Peptidomic analysis of the extensive array of host-defense peptides in skin secretions of

the dodecaploid frog Xenopus ruwenzoriensis (Pipidae)

Laurent Coquetab, Jolanta Kolodziejekc Thierry Jouenneab, Norbert Nowotnyc, Jay D. Kingd, J.

Michael Conlone*

aCNRS UMR 6270, University of Rouen, 76821 Mont-Saint-Aignan, France

bPISSARO, Institute for Research and Innovation in Biomedicine (IRIB), University of Rouen,

76821 Mont-Saint-Aignan, France

cViral Zoonoses, Emerging and Vector-Borne Infections Group, Institute of Virology,

University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria

dRare Species Conservatory Foundation, St. Louis, MO 63110, USA

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

Ulster, Coleraine BT52 1SA, U.K.

*Corresponding author. Tel: +44(0)7918526277; Fax: +44(0)2870124965;

E-mail address: m.conlon@ulster.ac.uk (J.M. Conlon)

1

ABSTRACT

The Uganda clawed frog Xenopus ruwenzoriensis with a karyotype of 2n = 108 is one of the

very few vertebrates with dodecaploid status. Peptidomic analysis of norepinephrine-

stimulated skin secretions from this species led to the isolation and structural characterization

of 23 host-defense peptides belonging to the following families: magainin (3 peptides),

peptide glycine-leucine-amide (PGLa; 6 peptides), xenopsin-precursor fragment (XPF; 3

peptides), caerulein precursor fragment (CPF; 8 peptides), and caerulein precursor fragment -

related peptide (CPF-RP; 3 peptides). In addition, the secretions contained caerulein, identical

to the peptide from Xenopus laevis, and two peptides that were identified as members of the

trefoil factor family (TFF). The data indicate that silencing of the host-defense peptide genes

following polyploidization has been appreciable and non-uniform. Consistent with data

derived from comparison of nucleotide sequences of mitochrondrial and nuclear genes,

cladistic analyses based upon the primary structures of the host-defense peptides provide

support for an evolutionary scenario in which X. ruwenzoriensis arose from an

allopolyploidization event involving an octoploid ancestor of the present-day frogs belonging

to the Xenopus amieti species group and a tetraploid ancestor of Xenopus pygmaeus.

Key words: antimicrobial peptide; frog skin; magainin; PGLa; procaerulein; proxenopsin;

allopolyploidy; Xenopus

2

1. Introduction

The taxonomy, evolutionary history, and cytogenetics of African clawed frogs within the

subfamily Xenopodinae of the family Pipidae are complex. Until relatively recently, these

frogs were assigned on the basis of morphology and karyotype either to the genus Xenopus or

to the sister-group genus Silurana. However, evidence from molecular phylogenetic studies

has established the monophyletic status of Xenopus + Silurana so that Silurana is now

generally described as a sub-genus of Xenopus. (de Sá and Hillis, 1990; Frost, 2015). The

evolutionary history of Xenopus involves both bifurcating speciation, such as allopatric

speciation when two populations of an ancestral species become geographically separated,

and reticulate sympatric speciation involving genome duplication by allopolyploidization that

has given rise to tetraploid, octoploid, and dodecaploid species (Kobel, 1996; Evans, 2008;

Evans et al., 2004). Xenopus tropicalis in the sub-genus Silurana with a karyotype of 2n =

20 is the only extant species to have preserved the ancestral diploid status (Tymowska and

Fischberg, 1982). Putative allopolyploidizations within this sub-genus have resulted in the

appearance of the tetraploids Xenopus calcaratus, Xenopus epitropicalis, and Xenopus

mellotropicalis (Evans et al., 2015).

Within the genus Xenopus, species retaining diploid status (karyotype 2n = 18) that is

thought to be related to the ancestral state existing prior to genome duplications have not yet

been described but a series of polyploidization events have led to the emergence of 14

tetraploids with 2n = 36 (Xenopus allofraseri, Xenopus borealis, Xenopus clivii, Xenopus

fischbergi Xenopus fraseri, Xenopus gilli, Xenopus laevis, Xenopus largeni, Xenopus

muelleri, Xenopus parafraseri, Xenopus petersii, Xenopus poweri, Xenopus pygmaeus, and

Xenopus victorianus), seven octoploids with 2n = 72 (Xenopus amieti, Xenopus andrei,

Xenopus boumbaensis, Xenopus itombwensis, Xenopus lenduensis, Xenopus vestitus and

3

Xenopus wittei), and four dodecaploids with 2n = 108 (Xenopus eysoole, Xenopus kobeli,

Xenopus longipes and Xenopus ruwenzoriensis) (Frost, 2015; Evans et al., 2015). Recent

work has clarified the taxonomic status of an incompletely characterized species previously

described by different authors as the nomen nudum “Xenopus alboventralis” (Salamone,

2006), “X. new tetraploid” (Evans et al., 2004), and “Xenopus muelleri West” (Mechkarska et

al., 2011). This species has now been assigned the name X. fischbergi (Evans et al., 2015).

