Peptidomic analysis of skin secretions of the Mexican burrowing toad Rhinophrynus

dorsalis (Rhinophrynidae): insight into the origin of host-defense peptides within the

Pipidae and characterization of a proline-arginine-rich peptide

J. Michael Conlona*, Laure Guilhaudisb, Jérôme Leprincec, Laurent Coquetd,

Maria Luisa Mangonie, Samir Attoubf, Thierry Jouenned, Jay D. Kingg

eSAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of

Ulster, Coleraine BT52 1SA, U.K.

b UNIROUEN, INSA Rouen, CNRS, COBRA, Normandy University 76000 Rouen, France

cInserm UU1239, PRIMACEN, Institute for Research and Innovation in Biomedicine (IRIB),

Normandy University, 76000 Rouen, France

dCNRS UMR 6270, PISSARO, Institute for Research and Innovation in Biomedicine (IRIB),

Normandy University, 76000 Rouen, France

eDepartment of Biochemical Sciences Instituto Pasteur-Fondazione Cenci Bolognetti, ,

Sapienza University of Rome, Rome, Italy

fDepartment of Pharmacology, College of Medicine and Health Sciences, United Arab

Emirates University, Al Ain, United Arab Emirates

g Rare Species Conservatory Foundation, St. Louis, MO 63110, U.S.A.

*Corresponding author. m.conlon@ulster.ac.uk

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ABSTRACT

The Mexican burrowing toad Rhinophrynus dorsalis is the sole extant representative of the

Rhinophrynidae. United in the superfamily Pipoidea, the Rhinophrynidae is considered to be

the sister-group to the extant Pipidae which comprises Hymenochirus, Pipa,

Pseudhymenochirus and Xenopus. Cationic, α-helical host-defense peptides of the type found

in Hymenochirus, Pseudhymenochirus, and Xenopus species (hymenochirins,

pseudhymenochirins, magainins, and peptides related to PGLa, XPF, and CPF) were not

detected in norepinephrine-stimulated skin secretions of R. dorsalis. Skin secretions of

representatives of the genus Pipa also do not contain cationic α-helical host-defense peptides

which suggests, as the most parsimonious hypothesis, that the ability to produce such

peptides by frogs within the Pipidae family arose in the common ancestor of (Hymenochirus

+ Pseudhymenochirus) + Xenopus after divergence from the line of evolution leading to

extant Pipa species. Peptidomic analysis of the R. dorsalis secretions led to the isolation of

rhinophrynin-27, a proline-arginine-rich peptide with the primary structure

ELRLPEIARPVPEVLPARLPLPALPRN, together with rhinophrynin-33 containing the C-

terminal extension KMAKNQ. Rhinophrynin-27 shows limited structural similarity to the

porcine multifunctional peptide PR-39 but it lacks antimicrobial and cytotoxic activities. Like

PR-39, the peptide adopts a poly-L-proline helix but some changes in the circular dichroism

spectrum were observed in the presence of anionic sodium dodecylsulfate micelles consistent

with the stabilization of turn structures.

.

Key words: Frog skin; Rhinophrynidae, Pipidae, Host-defense, Antimicrobial, PR-39

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1. Introduction

The phylogenetically ancient family Pipidae comprises four genera: Pipa, currently 7

species established in South America, and Hymenochirus (4 species), Pseudhymenochirus (1

species), and Xenopus (29 species) established in sub-Saharan Africa [1]. Until relatively

recently, the diploid frog Xenopus tropicalis and the tetraploid frog Xenopus epitropicalis

were assigned to the separate genus Silurana but the monophyletic status of Xenopus +

Silurana is well established so that Silurana is now generally described as a sub-genus of

Xenopus [2-4]. The family Rhinophrynidae, which comprises a single species, the Mexican

burrowing toad Rhinophrynus dorsalis Duméril and Bibron, 1841, is considered to be sister-

group to the extant Pipidae and it has been proposed that the two lines of evolution diverged

around the time of separation of North America from West Africa following the breakup of

Pangaea (approximately 175 - 190 MYA) [5,6]. The families Pipidae and Rhinophyrinidae

are united in the superfamily Pipoidea and both the fossil record and molecular analysis

provide strong evidence for the monophyly of the clade [6,7].

