Staphylococ-cus aureus (S aureus NCTC 10788) the Gram-negativebacterium Escherichia coli (E coli NCTC 10418) and thepathogenic yeast Candida albicans (C albicans NCPF 1467)The synthetic peptidewas initially dissolved in a small volumeof dimethylsulfoxide (DMSO) and made up to 1mL withsterile Muellar-Hinton broth (MHB) Doubling dilutions ofthis peptide stock solution peptide were made from 512ndash1mgL in sterile MHB and were incubated with microorgan-ism cultures (106 colony forming units (CFU)mL) in 96-wellmicrotiter cell culture plates for 18 h at 37∘C in a humidifiedatmosphere MHB alone was used as a negative control andeachmicroorganism inMHBwith no peptide addedwas usedas positive controls After incubation the growth of microor-ganisms was determined by means of measuring opticaldensity (OD) at 120582 = 550 nm using an ELISA plate reader(Biolise BioTek EL808) Minimal inhibitory concentrations(MICs) were defined as the lowest concentration at which nogrowth was detectable
29 Hemolysis Assay Peptide solutions in a range of con-centrations were prepared as described in Section 28 but in09 (wv) aqueous NaCl solution prior to performing thehemolysis assay A 2 suspension of washed horse red bloodcells in this solution was prepared from defibrinated horseblood (TCS Biosciences Ltd UK) and samples of this wereincubated at 37∘C for 120min with a range of peptide con-centrations similar to those employed for the antimicrobialassays Lysis of red cells was assessed by measurement ofthe optical density of supernatants following centrifugationat 120582 = 550 nm using an ELISA plate reader (Biolise BioTekEL808) Negative controls employed consisted of a 2 (vv)red cell suspension alone and positive controls consisted ofa 2 (vv) red cell suspension and an equal volume of salinecontaining 2 (vv) of the nonionic detergent Triton X-100(Sigma-Aldrich)
3 Results
31 Identification and Structural Characterization of the NovelPeptide Reverse phase HPLC fraction 33 of Agalychniscallidryas skin secretion (Figure 1(a)) was found to contain
4 The Scientific World Journal
Time (mmss)
36
218
400
583
765
2801 3048 3335 3622 3909 4156
AcT-2
Abso
rban
ce (120582
214
mA
)
(a)
1 Seq 21 5802 2952 G 72 18907 9504 M 83243 41672 63 34517 17309 R 70139 35120 54 44222 22162 P 54529 27315 45 53928 27014 P 44823 22462 36 72536 36318 W 35118 17609 27 F-
amidated16510 8305 1
b(1+) b(2+) y(1+) y(2+)
(b)
Figure 1 Region of reverse phase HPLC chromatogram of Agalychnis callidryas skin secretion indicating the elution positionretentiontime (arrow) of absorbance peak containing the peptide AcT-2 (a) Expected singly and doubly charged b-ion and y-ions arising fromfragmentation of AcT-2 as predicted using the MS-Product program available through Protein Prospector online Observed fragment ionsare indicated in bold type face and are underlined (b)
a myoactive peptide following preliminary smooth musclepharmacological screening and a sample of this fractionwas subjected to MALDI-TOF mass spectrometry whichindicated a relatively high degree of purity of a peptidewith an mz (M+H)+ of 88925 The doubly charged ion ofthis peptide was subsequently identified following electro-spray MS analysis and subjected to MSMS fragmentationsequencing (Figure 1(b)) This produced the tentative aminoacid sequence GMRPPWF based upon identification of b-and y-ion series The peptide was also deemed to be C-terminally amidated Bioinformatic analysis produced no hitswith any archived amphibian skin peptide but with twosynthetic antifungal peptides named PAF-26 and combi-1[12 13] (Figure 2(a)) Of note was the presence of residues3ndash8 of the novel myotropic peptide in a C-terminally-located domain of a prophenin-2-like protein from the killerwhale (Orcinus orca) that contains a cathelicidin sequence(accession no XP004284013) (Figure 2(a)) However theorigin and structural characteristics of the novel myotropicpeptide an internal -PPW- sequence and C-terminal amida-tion indicated that it was a member of the amphibian skintryptophyllin family subtype T-2 and thus it was namedAgalychnis callidryas tryptophyllin-2 (AcT-2) in accordance
32 Molecular Cloning of AcT-2 Biosynthetic Precursor-Encoding cDNA A cDNA encoding the AcT-2 precursorprotein was successfully and repeatedly cloned using theRACEPCR strategy employedThe sequencewas representedin at least 30 clones after employing repetitive PCR andcloning procedures The complete nucleotide and translatedopen-reading frame amino acid sequences of the clonedAcT-2 biosynthetic precursor-encoding cDNA are shown inFigure 2(b)The deduced open-reading frame consisted of 62amino acid residues and shared a similar architecture with
the previously reported precursors of the tryptophyllins PdT-1 and PdT-2 from Pachymedusa dacnicolor skin secretion(Chen et al 2004 Wang et al 2009) (Figure 2(c)) Thisconsisted of an N-terminal putative signal peptide an acidicamino acid residue-rich spacer peptide domain a single copyof a mature AcT-2 sequence and a C-terminal processingand amidation site (Figure 2(c)) As in the Pachymedusadacnicolor tryptophyllin precursors the mature peptide wasflanked N-terminally by a double basic amino acid residuepropeptide convertase processing site - -RR- cleavage ofwhich generated the N-terminus of mature AcT-2 The C-terminal region of themature AcT-2 peptide was flanked by atripeptide sequence -GKK in commonwith the precursor ofPdT-2 The double basic amino acid motif -KK is removedby propeptide convertase and the resulting C-terminal glycyl(G) residue serves as an amide donor through the action ofamidating enzyme complex
33 Smooth Muscle Activity of Synthetic AcT-2 SyntheticAcT-2 was found to be active in smooth muscle preparationsfrom both rat tail artery and urinary bladder but in differentways and at different potencies In rat tail artery smoothmus-cle preparations AcT-2 caused a dose-dependent relaxationwith an EC
50of 51 nM (Figure 3(a)) In contrast the peptide
induced a dose-dependent contraction of urinary bladdersmooth muscle with an EC
50of 93 120583M (Figure 3(b))
34 Antimicrobial and Hemolytic Activity of AcT-2 AcT-2was somewhat unexpectedly found to possess a broad spec-trum of antimicrobial activity MICs obtained with the threemodel test organisms were as follows S aureus (256mgL)E coli (512mgL) and C albicans (128mgL) (Figures 4(a)ndash4(c)) The order of sensitivity of the test organisms employedwas thus C albicans gt S aureus gt E coli which is a most
---------1--------- ---- --------2 ---------- - - - - - --3 4--AcT-2 M ASV KKS VFLVLFLGF ISISFC DEEKR EDDEEGNEREEKREIHEEG N Q E E R R GMRPPW F GK K
PdT-2 M DFL RKS LFLVLFLGF VSISFC DEEKR EDDDENHGSEEKREIHEEG N Q E E R R DMSPPW H GK K
PdT-1 M NFL KKS LFLVLFLGF VSISFC DEEKR QDDDEGNEREEKKEIQEDG N Q E E R R DKPPAW VP GK -
larr larr larr larrrarr rarr rarr rarr
(c)
