Peptidomic analysis of skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis

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 Conlon^a*, Laure Guilhaudis^b, Jérôme Leprince^c, Laurent Coquet^d,

Maria Luisa Mangoni^e, Samir Attoub^f, Thierry Jouenne^d, Jay D. King^g

^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. [e-mail]

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.

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

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 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

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. 

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. 
   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 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.

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).

3. 
   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.

3. 
   Antimicrobial and cytotoxicity assays
   

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.

3. 
   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 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].

2. 
   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 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.

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. 
   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].

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.

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 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 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.
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.

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.

Fig. 1

Fig.2

Fig. 3.

Fig. 4

Fig. 5

Rhinophrynin-33 ELRLPEIARPVPEVL*PARLPLPALPRNKMAKNQ

Rhinophrynin-27 ELRLPEIARPVPEVL*PARLPLPALPRN

PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP

Bac5 RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLRFP

Abaecin PYNPPRPGQSKPFPTFPGHGPFNPKIQWPYPLPNPGH

Fig. 6
Table 1. Complete or partial amino acid sequences, observed molecular masses ([M_r+H]_obs), and calculated molecular masses ([M_r+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-peptide
3	414.3			Non-peptide
4	430.2			Non-peptide
5	1411.8	1411.8	IPHEHRPRIQE	Laminin α-chain fragment
6	1510.9	1510.8	VIPHEHRPRIQE	Laminin α-chain fragment
7	398.2			Non-peptide
7	444.1			Non-peptide
8	383.2			Non-peptide
8	397.2			Non-peptide
9	412.3			Non-peptide
9	428.3			Non-peptide
10	5548.3		VIVPPNHKDA…..	Unknown
11	5663.6		LVIVPPNHKDA…..	unknown
12	3727.4	3727.2	ELRLPEIARPVPEVLPARLPLPALPRNKMAKNQ	Rhinophrynin-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-peptide
15	7266.2			Not determined
16	19,148			Not determined
17	18,903			Not-determined
18	19,017		YRTVYRCSTA,….	Unknown
19	5876.6			Non-determined

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