Skin secretions of Hymenochirus boettgeri [8,9], Pseudhymenochirus merlini [10], and a

wide range of Xenopus species (reviewed in [11,12]) contain extensive arrays of cationic,

amphipathic α-helical peptides. These peptides display growth inhibitory activity against

bacteria and fungi and so may be described as “antimicrobial”. However, many also possess

immunomodulatory, anti-tumor, anti-viral, and insulin-releasing activities (reviewed in

[13,14]) so that they are better described as host-defense peptides (HDPs). Skin secretions

from frogs of the Xenopus genus have proved to be a particularly rich source of such

peptides and five families have been identified on the basis of limited structural similarity:

magainin, peptide glycine-leucine-amide (PGLa), xenopsin-precursor fragment (XPF),

derived from the post-translational processing of proxenopsin, and both caerulein-precursor

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fragment (CPF) and caerulein-precursor fragment-related peptide (CPF-RP) derived from the

post-translational processing of procaeruleins (reviewed in [11,15]). In contrast, cytotoxic

HDPs were not detected in skin secretions of Pipa pipa ([11], Pipa carvalhoi [16]) and Pipa

parva (J.M. Conlon, unpublished data),

The New world frog R. dorsalis is found in coastal lowland regions (from sea-level to

500 m) in a range of habitats, such as tropical dry forests, grasslands, thorn scrub, and

cultivated fields. Although relatively rare in Texas, the species is common and widespread in

Mexico and northern Central America and is listed as Species of Least Concern by the

International Union for Conservation of Nature (IUCN) Red List [17]. The animal is strictly

fossorial, only emerging from its underground burrow to reproduce after the first rains with

the result that it is rarely seen in the wild. The present study uses peptidomic analysis

(reversed-phase HLPC coupled with MALDI-TOF mass spectrometry and automated Edman

degradation) to investigate the occurrence of HDPs in norepinephrine-stimulated skin

secretions from R. dorsalis.

2. Experimental

2.1. Collection of skin secretions

All experiments with live animals were approved by the Gladys Porter Zoo Scientific

and Research Committee and were carried out by authorized investigators. Four adult R.

dorsalis frogs (1 male, 16 g body weight; 3 female, 20, 20, and 30 g body weight) were

collected in the grounds of Rio Grande City High School, Rio Grande City, TX and were

housed in a vivarium at the Gladys Porter Zoo, Brownsville, TX. 

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Each frog was injected via the dorsal lymph sac with norepinephrine hydrochloride

(40 nmol/g body weight) and placed in a solution (100 ml) of distilled water for 15 min. The

injection did not produce the kind of copious, milky skin secretions often seen when Xenopus

species are stimulated with norepinephrine but the collection solution became more turbid

and foamy. The frog was removed and the collection solution was acidified by addition of

trifluoroacetic acid (TFA) (1 ml) and immediately frozen for shipment to Ulster University.

The solutions containing the secretions from each frog were pooled and passed at a flow rate

of 2 ml/min through 6 Sep-Pak C-18 cartridges (Waters Associates, Milford, MA) connected

in series. Bound material was eluted with acetonitrile/ water/TFA (70.0:29.9:0.1, v/v/v) and

freeze-dried. The material was redissolved in 0.1% (v/v) TFA/water (2 ml).

2.2. Peptide purification

The pooled skin secretions from R. dorsalis, after partial purification on Sep-Pak

cartridges, were injected onto a semipreparative (1 cm x 25 cm) Vydac 218TP510 (C-18)

reversed-phase HPLC column (Grace, Deerfield, IL) equilibrated with 0.1% (v/v) TFA/water

at a flow rate of 2.0 ml/min. The concentration of acetonitrile in the eluting solvent was

raised to 21% (v/v) over 10 min and to 63% (v/v) over 60 min using linear gradients.