Figure 2 Comparison of the primary structure of AcT-2 with those of similar peptides PAF26 and combi-1 are synthetic antifungal peptidesisolated from combinatorial peptide libraries [12 13] Prophenin-2-like is a region of an antimicrobial polypeptide cathelicidin sequencedfrom a genomic DNA template of the killer whale (Orcinus orca) (accession no XP004284013) Conserved amino acid residues are shadedblack and consensus residues are shaded grey (a) Nucleotide and translated open-reading frame amino acid sequence of a cloned cDNAencoding the biosynthetic precursor of AcT-2 The putative signal peptide is double-underlined the mature peptide is single-underlinedand the stop codon is indicated by an asterisk (b) Alignments of complete open-reading frame amino acid sequences of AcT-2 precursor-encoding cDNA with the two top hits obtained following BLAST analysis using the NCBI portal PdT-1 and PdT-2 represent Pachymedusadacnicolor tryptophyllin-1 and -2 respectively Conserved amino acids are shaded black and consensus amino acids are shaded grey Gapshave been inserted tomaximize alignments (1) Putative signal peptide domain (2) Acidic spacer peptide domain (3)Mature peptide domain(4) C-terminal processing site (c)
Figure 3 Dose-response curves obtained using synthetic AcT-2 on rat smooth muscle preparations (a) Effect of the peptide on tail arterysmoothmuscle preparations expressed asmeans and standard errors (119899 = 6) EC
50was found to be 51 nM (b) Effect of the peptide on urinary
bladder smooth muscle preparations expressed as means and standard errors (119899 = 6) EC50was found to be 93 120583M
unusual profile for previously reported typical amphibianskin cationic amphipathic 120572-helical antimicrobial peptidesAlso worthy of note was the virtual lack of hemolytic activityof AcT-2 even up to the highest concentration (512mgL)employed (Figure 4(d))
4 Discussion
Central-American red-eyed leaf frogs (Agalychnis callidryas)are probably the most universally recognized frogs in theworld having been used extensively by advertising agencies
Figure 4Minimal inhibitory concentration (MIC) curves obtained following incubation of synthetic AcT-2 with S aureus (a) E coli (b) andC albicans (c) The MIC value for each microorganism is indicated in respective panels by asterisks (d) The hemolytic activity of syntheticAcT-2 The arrow shows the highest concentration of peptide (512mgL) which was the only concentration at which hemolysis was observed(85)
due to their strikingly beautiful colors Although they areone of the most widely available of the phyllomedusine leaffrogs studies on their defensive skin secretion peptides havenot been carried out with the same degree of focus that hasbeen given to many of their more obscure relatives [2 14]Red-eyed leaf frogs are most vividly colored with vibrantlime-green dorsal surfaces white ventral surfaces cream andcobalt-blue striped sides and bright orange feet Despite thisfrogs are difficult to see as they press themselves againstleaves and pull their limbs tightly towards their bodies thusbecoming a green blob on the leaf surface Their brightcoloration however is only present during the day when thefrogs are largely inactive while during the hours of darknesswhenever the frogs are most active their colors are muchdrabber favouring shades of grey and brown [15]
The potent cocktails of defensive molecules presentin skin secretions effectively constitutes their front-line ofdefense against predators [1 2] To date several peptideswith a range of biological effects have been reported fromthese skin secretions and these include both antimicrobialand pharmacologically active peptides [1 2 16] HoweverAcT-2 described here represents the first skin secretiontryptophyllin described from this species despite severalhaving been reported from Phyllomedusa species over thepast decades [2] A recent report byWang et al [9] describeda simple rational scheme to classify the rather structurally-heterogenous group of amphibian skin peptides collectivelynamed tryptophyllins [9] In this peptideswere classified intoone of three groups named T-1 T-2 and T-3 tryptophyllinsbased on some common structural features On this basisAcT-2 shows the features of a type-2 (T-2) tryptophyllin incommon with PdT-2 [9] as both possess an internal -PW-sequence motif (Figure 2(c)) However despite the presence
of this common internal structural feature AcT-2 and PdT-2 display radically different potencies in stimulating thecontraction of rat uterus smooth muscle (EC
50values AcT-2
= 93 120583M PdT-2 = 4 nM) indicating that other features of theprimary structure influence their receptor interactions [9]However AcT-2 was found to possess a most potent arterialsmooth muscle relaxant effect (EC
50= 51 nM)mdashan effect not
observed with PdT-2 [9] The smooth muscle pharmacol-ogy and molecular targets for these Type-2 tryptophyllinsin mammalian tissues obviously warrant further in-depthinvestigation
As demonstrated in this study AcT-2 was unexpectedlyfound to possess antimicrobial activity albeit relatively lowpotency using three model test microorganisms with theorder of sensitivity being C albicans gt S aureus gt E coli orin other words yeast gt Gram-positive bacterium gt Gram-negative bacterium Related to this observation was thatfollowing database interrogation with the primary structureof AcT-2 three peptides were found which displayed somedegree of structural identity (Figure 2(a)) Two of thesenamed PAF26 and combi-1 were synthetic in nature andwere found through the screening of combinatorial peptidelibraries for antifungal activity [12 13] The third representeda cathelicidin domain of a prophenin-2-like protein deducedfrom a cloned cDNA of the killer whale (Orcinus orca)(accession no XP004284013) Interestingly all three AcT-2-like peptides were associated with antimicrobial functionswith the former two being specifically against fungi
While AcT-2 is of relatively low potency when comparedwith many other amphibian skin antimicrobial peptidesmost if not all of the latter act through a mechanism ofmicrobial target cell membrane lysis and some are stronglyhemolytic [17ndash19] To achieve this form of nonspecific
The Scientific World Journal 7
antimicrobial action such peptides are generallymuch longerin amino acid chain length than AcT-2 [17 19] ThusAcT-2 may act upon a different and possibly intracellulartarget rather than by direct lytic action on the cytoplasmicmembranemdasha mode of action that has actually been pro-posed for some ldquoclassicalrdquo antimicrobial peptides as well [1719] In support of this proposal to some degree is the lack ofhemolytic activity of AcT-2 observed following its incubationwith horse erythrocytes (Figure 4(d))
In conclusion the novel amphibian skin tryptophyllinnamed AcT-2 described here has been demonstrated tohave both selective mammalian smooth muscle myotropiceffects and antimicrobial activitymdashfindings which add toour increasing knowledge of the biological effects of thisenigmatic family of amphibian skin peptides
Thenucleotide sequence ofAcT-2 from the skin secretionof Agalychnis callidryas has been deposited in the EMBLNucleotide Sequence Database under the accession codeHG710094
Conflict of Interests
The authors declare that they have no conflict of interests
Authorsrsquo Contribution
Lilin Ge and Peng Lyu contributed equally to this work
References