Skin secretions of X. laevis contain a range of cationic, α-helical peptides that were

originally described as “antimicrobial” on the basis of their growth inhibitory activity against

bacteria and fungi. However, such compounds also display immunomodulatory, chemotactic,

and insulin-releasing activities and are cytotoxic to tumor cells and viruses [reviewed in

(Conlon et al., 2014b; Xu and Lai, 2015)] so that they are more accurately described as host-

defense peptides. Five families of such peptides have been identified on the basis of limited

structural similarity: magainin (Giovannini et al., 1987; Zasloff, 1987;), 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 (Gibson, et al., 1986).

During the past 6 years there has been a systematic program of investigation to

characterize the host-defense peptides present in the norepinephrine-stimulated skin

secretions of other species frogs belonging to the family Pipidae. This has led to the isolation

of magainin, PGLa, CPF, CPF-RP and XPF peptides from X. amieti (Conlon et al., 2010), X.

andrei (Mechkarska et al., 2011b), X. boumbaensis (Conlon et al., 2015b), X. epitropicalis

(Conlon et al., 2012), X. borealis (Mechkarska et al., 2010), X. clivii (Conlon et al., 2011), X.

fischbergi (Mechkarska et al., 2011a), X. fraseri (Conlon et al., 2014a), X. gilli (Conlon et al.,

2015a), X. lenduensis (King et al., 2012), X. muelleri (Mechkarska et al., 2011a), X. petersii

4

(King et al., 2012), X. pygmaeus (King et al., 2012), X. tropicalis (Ali et al., 2001; Roelants

et al., 2013), X. victorianus (King et al., 2013), X. vestitus (Mechkarska et al., 2014), and X.

wittei (Mechkarska et al., 2014). Host-defense peptides have also been isolated from

laboratory-generated F1 hybrids of X. laevis × X. muelleri (Mechkarska et al., 2012) and X.

laevis × X. borealis (Mechkarska et al., 2013).

The aim of the present study was to extend this program by using peptidomic analysis

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

degradation) to identify and characterize host-defense peptides in norepinephrine-stimulated

skin secretions from a dodecaploid frog, the Uganda clawed frog X. ruwenzoriensis

Tymowska and Fischberg, 1973 (also known as the Ruwenzori clawed frog). In common

with all members of the genus, X. ruwenzoriensis is a predominantly aquatic species that

occupies a restricted range in the foothills of the Ruwenzori Mountains in Uganda extending

into Kivu and Orientale in the Democratic Republic of the Congo (Greenbaum and Kusamba.

2010; Frost 2015). Its natural habitats are subtropical or tropical moist lowland forests,

freshwater marshes, intermittent freshwater marshes, rural gardens, and heavily degraded

former forest. X. ruwenzoriensis is listed as Data Deficient by the International Union for

Conservation of Nature (IUCN) Red List in view of uncertainties as to its extent of

occurrence, status and ecological requirements but it is believed to be rare within its range

and threatened by habitat loss and by human consumption (IUCN SSC Amphibian Specialist

Group 2014).

2. Experimental

2.1. Collection of skin secretions

All experiments with live animals were approved by the Animal Research Ethics

committee of Université Paris-Sud and were carried out by authorized investigators. The X.

5

ruwenzoriensis individuals were laboratory bred from parent stock that was collected in 1972

by Professor M. Fischberg near the Ruwenzori massif, close to the Semliki River in the

Democratic Republic of the Congo. All animals were housed in a vivarium at the Université

Paris-Sud 

Each animal (one male, body weight 22g and one female, body weight 29g) 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 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 group were pooled and separately passed at a flow rate of 2 ml/min through 8 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 skin secretions from X. ruwenzoriensis, 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, USA) 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 280 nm, and fractions (1 min) were

collected. All major peaks with retention times > 35 min were collected and 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

6

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 208TP510 (C-8) columns. The concentration of acetonitrile in

the eluting solvent was raised from 21% to 56% over 60 min and the flow rate was 2.0

ml/min.

Identification of caerulein (sulphated and desulfated forms) was facilitated by the fact that

the peptides contain a tryptophan residue and so shows strong absorbance at 280nm. These

components were purified to near homogeneity by chromatography on (1.0 cm x 25 cm)

Vydac 214TP510 (C-4) and (1.0 cm x 25 cm) Vydac 208TP510 (C-8) columns. The

concentration of acetonitrile in the eluting solvent was raised from 14% to 42% over 60 min

and the flow rate was 2.0 ml/min.

2.3. Structural characterization

The primary structures of the peptides were determined by automated Edman

degradation using a model 494 Procise sequenator (Applied Biosystems, Courtaboeuf,

France). MALDI-TOF mass spectrometry was carried out using a MALDI-TOF-TOF

Ultraflex instrument (Bruker Daltonique S.A., Wissembourg, France) 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%.