Absorbance was monitored at 214 nm and peak fractions were collected by hand. The

peptides within the peaks that were present in major abundance were subjected to further

purification. These components were purified to near homogeneity, as assessed by a

symmetrical peak shape and mass spectrometry, by chromatography on (1.0 cm x 25 cm)

Vydac 214TP510 (C-4) and (1.0 cm x 25 cm) Vydac 219TP510 (phenyl) columns. The

concentration of acetonitrile in the eluting solvent was raised from 7% to 35% over 50 min

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for the more hydrophilic components and from 21% to 49% over 50 min for the more

hydrophobic components. The flow rate was 2.0 ml/min.

2.3. Structural characterization

MALDI-TOF mass spectrometry was carried out using a Voyager DE-PRO

instrument (Applied Biosystems, Foster City, CA) that was operated in reflector mode with

delayed extraction and the accelerating voltage in the ion source was 20 kV. The instrument

was calibrated with peptides of known molecular mass in the 2 - 4 kDa range. The accuracy

of mass determinations was 0.02%. Spectra were recorded using both -cyano-4-

hydroxycinnamic acid and sinapinic acid as matrix solutions. The complete primary

structures of peptides in the mass range 1 - 4 kDa were determined by automated Edman

degradation using a model 494 Procise sequenator (Applied Biosystems). For larger

peptides/proteins (molecular mass > 4kDa), only the amino acid sequence at N-terminus

(residues 1-10) were determined in order to permit identification. Amino acid composition

analyses were performed by the University of Nebraska Medical Center Protein Structure

Core Facility (Omaha, NE).

2.4. Peptide synthesis

Rhinophrynin-27 (ELRLPEIARPVPEVLPARLPLPALPRN) was supplied at a purity

> 95% by Synpeptide Co., Ltd (Shanghai, China). Its identity and purity were confirmed by

electrospray mass spectrometry.

2.5. Antimicrobial and cytotoxicity assays

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Reference strains of microorganisms were purchased from the American Type Culture

Collection (Rockville, MD, USA). Minimum inhibitory concentrations (MIC) of

rhinophrynin-27 against reference strains of Staphylococcus epidermidis (ATCC 12228),

Bacillus megaterium (Bm11), Escherichia coli (ATCC 25922) and Candida parapsilosis

(ATCC 22019) were measured by standard microdilution methods [18,19] as previously

described [20]. Hemolyic activity was determined by incubation of washed erythrocytes (2 x

107 cells) from male NIH male Swiss mice (Harlan Ltd, Bicester, UK) with rhinophrynin-27 (

31.3 - 500 μM ) for 60 min at 37 oC as previously described [8]. Cytotoxicity of

rhinophrynin-27 against human non-small cell lung adenocarcinoma A549 cells was

measured as previously described [21]. The effects of the peptide (1 - 100 μM) on cell

viability were determined by measurement of ATP concentrations using a CellTiter-Glo

Luminescent Cell Viability assay (Promega Corporation, Madison, WI, USA). All animal

experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act

1986 and EU Directive 2010/63EU for animal experiments.

2.6. CD spectra

Spectra were obtained using a MOS-500 Circular Dichroism Spectrometer (Bio-Logic,

Claix, France). Data points were collected from 260 nm to 185 nm, with an integration time

of 2 s per point and a step size of 1 nm, using a 1.0 mm path length rectangular quartz cell.

Measurements were carried out at room temperature, 20°C and 5.5°C. Rhinophrynin-27 was

dissolved in water, in 2,2,2-trifluoroethanol (TFE)-water (25% and 50%, v/v), in 20 mM

sodium dodecyl sulfate (SDS) aqueous solution, and in 20 mM dodecylphosphocholine

(DPC) aqueous solution at a final concentration of 0.18 - 0.21 mg/ml. The concentration of

20 mM for detergents was chosen to ensure micelle formation. For each spectrum, three scans

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were accumulated and then averaged. The baseline was obtained by recording a spectrum of

the solvent, and the mean residue molar ellipticity ([θ]MRE), deg cm2 dmol−1, was calculated

from the observed ellipticity after baseline correction. The -helical content was estimated by

using the Forood formula [22].