[1] B T Clarke ldquoThe natural history of amphibian skin secretionstheir normal functioning and potential medical applicationsrdquoBiological Reviews of the Cambridge Philosophical Society vol72 no 3 pp 365ndash379 1997
[2] V Erspamer ldquoBioactive secretions of the integumentrdquo inAmphibian Biologymdashvol 1 the Integument H Heatwole andG T Barthalmus Eds pp 179ndash350 Surrey Beatty amp SonsChipping Norton 1994
[3] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[4] J W Daly T F Spande and H M Garraffo ldquoAlkaloidsfrom amphibian skin a tabulation of over eight-hundredcompoundsrdquo Journal of Natural Products vol 68 no 10 pp1556ndash1575 2005
[5] F E Koehn ldquoHigh impact technologies for natural productsscreeningrdquo Progress in Drug Research vol 65 pp 176ndash210 2008
[6] R P Borris ldquoNatural products research perspectives from amajor pharmaceutical companyrdquo Journal of Ethnopharmacol-ogy vol 51 no 1ndash3 pp 29ndash38 1996
[7] GM Cragg andD J Newman ldquoNatural products a continuingsource of novel drug leadsrdquo Biochimica et Biophysica Acta vol1830 pp 3670ndash3695 2013
[8] T ChenD F OrrMOrsquoRourke et al ldquoPachymedusa dacnicolortryptophyllin-1 structural characterization pharmacologicalactivity and cloning of precursor cDNArdquo Regulatory Peptidesvol 117 no 1 pp 25ndash32 2004
[9] L Wang M Zhou T Chen B Walker and C Shaw ldquoPdT-2 anovelmyotropic Type-2 tryptophyllin from the skin secretion of
the Mexican giant leaf frog Pachymedusa dacnicolorrdquo Peptidesvol 30 no 8 pp 1557ndash1561 2009
[10] RWang T ChenM Zhou LWang andC Shaw ldquoPsT-1 a newtryptophyllin peptide from the skin secretion of Waxy MonkeyLeaf Frog Phyllomedusa sauvageirdquoRegulatory Peptides vol 184pp 14ndash21 2013
[11] M J Tyler D J M Stone and J H Bowie ldquoA novel method forthe release and collection of dermal glandular secretions fromthe skin of frogsrdquo Journal of Pharmacological and ToxicologicalMethods vol 28 no 4 pp 199ndash200 1992
[12] D I Chan E J Prenner and H J Vogel ldquoTryptophan- andarginine-rich antimicrobial peptides structures and mecha-nisms of actionrdquo Biochimica et Biophysica Acta vol 1758 no9 pp 1184ndash1202 2006
[13] A Munoz B Lopez-Garcıa and J F Marcos ldquoStudies onthe mode of action of the antifungal hexapeptide PAF26rdquoAntimicrobial Agents and Chemotherapy vol 50 no 11 pp3847ndash3855 2006
[14] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[15] J G Walls Red-Eyes and Other Leaf-Frogs TFH Publications1996
[16] J M Conlon J Kolodziejek and N Nowotny ldquoAntimicrobialpeptides from ranid frogs taxonomic and phylogeneticmarkersand a potential source of new therapeutic agentsrdquo Biochimica etBiophysica Acta vol 1696 no 1 pp 1ndash14 2004
[17] J M Conlon ldquoThe contribution of skin antimicrobial peptidesto the system of innate immunity in anuransrdquo Cell and TissueResearch vol 343 no 1 pp 201ndash212 2011
[18] R M Epand and H J Vogel ldquoDiversity of antimicrobial pep-tides and their mechanisms of actionrdquo Biochimica et BiophysicaActa vol 1462 no 1-2 pp 11ndash28 1999
[19] M Simmaco G Mignogna and D Barra ldquoAntimicrobial pep-tides from amphibian skin what do they tell usrdquo Biopolymersvol 47 no 6 pp 435ndash450 1998
1 Seq 21 5802 2952 G 72 18907 9504 M 83243 41672 63 34517 17309 R 70139 35120 54 44222 22162 P 54529 27315 45 53928 27014 P 44823 22462 36 72536 36318 W 35118 17609 27 F-
amidated16510 8305 1
b(1+) b(2+) y(1+) y(2+)
(b)
Figure 1 Region of reverse phase HPLC chromatogram of Agalychnis callidryas skin secretion indicating the elution positionretentiontime (arrow) of absorbance peak containing the peptide AcT-2 (a) Expected singly and doubly charged b-ion and y-ions arising fromfragmentation of AcT-2 as predicted using the MS-Product program available through Protein Prospector online Observed fragment ionsare indicated in bold type face and are underlined (b)
a myoactive peptide following preliminary smooth musclepharmacological screening and a sample of this fractionwas subjected to MALDI-TOF mass spectrometry whichindicated a relatively high degree of purity of a peptidewith an mz (M+H)+ of 88925 The doubly charged ion ofthis peptide was subsequently identified following electro-spray MS analysis and subjected to MSMS fragmentationsequencing (Figure 1(b)) This produced the tentative aminoacid sequence GMRPPWF based upon identification of b-and y-ion series The peptide was also deemed to be C-terminally amidated Bioinformatic analysis produced no hitswith any archived amphibian skin peptide but with twosynthetic antifungal peptides named PAF-26 and combi-1[12 13] (Figure 2(a)) Of note was the presence of residues3ndash8 of the novel myotropic peptide in a C-terminally-located domain of a prophenin-2-like protein from the killerwhale (Orcinus orca) that contains a cathelicidin sequence(accession no XP004284013) (Figure 2(a)) However theorigin and structural characteristics of the novel myotropicpeptide an internal -PPW- sequence and C-terminal amida-tion indicated that it was a member of the amphibian skintryptophyllin family subtype T-2 and thus it was namedAgalychnis callidryas tryptophyllin-2 (AcT-2) in accordance
32 Molecular Cloning of AcT-2 Biosynthetic Precursor-Encoding cDNA A cDNA encoding the AcT-2 precursorprotein was successfully and repeatedly cloned using theRACEPCR strategy employedThe sequencewas representedin at least 30 clones after employing repetitive PCR andcloning procedures The complete nucleotide and translatedopen-reading frame amino acid sequences of the clonedAcT-2 biosynthetic precursor-encoding cDNA are shown inFigure 2(b)The deduced open-reading frame consisted of 62amino acid residues and shared a similar architecture with
the previously reported precursors of the tryptophyllins PdT-1 and PdT-2 from Pachymedusa dacnicolor skin secretion(Chen et al 2004 Wang et al 2009) (Figure 2(c)) Thisconsisted of an N-terminal putative signal peptide an acidicamino acid residue-rich spacer peptide domain a single copyof a mature AcT-2 sequence and a C-terminal processingand amidation site (Figure 2(c)) As in the Pachymedusadacnicolor tryptophyllin precursors the mature peptide wasflanked N-terminally by a double basic amino acid residuepropeptide convertase processing site - -RR- cleavage ofwhich generated the N-terminus of mature AcT-2 The C-terminal region of themature AcT-2 peptide was flanked by atripeptide sequence -GKK in commonwith the precursor ofPdT-2 The double basic amino acid motif -KK is removedby propeptide convertase and the resulting C-terminal glycyl(G) residue serves as an amide donor through the action ofamidating enzyme complex
33 Smooth Muscle Activity of Synthetic AcT-2 SyntheticAcT-2 was found to be active in smooth muscle preparationsfrom both rat tail artery and urinary bladder but in differentways and at different potencies In rat tail artery smoothmus-cle preparations AcT-2 caused a dose-dependent relaxationwith an EC
50of 51 nM (Figure 3(a)) In contrast the peptide
induced a dose-dependent contraction of urinary bladdersmooth muscle with an EC
50of 93 120583M (Figure 3(b))