3. Results

3.1. Purification of the peptides

The pooled skin secretions from R. dorsalis, after partial purification on Sep-Pak C-18

cartridges, were chromatographed on a Vydac C-18 semipreparative reversed-phase HPLC

column (Fig. 1). The prominent peaks designated 1 - 19 were collected by hand and subjected

to further purification. The major components present in each peak were purified to near

homogeneity, as assessed by a symmetrical peak shape and mass spectrometry, by further

chromatography on semipreparative Vydac C-4 and Vydac phenyl columns. The

methodology is illustrated by the purification of rhinophrynin-27 (Fig. 2).

3.2. Structural characterization

The molecular masses of the components purified from R. dorsalis skin secretions are

shown in Table 1. The compounds from peaks 1-4, 7-9, and 15 with masses < 600 kDa were

shown by Edman degradation and amino acid composition analysis not to be peptides. The

nature of these substances, which are present in relatively high concentrations in the

secretions, remains to be determined. The complete primary structures of the peptides present

in peaks 5, 6, 12, and 13 were determined by Edman degradation. A BLAST search (National

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Center for Biotechnology Information, Bethesda, MD, USA) indicated that the structurally

related peptides in peaks 5 and 6 may represent fragments of the α-chain of the structural

protein laminin. The peptides in peaks 12 and 13 are structurally related peptides with 33 and

27 amino acid residues respectively that are rich in arginine and proline residues and have

been termed rhinophrynin-33 and rhinophrynin-27. The molecular masses of these peptides

determined by MALDI-TOF mass spectrometry are consistent with their proposed structures

(Table 1). A minor component in peak 12 was provisionally identified as a fragment of a zinc

finger protein.

Peaks 10-11 and 15-19 (Fig. 1) contained peptides with substantially higher molecular

masses than those of the host-defense peptides generally found in skin secretions of frogs

from the Pipidae family (2 - 4 kDa). When skin secretions from the dodecaploid frog

Xenopus ruwenzoriensis were chromatographed on a Vydac C-18 semipreparative HPLC

column under the same conditions shown in Fig. 1, the extensive array of host-defense

peptides belonging to the magainin, PGLa, XPF, CPF, and CPF-RP families were eluted with

retention times between 36 and 65 min [12]. Similarly, the host-defense peptides in H.

boettgeri skin secretions belonging to the hymenochirin family and in P. merlini skin

secretions belonging to the hymenochirin and pseudhymenochirin families were eluted under

the same conditions with retention times between 43 and 68 min [10]. The components in R.

dorsalis skin secretions with retention times between 40 and 68 min had molecular masses

>10 kDa. The amino acid sequence YRTVYRCSTA… at the N-terminus of the most

abundant component in this region of the chromatogram (peak 18) did not show sufficient

sequence identity with previously described proteins to permit identification. It is concluded,

therefore, that the type of cationic, α-helical host-defense peptides produced in the skins of

representatives of the Hymenochirus, Peudhymenochirus, and Xenopus genera are either

absent from R. dorsalis skin secretions or present only in very low concentration.

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3.3. Antimicrobial and cytotoxic activities

Rhinophrynin-27 produced < 5% hemolysis during a 60 min incubation with freshly-

prepared mouse erythrocytes at concentrations up to 500 μM and showed <5% cytolytic

activity against human non-small cell lung adenocarcinoma A549 cells at concentrations up

to 100 μM (data not shown). Rhinophrynin-27 did not significantly inhibit the growth of the

Gram-negative bacterium E. coli, the Gram-positive bacteria S. epidermidis and B.

megaterium, and the opportunist yeast pathogen C. parapsilosis at concentrations up to 128

μM.