34 Antimicrobial and Hemolytic Activity of AcT-2 AcT-2was somewhat unexpectedly found to possess a broad spec-trum of antimicrobial activity MICs obtained with the threemodel test organisms were as follows S aureus (256mgL)E coli (512mgL) and C albicans (128mgL) (Figures 4(a)ndash4(c)) The order of sensitivity of the test organisms employedwas thus C albicans gt S aureus gt E coli which is a most
---------1--------- ---- --------2 ---------- - - - - - --3 4--AcT-2 M ASV KKS VFLVLFLGF ISISFC DEEKR EDDEEGNEREEKREIHEEG N Q E E R R GMRPPW F GK K
PdT-2 M DFL RKS LFLVLFLGF VSISFC DEEKR EDDDENHGSEEKREIHEEG N Q E E R R DMSPPW H GK K
PdT-1 M NFL KKS LFLVLFLGF VSISFC DEEKR QDDDEGNEREEKKEIQEDG N Q E E R R DKPPAW VP GK -
larr larr larr larrrarr rarr rarr rarr
(c)
Figure 2 Comparison of the primary structure of AcT-2 with those of similar peptides PAF26 and combi-1 are synthetic antifungal peptidesisolated from combinatorial peptide libraries [12 13] Prophenin-2-like is a region of an antimicrobial polypeptide cathelicidin sequencedfrom a genomic DNA template of the killer whale (Orcinus orca) (accession no XP004284013) Conserved amino acid residues are shadedblack and consensus residues are shaded grey (a) Nucleotide and translated open-reading frame amino acid sequence of a cloned cDNAencoding the biosynthetic precursor of AcT-2 The putative signal peptide is double-underlined the mature peptide is single-underlinedand the stop codon is indicated by an asterisk (b) Alignments of complete open-reading frame amino acid sequences of AcT-2 precursor-encoding cDNA with the two top hits obtained following BLAST analysis using the NCBI portal PdT-1 and PdT-2 represent Pachymedusadacnicolor tryptophyllin-1 and -2 respectively Conserved amino acids are shaded black and consensus amino acids are shaded grey Gapshave been inserted tomaximize alignments (1) Putative signal peptide domain (2) Acidic spacer peptide domain (3)Mature peptide domain(4) C-terminal processing site (c)
Figure 3 Dose-response curves obtained using synthetic AcT-2 on rat smooth muscle preparations (a) Effect of the peptide on tail arterysmoothmuscle preparations expressed asmeans and standard errors (119899 = 6) EC
50was found to be 51 nM (b) Effect of the peptide on urinary
bladder smooth muscle preparations expressed as means and standard errors (119899 = 6) EC50was found to be 93 120583M
unusual profile for previously reported typical amphibianskin cationic amphipathic 120572-helical antimicrobial peptidesAlso worthy of note was the virtual lack of hemolytic activityof AcT-2 even up to the highest concentration (512mgL)employed (Figure 4(d))
4 Discussion
Central-American red-eyed leaf frogs (Agalychnis callidryas)are probably the most universally recognized frogs in theworld having been used extensively by advertising agencies
Figure 4Minimal inhibitory concentration (MIC) curves obtained following incubation of synthetic AcT-2 with S aureus (a) E coli (b) andC albicans (c) The MIC value for each microorganism is indicated in respective panels by asterisks (d) The hemolytic activity of syntheticAcT-2 The arrow shows the highest concentration of peptide (512mgL) which was the only concentration at which hemolysis was observed(85)
due to their strikingly beautiful colors Although they areone of the most widely available of the phyllomedusine leaffrogs studies on their defensive skin secretion peptides havenot been carried out with the same degree of focus that hasbeen given to many of their more obscure relatives [2 14]Red-eyed leaf frogs are most vividly colored with vibrantlime-green dorsal surfaces white ventral surfaces cream andcobalt-blue striped sides and bright orange feet Despite thisfrogs are difficult to see as they press themselves againstleaves and pull their limbs tightly towards their bodies thusbecoming a green blob on the leaf surface Their brightcoloration however is only present during the day when thefrogs are largely inactive while during the hours of darknesswhenever the frogs are most active their colors are muchdrabber favouring shades of grey and brown [15]
The potent cocktails of defensive molecules presentin skin secretions effectively constitutes their front-line ofdefense against predators [1 2] To date several peptideswith a range of biological effects have been reported fromthese skin secretions and these include both antimicrobialand pharmacologically active peptides [1 2 16] HoweverAcT-2 described here represents the first skin secretiontryptophyllin described from this species despite severalhaving been reported from Phyllomedusa species over thepast decades [2] A recent report byWang et al [9] describeda simple rational scheme to classify the rather structurally-heterogenous group of amphibian skin peptides collectivelynamed tryptophyllins [9] In this peptideswere classified intoone of three groups named T-1 T-2 and T-3 tryptophyllinsbased on some common structural features On this basisAcT-2 shows the features of a type-2 (T-2) tryptophyllin incommon with PdT-2 [9] as both possess an internal -PW-sequence motif (Figure 2(c)) However despite the presence
of this common internal structural feature AcT-2 and PdT-2 display radically different potencies in stimulating thecontraction of rat uterus smooth muscle (EC
50values AcT-2
= 93 120583M PdT-2 = 4 nM) indicating that other features of theprimary structure influence their receptor interactions [9]However AcT-2 was found to possess a most potent arterialsmooth muscle relaxant effect (EC
50= 51 nM)mdashan effect not
observed with PdT-2 [9] The smooth muscle pharmacol-ogy and molecular targets for these Type-2 tryptophyllinsin mammalian tissues obviously warrant further in-depthinvestigation
As demonstrated in this study AcT-2 was unexpectedlyfound to possess antimicrobial activity albeit relatively lowpotency using three model test microorganisms with theorder of sensitivity being C albicans gt S aureus gt E coli orin other words yeast gt Gram-positive bacterium gt Gram-negative bacterium Related to this observation was thatfollowing database interrogation with the primary structureof AcT-2 three peptides were found which displayed somedegree of structural identity (Figure 2(a)) Two of thesenamed PAF26 and combi-1 were synthetic in nature andwere found through the screening of combinatorial peptidelibraries for antifungal activity [12 13] The third representeda cathelicidin domain of a prophenin-2-like protein deducedfrom a cloned cDNA of the killer whale (Orcinus orca)(accession no XP004284013) Interestingly all three AcT-2-like peptides were associated with antimicrobial functionswith the former two being specifically against fungi
While AcT-2 is of relatively low potency when comparedwith many other amphibian skin antimicrobial peptidesmost if not all of the latter act through a mechanism ofmicrobial target cell membrane lysis and some are stronglyhemolytic [17ndash19] To achieve this form of nonspecific
The Scientific World Journal 7