3.4. Conformational analysis

In water, the CD spectrum of rhinophrynin-27 exhibited a strong negative band at 198 nm

with a slight shoulder around 225 nm (Fig. 3). This spectrum resembles those of other proline

rich peptides that have been reported to adopt a left handed polyproline type II (PPII) helical

structure [23-27]. The essential features of this structure are a strong negative band in the

vicinity of 200 nm and a weak positive band around 220 nm. In contrast, disordered

structures tend to exhibit a negative peak around 195 nm, and a positive peak around 220 nm,

is absent. Although a positive peak around 220 nm was not observed in the spectrum of

rhinophrynin-27, the strong intensity of the negative band as well as its high proline content

(26%) suggest the presence of a PPII conformation in the peptide. Similar conclusions were

drawn from CD analyses of several Pro-rich peptides, for which it was reported that the

absence of the weak positive peak was attributable to a relative small number of proline

residues or to deviation from the ideal PP-II structure [27-29].

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To determine whether the solution environment played a role in the conformation of the

peptide, additional CD spectra were recorded in the secondary structure-inducing solvent

TFE and in two membrane-mimetic environments, SDS and DPC micelles (Fig.3).

Zwitterionic detergent micelles such as DPC are used to mimic eukaryote membranes while

the negatively charged SDS micelles resemble bacterial membranes. The CD spectrum of

rhinophyrinin-27 in the presence of DPC micelles was very similar to the one obtained in

water (data not shown). In the presence of TFE or SDS micelles, the dominant negative band

shifted to 199 nm and decreased in intensity while the weak shoulder around 225 nm

increased in intensity. The low ratio of the 222 nm:208 nm band intensity excluded the

possibility of any appreciable -helical structural content. In support of this, the direct

calculation of the -helical content using the Forood formula [22] did not reach 15% even in

the presence of 50% TFE. This suggests that in the presence of TFE or SDS micelles

rhinophrynin-27 adopts a conformation composed of a PP-II helix and turn structures.

The effect of temperature was examined to confirm the presence of a polyproline helical

structure as it has been shown that PII propensity is observed to decrease with an increase in

temperature [24,27,30]. In water and in the presence of DPC micelles, an increase in

magnitude of the peak around 200 nm was observed with the decrease of temperature as

expected for a PPII structure (Figs 4A and B). In contrast, in the presence of SDS micelles, a

small overall decrease of intensity was observed with lowering of temperature (Fig. 4C). This

unexpected behavior could be due to the additional presence of turns stabilized by the

interaction of rhinophrynin-27 with SDS micelles.

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Discussion

The present study has provided insight into the origin of the extensive array of cationic,

α-helical peptides present in skin secretions of certain species within the family Pipidae.

Phylogenetic relationships among the Pipidaee are not fully resolved but it is generally

accepted on the basis of morphological and molecular evidence that Hymenochirus +

Pseudhymenochirus form a clade with Pipa as sister-group to the combined assemblage of

Xenopus + (Hymenochirus + Pseudhymenochirus) [6, 31-33]. The alternative hypothesis that

Pipa + Hymenochirus + Pseudhymenochirus form a monophyletic clade [34, 35] has been

rejected. Cationic, α-helical peptides of the kind synthesized by species belonging to the

Hymenochirus, Pseudhymenochirus, and Xenopus genera were either absent from R. dorsalis

skin secretions or were present only in very low concentrations. In the light of the absence of

such peptides in skin secretions of three species within the genus Pipa [11,16], a number of

possible scenarios may be proposed a priori to account for the observed distribution of HDPs

within the Pipoidea. The ability to synthesize HDPs arose in (A) the common ancestor of the

Rhinophrynidae and the Pipidae but was lost in the lines leading to the extant Pipa and

Rhinophrynus species, (B) the common ancestor of the Pipidae after divergence from

Rhinophrynidae but was lost in the line leading to the extant Pipa species, (C) the common

ancestor of Hymenochirus, Pseudhymenochirus and Xenopus, after divergence from the line

leading to the extant Pipa species, and (D) independently in the lines leading to extant

Xenopus and to the (Hymenochirus + Pseudhymenochirus) species. The most parsimonious

hypothesis to explain the distribution of the HDPs is hypothesis C, that is the ability to

synthesize such peptides arose in common ancestor of the present-day African species

belonging to the genera Hymenochirus, Pseudhymenochirus and Xenopus after divergence

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from the lines of evolution leading to the extant New World species that belong to the genera

Rhinophyrinus and Pipa. This scenario is illustrated schematically in Fig. 5.