antimicrobial action such peptides are generallymuch longerin amino acid chain length than AcT-2 [17 19] ThusAcT-2 may act upon a different and possibly intracellulartarget rather than by direct lytic action on the cytoplasmicmembranemdasha mode of action that has actually been pro-posed for some ldquoclassicalrdquo antimicrobial peptides as well [1719] In support of this proposal to some degree is the lack ofhemolytic activity of AcT-2 observed following its incubationwith horse erythrocytes (Figure 4(d))
In conclusion the novel amphibian skin tryptophyllinnamed AcT-2 described here has been demonstrated tohave both selective mammalian smooth muscle myotropiceffects and antimicrobial activitymdashfindings which add toour increasing knowledge of the biological effects of thisenigmatic family of amphibian skin peptides
Thenucleotide sequence ofAcT-2 from the skin secretionof Agalychnis callidryas has been deposited in the EMBLNucleotide Sequence Database under the accession codeHG710094
Conflict of Interests
The authors declare that they have no conflict of interests
Authorsrsquo Contribution
Lilin Ge and Peng Lyu contributed equally to this work
References
[1] B T Clarke ldquoThe natural history of amphibian skin secretionstheir normal functioning and potential medical applicationsrdquoBiological Reviews of the Cambridge Philosophical Society vol72 no 3 pp 365ndash379 1997
[2] V Erspamer ldquoBioactive secretions of the integumentrdquo inAmphibian Biologymdashvol 1 the Integument H Heatwole andG T Barthalmus Eds pp 179ndash350 Surrey Beatty amp SonsChipping Norton 1994
[3] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[4] J W Daly T F Spande and H M Garraffo ldquoAlkaloidsfrom amphibian skin a tabulation of over eight-hundredcompoundsrdquo Journal of Natural Products vol 68 no 10 pp1556ndash1575 2005
[5] F E Koehn ldquoHigh impact technologies for natural productsscreeningrdquo Progress in Drug Research vol 65 pp 176ndash210 2008
[6] R P Borris ldquoNatural products research perspectives from amajor pharmaceutical companyrdquo Journal of Ethnopharmacol-ogy vol 51 no 1ndash3 pp 29ndash38 1996
[7] GM Cragg andD J Newman ldquoNatural products a continuingsource of novel drug leadsrdquo Biochimica et Biophysica Acta vol1830 pp 3670ndash3695 2013
[8] T ChenD F OrrMOrsquoRourke et al ldquoPachymedusa dacnicolortryptophyllin-1 structural characterization pharmacologicalactivity and cloning of precursor cDNArdquo Regulatory Peptidesvol 117 no 1 pp 25ndash32 2004
[9] L Wang M Zhou T Chen B Walker and C Shaw ldquoPdT-2 anovelmyotropic Type-2 tryptophyllin from the skin secretion of
the Mexican giant leaf frog Pachymedusa dacnicolorrdquo Peptidesvol 30 no 8 pp 1557ndash1561 2009
[10] RWang T ChenM Zhou LWang andC Shaw ldquoPsT-1 a newtryptophyllin peptide from the skin secretion of Waxy MonkeyLeaf Frog Phyllomedusa sauvageirdquoRegulatory Peptides vol 184pp 14ndash21 2013
[11] M J Tyler D J M Stone and J H Bowie ldquoA novel method forthe release and collection of dermal glandular secretions fromthe skin of frogsrdquo Journal of Pharmacological and ToxicologicalMethods vol 28 no 4 pp 199ndash200 1992
[12] D I Chan E J Prenner and H J Vogel ldquoTryptophan- andarginine-rich antimicrobial peptides structures and mecha-nisms of actionrdquo Biochimica et Biophysica Acta vol 1758 no9 pp 1184ndash1202 2006
[13] A Munoz B Lopez-Garcıa and J F Marcos ldquoStudies onthe mode of action of the antifungal hexapeptide PAF26rdquoAntimicrobial Agents and Chemotherapy vol 50 no 11 pp3847ndash3855 2006
[14] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[15] J G Walls Red-Eyes and Other Leaf-Frogs TFH Publications1996
[16] J M Conlon J Kolodziejek and N Nowotny ldquoAntimicrobialpeptides from ranid frogs taxonomic and phylogeneticmarkersand a potential source of new therapeutic agentsrdquo Biochimica etBiophysica Acta vol 1696 no 1 pp 1ndash14 2004
[17] J M Conlon ldquoThe contribution of skin antimicrobial peptidesto the system of innate immunity in anuransrdquo Cell and TissueResearch vol 343 no 1 pp 201ndash212 2011
[18] R M Epand and H J Vogel ldquoDiversity of antimicrobial pep-tides and their mechanisms of actionrdquo Biochimica et BiophysicaActa vol 1462 no 1-2 pp 11ndash28 1999
[19] M Simmaco G Mignogna and D Barra ldquoAntimicrobial pep-tides from amphibian skin what do they tell usrdquo Biopolymersvol 47 no 6 pp 435ndash450 1998
---------1--------- ---- --------2 ---------- - - - - - --3 4--AcT-2 M ASV KKS VFLVLFLGF ISISFC DEEKR EDDEEGNEREEKREIHEEG N Q E E R R GMRPPW F GK K
PdT-2 M DFL RKS LFLVLFLGF VSISFC DEEKR EDDDENHGSEEKREIHEEG N Q E E R R DMSPPW H GK K
PdT-1 M NFL KKS LFLVLFLGF VSISFC DEEKR QDDDEGNEREEKKEIQEDG N Q E E R R DKPPAW VP GK -
larr larr larr larrrarr rarr rarr rarr
(c)
Figure 2 Comparison of the primary structure of AcT-2 with those of similar peptides PAF26 and combi-1 are synthetic antifungal peptidesisolated from combinatorial peptide libraries [12 13] Prophenin-2-like is a region of an antimicrobial polypeptide cathelicidin sequencedfrom a genomic DNA template of the killer whale (Orcinus orca) (accession no XP004284013) Conserved amino acid residues are shadedblack and consensus residues are shaded grey (a) Nucleotide and translated open-reading frame amino acid sequence of a cloned cDNAencoding the biosynthetic precursor of AcT-2 The putative signal peptide is double-underlined the mature peptide is single-underlinedand the stop codon is indicated by an asterisk (b) Alignments of complete open-reading frame amino acid sequences of AcT-2 precursor-encoding cDNA with the two top hits obtained following BLAST analysis using the NCBI portal PdT-1 and PdT-2 represent Pachymedusadacnicolor tryptophyllin-1 and -2 respectively Conserved amino acids are shaded black and consensus amino acids are shaded grey Gapshave been inserted tomaximize alignments (1) Putative signal peptide domain (2) Acidic spacer peptide domain (3)Mature peptide domain(4) C-terminal processing site (c)
Figure 3 Dose-response curves obtained using synthetic AcT-2 on rat smooth muscle preparations (a) Effect of the peptide on tail arterysmoothmuscle preparations expressed asmeans and standard errors (119899 = 6) EC
50was found to be 51 nM (b) Effect of the peptide on urinary
bladder smooth muscle preparations expressed as means and standard errors (119899 = 6) EC50was found to be 93 120583M
unusual profile for previously reported typical amphibianskin cationic amphipathic 120572-helical antimicrobial peptidesAlso worthy of note was the virtual lack of hemolytic activityof AcT-2 even up to the highest concentration (512mgL)employed (Figure 4(d))
4 Discussion
Central-American red-eyed leaf frogs (Agalychnis callidryas)are probably the most universally recognized frogs in theworld having been used extensively by advertising agencies
Figure 4Minimal inhibitory concentration (MIC) curves obtained following incubation of synthetic AcT-2 with S aureus (a) E coli (b) andC albicans (c) The MIC value for each microorganism is indicated in respective panels by asterisks (d) The hemolytic activity of syntheticAcT-2 The arrow shows the highest concentration of peptide (512mgL) which was the only concentration at which hemolysis was observed(85)
due to their strikingly beautiful colors Although they areone of the most widely available of the phyllomedusine leaffrogs studies on their defensive skin secretion peptides havenot been carried out with the same degree of focus that hasbeen given to many of their more obscure relatives [2 14]Red-eyed leaf frogs are most vividly colored with vibrantlime-green dorsal surfaces white ventral surfaces cream andcobalt-blue striped sides and bright orange feet Despite thisfrogs are difficult to see as they press themselves againstleaves and pull their limbs tightly towards their bodies thusbecoming a green blob on the leaf surface Their brightcoloration however is only present during the day when thefrogs are largely inactive while during the hours of darknesswhenever the frogs are most active their colors are muchdrabber favouring shades of grey and brown [15]
The potent cocktails of defensive molecules presentin skin secretions effectively constitutes their front-line ofdefense against predators [1 2] To date several peptideswith a range of biological effects have been reported fromthese skin secretions and these include both antimicrobialand pharmacologically active peptides [1 2 16] HoweverAcT-2 described here represents the first skin secretiontryptophyllin described from this species despite severalhaving been reported from Phyllomedusa species over thepast decades [2] A recent report byWang et al [9] describeda simple rational scheme to classify the rather structurally-heterogenous group of amphibian skin peptides collectivelynamed tryptophyllins [9] In this peptideswere classified intoone of three groups named T-1 T-2 and T-3 tryptophyllinsbased on some common structural features On this basisAcT-2 shows the features of a type-2 (T-2) tryptophyllin incommon with PdT-2 [9] as both possess an internal -PW-sequence motif (Figure 2(c)) However despite the presence
of this common internal structural feature AcT-2 and PdT-2 display radically different potencies in stimulating thecontraction of rat uterus smooth muscle (EC
50values AcT-2
= 93 120583M PdT-2 = 4 nM) indicating that other features of theprimary structure influence their receptor interactions [9]However AcT-2 was found to possess a most potent arterialsmooth muscle relaxant effect (EC
50= 51 nM)mdashan effect not
observed with PdT-2 [9] The smooth muscle pharmacol-ogy and molecular targets for these Type-2 tryptophyllinsin mammalian tissues obviously warrant further in-depthinvestigation
As demonstrated in this study AcT-2 was unexpectedlyfound to possess antimicrobial activity albeit relatively lowpotency using three model test microorganisms with theorder of sensitivity being C albicans gt S aureus gt E coli orin other words yeast gt Gram-positive bacterium gt Gram-negative bacterium Related to this observation was thatfollowing database interrogation with the primary structureof AcT-2 three peptides were found which displayed somedegree of structural identity (Figure 2(a)) Two of thesenamed PAF26 and combi-1 were synthetic in nature andwere found through the screening of combinatorial peptidelibraries for antifungal activity [12 13] The third representeda cathelicidin domain of a prophenin-2-like protein deducedfrom a cloned cDNA of the killer whale (Orcinus orca)(accession no XP004284013) Interestingly all three AcT-2-like peptides were associated with antimicrobial functionswith the former two being specifically against fungi
While AcT-2 is of relatively low potency when comparedwith many other amphibian skin antimicrobial peptidesmost if not all of the latter act through a mechanism ofmicrobial target cell membrane lysis and some are stronglyhemolytic [17ndash19] To achieve this form of nonspecific
The Scientific World Journal 7
antimicrobial action such peptides are generallymuch longerin amino acid chain length than AcT-2 [17 19] ThusAcT-2 may act upon a different and possibly intracellulartarget rather than by direct lytic action on the cytoplasmicmembranemdasha mode of action that has actually been pro-posed for some ldquoclassicalrdquo antimicrobial peptides as well [1719] In support of this proposal to some degree is the lack ofhemolytic activity of AcT-2 observed following its incubationwith horse erythrocytes (Figure 4(d))
In conclusion the novel amphibian skin tryptophyllinnamed AcT-2 described here has been demonstrated tohave both selective mammalian smooth muscle myotropiceffects and antimicrobial activitymdashfindings which add toour increasing knowledge of the biological effects of thisenigmatic family of amphibian skin peptides
Thenucleotide sequence ofAcT-2 from the skin secretionof Agalychnis callidryas has been deposited in the EMBLNucleotide Sequence Database under the accession codeHG710094
Conflict of Interests
The authors declare that they have no conflict of interests
Authorsrsquo Contribution
Lilin Ge and Peng Lyu contributed equally to this work
References
[1] B T Clarke ldquoThe natural history of amphibian skin secretionstheir normal functioning and potential medical applicationsrdquoBiological Reviews of the Cambridge Philosophical Society vol72 no 3 pp 365ndash379 1997
[2] V Erspamer ldquoBioactive secretions of the integumentrdquo inAmphibian Biologymdashvol 1 the Integument H Heatwole andG T Barthalmus Eds pp 179ndash350 Surrey Beatty amp SonsChipping Norton 1994
[3] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[4] J W Daly T F Spande and H M Garraffo ldquoAlkaloidsfrom amphibian skin a tabulation of over eight-hundredcompoundsrdquo Journal of Natural Products vol 68 no 10 pp1556ndash1575 2005
[5] F E Koehn ldquoHigh impact technologies for natural productsscreeningrdquo Progress in Drug Research vol 65 pp 176ndash210 2008
[6] R P Borris ldquoNatural products research perspectives from amajor pharmaceutical companyrdquo Journal of Ethnopharmacol-ogy vol 51 no 1ndash3 pp 29ndash38 1996
[7] GM Cragg andD J Newman ldquoNatural products a continuingsource of novel drug leadsrdquo Biochimica et Biophysica Acta vol1830 pp 3670ndash3695 2013
[8] T ChenD F OrrMOrsquoRourke et al ldquoPachymedusa dacnicolortryptophyllin-1 structural characterization pharmacologicalactivity and cloning of precursor cDNArdquo Regulatory Peptidesvol 117 no 1 pp 25ndash32 2004
[9] L Wang M Zhou T Chen B Walker and C Shaw ldquoPdT-2 anovelmyotropic Type-2 tryptophyllin from the skin secretion of
the Mexican giant leaf frog Pachymedusa dacnicolorrdquo Peptidesvol 30 no 8 pp 1557ndash1561 2009
[10] RWang T ChenM Zhou LWang andC Shaw ldquoPsT-1 a newtryptophyllin peptide from the skin secretion of Waxy MonkeyLeaf Frog Phyllomedusa sauvageirdquoRegulatory Peptides vol 184pp 14ndash21 2013
[11] M J Tyler D J M Stone and J H Bowie ldquoA novel method forthe release and collection of dermal glandular secretions fromthe skin of frogsrdquo Journal of Pharmacological and ToxicologicalMethods vol 28 no 4 pp 199ndash200 1992
[12] D I Chan E J Prenner and H J Vogel ldquoTryptophan- andarginine-rich antimicrobial peptides structures and mecha-nisms of actionrdquo Biochimica et Biophysica Acta vol 1758 no9 pp 1184ndash1202 2006
[13] A Munoz B Lopez-Garcıa and J F Marcos ldquoStudies onthe mode of action of the antifungal hexapeptide PAF26rdquoAntimicrobial Agents and Chemotherapy vol 50 no 11 pp3847ndash3855 2006