The skin secretions of R. dorsalis contain relatively high concentrations of the Arg-

Pro-rich peptide rhinophrynin-33, which contains five copies of the dipeptide sequence Leu-

Pro, together with the truncated form rhinophrynin-27 which presumably arises from

proteolytic cleavage at the Lys28 processing site. Although not necessarily related

evolutionarily, Arg-Pro-rich peptides with antimicrobial activity have been identified in

artiodactyls (cattle, goat, pig, sheep) and a range of insects, crustaceans, and molluscs

(reviewed in [36]). No such peptide has yet been described in skin secretions of any

representative of the Pipidae. As shown in Fig.6, rhinophyrinin-27shows limited structural

similarity to the multifunctional cathelicidin peptide PR-39 that was first isolated from

porcine small intestine [37] and subequently identified in bone marrow, thymus, spleen, and

leucocytes [38]. There is no significant amino acid sequence identity between the

rhinophyrinins and the bactenecins such as bac-5 isolated from from bovine neutrophils [39]

or the abecins such as the component from the bumblebee, Bombus pascuorum [40]. PR-39 is

an important component of the system of innate immunity in artiodactyls and shows potent

antimicrobial activity against a range of enteric bacterial pathogens by a mechanism that

involves inhibition of cDNA replication and protein synthesis rather than cell lysis, as is the

case with the cationic α-helical peptides from Pipidae species [41]. However, rhinophrynin-

27, while lacking cytotoxic activity against erythrocyes and A549 cells, also lacked growth-

inhibitory activity the Gram-negative E. coli and the Gram-positive S. epidermidis and B.

megaterium. Structure-activity studies have demonstrated that the strongly cationic N-

terminal domain (residues 1-15) of PR-39 is a critically important determinant of

antimicrobial activity [42] so that the presence of two glutamic acid residues in this region of

rhinophrynin-27 is probably responsible for observed abolition of antibacterial activity. The

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biological role of the rhinophrynins in the frog, if any, is unknown. Like the cationic α-helical

peptides from representatives of the Pipidae [13]. PR-39 is a multifunction peptide displaying

immunomodulatory, anti-apoptopic, and chemoattractive properies and promoting

angiogenesis and wound healing (reviewed in [43]. Preliminary data show that rhinophrynin-

27 has complex effects on the production of pro-inflammatory cytokines by mouse peritoneal

macrophages (M. Lukic, University of Kragujevac, unpublished data) suggesting the

possibility of an immunomodulatory role. The possibility that the rhinophrynins have an

antipredator function remains open.

The circular dichroism spectrum of porcine PR-39 in water indicates that the peptide

adopts a left handed polyproline II helical conformation that is unaffected by the presence of

liposomes, thereby suggesting that interaction with cell membranes does not modify its

conformation appreciably [44]. Although rhinophrynin-27 exhibited a CD spectrum similar to

the one of PR-39 in water at room temperature, some differences were observed when

recording spectra at different temperatures in the presence of membrane mimetic micelles. In

particular, in the presence of anionic SDS micelles, the CD spectra of rhinophrynin-27

reflected the presence of turn structures in addition to a polyproline II helix indicating that, in

contrast to PR-39, the conformation of the peptide was modified by the interaction with

negatively charged micelles. Although the reason for this different behaviour is unknown, it

suggests a different mode of interaction between rhinophrynin-27 and bacteria membranes

which might contribute to the observed lack of antimicrobial activity of the peptide.

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Acknowledgments

The authors thank Clint Guadiana and Colette Hairston Adams, Gladys Porter Zoo for help in

collecting the frog species and Laurey Steinke and Michele Fontaine, University of Nebraska

Medical Center, Omaha, NE for amino acid composition analysis and Per F. Nielsen, Novo

Nordisk for sequence analysis of peak 18 (Fig. 1). They also thank Labex Synorg (ANR-11-

LABX-0029) for financial support.