[14] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[15] J G Walls Red-Eyes and Other Leaf-Frogs TFH Publications1996
[16] J M Conlon J Kolodziejek and N Nowotny ldquoAntimicrobialpeptides from ranid frogs taxonomic and phylogeneticmarkersand a potential source of new therapeutic agentsrdquo Biochimica etBiophysica Acta vol 1696 no 1 pp 1ndash14 2004
[17] J M Conlon ldquoThe contribution of skin antimicrobial peptidesto the system of innate immunity in anuransrdquo Cell and TissueResearch vol 343 no 1 pp 201ndash212 2011
[18] R M Epand and H J Vogel ldquoDiversity of antimicrobial pep-tides and their mechanisms of actionrdquo Biochimica et BiophysicaActa vol 1462 no 1-2 pp 11ndash28 1999
[19] M Simmaco G Mignogna and D Barra ldquoAntimicrobial pep-tides from amphibian skin what do they tell usrdquo Biopolymersvol 47 no 6 pp 435ndash450 1998
Figure 4Minimal inhibitory concentration (MIC) curves obtained following incubation of synthetic AcT-2 with S aureus (a) E coli (b) andC albicans (c) The MIC value for each microorganism is indicated in respective panels by asterisks (d) The hemolytic activity of syntheticAcT-2 The arrow shows the highest concentration of peptide (512mgL) which was the only concentration at which hemolysis was observed(85)
due to their strikingly beautiful colors Although they areone of the most widely available of the phyllomedusine leaffrogs studies on their defensive skin secretion peptides havenot been carried out with the same degree of focus that hasbeen given to many of their more obscure relatives [2 14]Red-eyed leaf frogs are most vividly colored with vibrantlime-green dorsal surfaces white ventral surfaces cream andcobalt-blue striped sides and bright orange feet Despite thisfrogs are difficult to see as they press themselves againstleaves and pull their limbs tightly towards their bodies thusbecoming a green blob on the leaf surface Their brightcoloration however is only present during the day when thefrogs are largely inactive while during the hours of darknesswhenever the frogs are most active their colors are muchdrabber favouring shades of grey and brown [15]
The potent cocktails of defensive molecules presentin skin secretions effectively constitutes their front-line ofdefense against predators [1 2] To date several peptideswith a range of biological effects have been reported fromthese skin secretions and these include both antimicrobialand pharmacologically active peptides [1 2 16] HoweverAcT-2 described here represents the first skin secretiontryptophyllin described from this species despite severalhaving been reported from Phyllomedusa species over thepast decades [2] A recent report byWang et al [9] describeda simple rational scheme to classify the rather structurally-heterogenous group of amphibian skin peptides collectivelynamed tryptophyllins [9] In this peptideswere classified intoone of three groups named T-1 T-2 and T-3 tryptophyllinsbased on some common structural features On this basisAcT-2 shows the features of a type-2 (T-2) tryptophyllin incommon with PdT-2 [9] as both possess an internal -PW-sequence motif (Figure 2(c)) However despite the presence
of this common internal structural feature AcT-2 and PdT-2 display radically different potencies in stimulating thecontraction of rat uterus smooth muscle (EC
50values AcT-2
= 93 120583M PdT-2 = 4 nM) indicating that other features of theprimary structure influence their receptor interactions [9]However AcT-2 was found to possess a most potent arterialsmooth muscle relaxant effect (EC
50= 51 nM)mdashan effect not
observed with PdT-2 [9] The smooth muscle pharmacol-ogy and molecular targets for these Type-2 tryptophyllinsin mammalian tissues obviously warrant further in-depthinvestigation
As demonstrated in this study AcT-2 was unexpectedlyfound to possess antimicrobial activity albeit relatively lowpotency using three model test microorganisms with theorder of sensitivity being C albicans gt S aureus gt E coli orin other words yeast gt Gram-positive bacterium gt Gram-negative bacterium Related to this observation was thatfollowing database interrogation with the primary structureof AcT-2 three peptides were found which displayed somedegree of structural identity (Figure 2(a)) Two of thesenamed PAF26 and combi-1 were synthetic in nature andwere found through the screening of combinatorial peptidelibraries for antifungal activity [12 13] The third representeda cathelicidin domain of a prophenin-2-like protein deducedfrom a cloned cDNA of the killer whale (Orcinus orca)(accession no XP004284013) Interestingly all three AcT-2-like peptides were associated with antimicrobial functionswith the former two being specifically against fungi
While AcT-2 is of relatively low potency when comparedwith many other amphibian skin antimicrobial peptidesmost if not all of the latter act through a mechanism ofmicrobial target cell membrane lysis and some are stronglyhemolytic [17ndash19] To achieve this form of nonspecific
The Scientific World Journal 7
antimicrobial action such peptides are generallymuch longerin amino acid chain length than AcT-2 [17 19] ThusAcT-2 may act upon a different and possibly intracellulartarget rather than by direct lytic action on the cytoplasmicmembranemdasha mode of action that has actually been pro-posed for some ldquoclassicalrdquo antimicrobial peptides as well [1719] In support of this proposal to some degree is the lack ofhemolytic activity of AcT-2 observed following its incubationwith horse erythrocytes (Figure 4(d))
In conclusion the novel amphibian skin tryptophyllinnamed AcT-2 described here has been demonstrated tohave both selective mammalian smooth muscle myotropiceffects and antimicrobial activitymdashfindings which add toour increasing knowledge of the biological effects of thisenigmatic family of amphibian skin peptides
Thenucleotide sequence ofAcT-2 from the skin secretionof Agalychnis callidryas has been deposited in the EMBLNucleotide Sequence Database under the accession codeHG710094
Conflict of Interests
The authors declare that they have no conflict of interests
Authorsrsquo Contribution
Lilin Ge and Peng Lyu contributed equally to this work
References
[1] B T Clarke ldquoThe natural history of amphibian skin secretionstheir normal functioning and potential medical applicationsrdquoBiological Reviews of the Cambridge Philosophical Society vol72 no 3 pp 365ndash379 1997
[2] V Erspamer ldquoBioactive secretions of the integumentrdquo inAmphibian Biologymdashvol 1 the Integument H Heatwole andG T Barthalmus Eds pp 179ndash350 Surrey Beatty amp SonsChipping Norton 1994
[3] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[4] J W Daly T F Spande and H M Garraffo ldquoAlkaloidsfrom amphibian skin a tabulation of over eight-hundredcompoundsrdquo Journal of Natural Products vol 68 no 10 pp1556ndash1575 2005
[5] F E Koehn ldquoHigh impact technologies for natural productsscreeningrdquo Progress in Drug Research vol 65 pp 176ndash210 2008
[6] R P Borris ldquoNatural products research perspectives from amajor pharmaceutical companyrdquo Journal of Ethnopharmacol-ogy vol 51 no 1ndash3 pp 29ndash38 1996
[7] GM Cragg andD J Newman ldquoNatural products a continuingsource of novel drug leadsrdquo Biochimica et Biophysica Acta vol1830 pp 3670ndash3695 2013
[8] T ChenD F OrrMOrsquoRourke et al ldquoPachymedusa dacnicolortryptophyllin-1 structural characterization pharmacologicalactivity and cloning of precursor cDNArdquo Regulatory Peptidesvol 117 no 1 pp 25ndash32 2004
[9] L Wang M Zhou T Chen B Walker and C Shaw ldquoPdT-2 anovelmyotropic Type-2 tryptophyllin from the skin secretion of
the Mexican giant leaf frog Pachymedusa dacnicolorrdquo Peptidesvol 30 no 8 pp 1557ndash1561 2009
[10] RWang T ChenM Zhou LWang andC Shaw ldquoPsT-1 a newtryptophyllin peptide from the skin secretion of Waxy MonkeyLeaf Frog Phyllomedusa sauvageirdquoRegulatory Peptides vol 184pp 14ndash21 2013
[11] M J Tyler D J M Stone and J H Bowie ldquoA novel method forthe release and collection of dermal glandular secretions fromthe skin of frogsrdquo Journal of Pharmacological and ToxicologicalMethods vol 28 no 4 pp 199ndash200 1992
[12] D I Chan E J Prenner and H J Vogel ldquoTryptophan- andarginine-rich antimicrobial peptides structures and mecha-nisms of actionrdquo Biochimica et Biophysica Acta vol 1758 no9 pp 1184ndash1202 2006
[13] A Munoz B Lopez-Garcıa and J F Marcos ldquoStudies onthe mode of action of the antifungal hexapeptide PAF26rdquoAntimicrobial Agents and Chemotherapy vol 50 no 11 pp3847ndash3855 2006
[14] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[15] J G Walls Red-Eyes and Other Leaf-Frogs TFH Publications1996
[16] J M Conlon J Kolodziejek and N Nowotny ldquoAntimicrobialpeptides from ranid frogs taxonomic and phylogeneticmarkersand a potential source of new therapeutic agentsrdquo Biochimica etBiophysica Acta vol 1696 no 1 pp 1ndash14 2004
[17] J M Conlon ldquoThe contribution of skin antimicrobial peptidesto the system of innate immunity in anuransrdquo Cell and TissueResearch vol 343 no 1 pp 201ndash212 2011
[18] R M Epand and H J Vogel ldquoDiversity of antimicrobial pep-tides and their mechanisms of actionrdquo Biochimica et BiophysicaActa vol 1462 no 1-2 pp 11ndash28 1999
[19] M Simmaco G Mignogna and D Barra ldquoAntimicrobial pep-tides from amphibian skin what do they tell usrdquo Biopolymersvol 47 no 6 pp 435ndash450 1998
antimicrobial action such peptides are generallymuch longerin amino acid chain length than AcT-2 [17 19] ThusAcT-2 may act upon a different and possibly intracellulartarget rather than by direct lytic action on the cytoplasmicmembranemdasha mode of action that has actually been pro-posed for some ldquoclassicalrdquo antimicrobial peptides as well [1719] In support of this proposal to some degree is the lack ofhemolytic activity of AcT-2 observed following its incubationwith horse erythrocytes (Figure 4(d))
In conclusion the novel amphibian skin tryptophyllinnamed AcT-2 described here has been demonstrated tohave both selective mammalian smooth muscle myotropiceffects and antimicrobial activitymdashfindings which add toour increasing knowledge of the biological effects of thisenigmatic family of amphibian skin peptides
Thenucleotide sequence ofAcT-2 from the skin secretionof Agalychnis callidryas has been deposited in the EMBLNucleotide Sequence Database under the accession codeHG710094
Conflict of Interests
The authors declare that they have no conflict of interests
Authorsrsquo Contribution
Lilin Ge and Peng Lyu contributed equally to this work
References
[1] B T Clarke ldquoThe natural history of amphibian skin secretionstheir normal functioning and potential medical applicationsrdquoBiological Reviews of the Cambridge Philosophical Society vol72 no 3 pp 365ndash379 1997
[2] V Erspamer ldquoBioactive secretions of the integumentrdquo inAmphibian Biologymdashvol 1 the Integument H Heatwole andG T Barthalmus Eds pp 179ndash350 Surrey Beatty amp SonsChipping Norton 1994
[3] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[4] J W Daly T F Spande and H M Garraffo ldquoAlkaloidsfrom amphibian skin a tabulation of over eight-hundredcompoundsrdquo Journal of Natural Products vol 68 no 10 pp1556ndash1575 2005
[5] F E Koehn ldquoHigh impact technologies for natural productsscreeningrdquo Progress in Drug Research vol 65 pp 176ndash210 2008
[6] R P Borris ldquoNatural products research perspectives from amajor pharmaceutical companyrdquo Journal of Ethnopharmacol-ogy vol 51 no 1ndash3 pp 29ndash38 1996
[7] GM Cragg andD J Newman ldquoNatural products a continuingsource of novel drug leadsrdquo Biochimica et Biophysica Acta vol1830 pp 3670ndash3695 2013
[8] T ChenD F OrrMOrsquoRourke et al ldquoPachymedusa dacnicolortryptophyllin-1 structural characterization pharmacologicalactivity and cloning of precursor cDNArdquo Regulatory Peptidesvol 117 no 1 pp 25ndash32 2004
[9] L Wang M Zhou T Chen B Walker and C Shaw ldquoPdT-2 anovelmyotropic Type-2 tryptophyllin from the skin secretion of
the Mexican giant leaf frog Pachymedusa dacnicolorrdquo Peptidesvol 30 no 8 pp 1557ndash1561 2009
[10] RWang T ChenM Zhou LWang andC Shaw ldquoPsT-1 a newtryptophyllin peptide from the skin secretion of Waxy MonkeyLeaf Frog Phyllomedusa sauvageirdquoRegulatory Peptides vol 184pp 14ndash21 2013
[11] M J Tyler D J M Stone and J H Bowie ldquoA novel method forthe release and collection of dermal glandular secretions fromthe skin of frogsrdquo Journal of Pharmacological and ToxicologicalMethods vol 28 no 4 pp 199ndash200 1992
[12] D I Chan E J Prenner and H J Vogel ldquoTryptophan- andarginine-rich antimicrobial peptides structures and mecha-nisms of actionrdquo Biochimica et Biophysica Acta vol 1758 no9 pp 1184ndash1202 2006
[13] A Munoz B Lopez-Garcıa and J F Marcos ldquoStudies onthe mode of action of the antifungal hexapeptide PAF26rdquoAntimicrobial Agents and Chemotherapy vol 50 no 11 pp3847ndash3855 2006
[14] V Erspamer G Falconieri Erspamer and J M Cei ldquoActivepeptides in the skins of two hundred and thirty Americanamphibian speciesrdquo Comparative Biochemistry and PhysiologyC vol 85 no 1 pp 125ndash137 1986
[15] J G Walls Red-Eyes and Other Leaf-Frogs TFH Publications1996
[16] J M Conlon J Kolodziejek and N Nowotny ldquoAntimicrobialpeptides from ranid frogs taxonomic and phylogeneticmarkersand a potential source of new therapeutic agentsrdquo Biochimica etBiophysica Acta vol 1696 no 1 pp 1ndash14 2004
[17] J M Conlon ldquoThe contribution of skin antimicrobial peptidesto the system of innate immunity in anuransrdquo Cell and TissueResearch vol 343 no 1 pp 201ndash212 2011
[18] R M Epand and H J Vogel ldquoDiversity of antimicrobial pep-tides and their mechanisms of actionrdquo Biochimica et BiophysicaActa vol 1462 no 1-2 pp 11ndash28 1999
[19] M Simmaco G Mignogna and D Barra ldquoAntimicrobial pep-tides from amphibian skin what do they tell usrdquo Biopolymersvol 47 no 6 pp 435ndash450 1998
![Current Medicinal Chemistry · development of novel treatment options and alternative antimicrobial therapies [2]. Therefore, the development of new effective antimicrobial agents](https://static.documents.pub/doc/80x56/6047d801b50d2932fe4da5b0/current-medicinal-chemistry-development-of-novel-treatment-options-and-alternative.jpg)