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Legend to Figures

Fig. 1. Reversed-phase HPLC on a semipreparative Vydac C-18 column of skin secretions

from R. dorsalis after partial purification on Sep-Pak cartridges. The major components in the

peaks designated 1 - 19 were purified to near homogeneity by further chromatography on

semi-preparative Vydac C-4 and phenyl columns. The dashed line shows the concentration of

acetonitrile in the eluting solvent.

Fig, 2. Purification to near homogeneity of rhinophrynin-27 on (A ) a semipreparative Vydac

C-4 column and (B) a semipreparative Vydac phenyl column. The dashed line shows the

concentration of acetonitrile in the eluting solvent and the arrowheads show where peak

collection began and ended.

Fig, 3. CD spectra of rhinophrynin-27 at room temperature in water (solid black), 25 %

trifluoroethanol (TFE) (solid gray), 50% TFE (dashed gray) and in the presence of 20 mM

sodum dodecylsulfate (SDS) (dashed black)

Fig, 4. Effect of temperature on the CD spectra of rhinophrynin-27. Black solid lines indicate

the spectra at 20°C and black dashed lines the spectra at 5.5°C (A) in water, (B) in the

presence of 20 mM dodecylphosphocholine (DPC) micelles, and (C) in the presence of 20

mM sodium dodecylsulfate (SDS) micelles.

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Fig. 5. A simplified schematic representation of the proposed time, denoted by the asterisk,

when representatives of the Pipoidea developed the ability to synthesize cationic, α-helical

host-defense peptides in their skins . Hypothesis C is described in the text.

Fig. 6. A comparison of the primary structures of rhinophrynin-33 and rhinophrynin-27 from

R. dorsalis, porcine PR-39, bovine bac-5, and abecin from the bumble bee Bombus

pascuorum. Regions of structural similarity between the rhinophrynins and PR-39 are

highlighted in grey.

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Fig. 1

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Fig.2

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Fig. 3.

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Fig. 4

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Fig. 5

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Rhinophrynin-33 ELRLPEIARPVPEVL*PARLPLPALPRNKMAKNQ

Rhinophrynin-27 ELRLPEIARPVPEVL*PARLPLPALPRN

PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP

Bac5 RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLRFP

Abaecin PYNPPRPGQSKPFPTFPGHGPFNPKIQWPYPLPNPGH

Fig. 6

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Table 1. Complete or partial amino acid sequences, observed molecular masses ([Mr+H]obs),

and calculated molecular masses ([Mr+H]calc) of components isolated from skin secretions of

R. dorsalis

Peak no. [M + H]+obs [M + H]+

calc Amino acid sequence Putative assignment

1 279.2 Non-peptide

2 321.2 Non-peptide3 414.3 Non-peptide4 430.2 Non-peptide5 1411.8 1411.8 IPHEHRPRIQE Laminin α-chain fragment6 1510.9 1510.8 VIPHEHRPRIQE Laminin α-chain fragment7 398.2 Non-peptide7 444.1 Non-peptide8 383.2 Non-peptide8 397.2 Non-peptide9 412.3 Non-peptide9 428.3 Non-peptide10 5548.3 VIVPPNHKDA….. Unknown11 5663.6 LVIVPPNHKDA….. unknown12 3727.4 3727.2 ELRLPEIARPVPEVLPARL

PLPALPRNKMAKNQRhinophrynin-33

12 7348.2 LKCNYCKNGRSF….. Zinc finger MYM-type protein 2 isoform X1 fragment

13 3027.2 3026.8 ELRLPEIARPVPEVLPARLPLPALPRN

Rhinophrynin-27

14 563.4 Non-peptide15 7266.2 Not determined16 19,148 Not determined17 18,903 Not-determined18 19,017 YRTVYRCSTA,…. Unknown19 5876.6 Non-determined

Peak No. refers to the chromatogram shown in Figure 1.

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