2.4. Cladistic analysis

Phylogenetic trees based upon the amino acid sequences of the known host-defense

peptides from Xenopus species were generated using the maximum likelihood method (Yang

7

1994) in the MEGA6 program (Tamura et al., 2013). The primary structures of the peptides

from which the trees are derived are shown in previous publications (Conlon and

Mechkarska, 2014; Conlon et al., 2015a; Conlon et al., 2015b). Evolutionary distances were

computed using the Dayhoff matrix-based substitution model (Schwarz and Dayhoff, 1979)

and are in the units of the number of amino acid substitutions per site. The primary structures

of the peptides from X. tropicalis (Ali et al., 2001; Roelants et al., 2013) were used as

outgroups to polarize the ingroup taxa.

3. Results

3.1. Purification of the peptides

The peptides described in this study are classified according to the terminology used

previously for structurally related peptides from other Xenopus species (Conlon and

Mechkarska, 2014). The magainin, PGLa, CPF, CPF-RP, and XPF peptide families are

recognized. The species origin is denoted by R for ruwenzoriensis. Paralogs are differentiated

by numerals e.g. PGLa-R1 and PGLa-R2.

The pooled skin secretions from X. ruwenzoriensis, 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 - 17 were collected and subjected

to further purification. The major components present in each peak, identified by subsequent

structural analysis, are shown in Fig. 2. The peak denoted by A contained caerulein and the

peak denoted by B contained desulfated caerulein. All the peptides 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 C-8 columns (chromatograms not

shown).

8

3.2. Structural characterization

The primary structures of the peptides isolated from X. ruwenzoriensis skin secretions

were established by automated Edman degradation and their amino acid sequences are shown

in Fig. 2. MALDI-TOF mass spectrometry was used to demonstrate that all PGLa peptides

and the CPF-RP peptides are C-terminally α-amidated. PGLa-R3 and PGLa-R4 differ by a

single amino acid (Ser4 Thr) (Fig. 2) and it was not possible to separate the two peptides

under the chromatographic conditions used in this study. During Edman degradation

phenylthiohydantoin (PTH)-derivatives of Ser and Thr were observed during cycle 4 whereas

single PTH-derivatives were detected during all other cycles. Signals corresponding to the

molecular masses [M + H]+ of both peptides were detected during MALDI-TOF mass

spectrometry (Fig. 2). The primary structure of major component of caerulein from X.

ruwnzoriensis skin secretions (peak A) was shown to be identical to that of X. laevis caerulein

(Anastasi et al., 1970) by mass spectrometry (Fig. 2) and by the observation that a mixture of

peptides from both species was eluted from a C-18 reversed-phase HPLC column as a single,

sharp peak. The presence of a sulphated tyrosine residue was demonstrated by electrospray

mass spectrometry operated in negative ion mode as previously described (Zahid et al.,

2011). The minor component (peak B) represented desulfated caerulein.

Peaks 6 and 7 (Fig. 2) contained peptides with substantially higher molecular masses

(15,259 and 14,505) than those of the host-defense peptides generally found in skin

secretions of frogs from the Xenopus genus. There was insufficient material to permit full

structural characterization of these peptides but determination of the common amino acid

sequence at the N-terminal region (YSTIYRCTSQNPSGR. . ..) established that they are

members of members of the trefoil factor family (TFF) of peptides. This sequence is identical

9

to that present at the N-terminal region of three peptides (TFF-BM1, TFF-BM2, and TFF-

BM3) isolated from skin secretion of X. boumbaensis (Conlon et al., 2015b) and at the N-

terminus of a predicted protein of unknown function designated LOC 100488906 in the X.

tropicalis genome database.

3.3. Cladistic analysis

Optimal phylogenetic trees based upon the amino acid sequences of the host-defense

peptides were constructed using the maximum likelihood method with the sequences of

corresponding peptides from X. tropicalis as outgroups, The trees are drawn to scale, with

branch lengths in the same units as those of the evolutionary distances used to infer the

phylogenetic tree. The following optimal trees derived from the primary structures of 37

magainin peptides from 18 species (Fig. 3A), 39 PGLa peptides from 16 species (Fig. 3B), 32

XPF peptides from 15 species (Fig. 3C), 45 CPF peptides from 13 species (Fig. 3D), and 33

peptides from 12 species (Fig. 3E) are shown.

4. Discussion

Although relatively common in plants, polyploidization in sexually reproducing

animals is rare (Otto and Whitton, 2000). Polyploids can originate by autopolyploidization

(genome duplication within a species) but, in the case of Xenopus, allopolyploidization

(genome duplication associated with hybridization between different species) is believed to

be exclusive mechanism (Kobel 1996). The extant species have probably arisen from

multiple independent allopolyploidization events (Evans, 2008; Schmid et al., 2015).

Although it is not always clear what advantage polyploid status confers on the organism, it

10

has been proposed that polyploidy accelerates the rate of evolutionary adaptation through

complex effects on the frequency or fitness of beneficial mutations (Selmecki et al., 2015).

While conclusive evidence is lacking, an increase in the diversity of host-defense peptides

synthesized in the skin resulting from polyploidization may provide frogs with increased

protection against invasion by parasites (Jackson and Tinsely, 2003) and pathogenic bacteria

(Conlon, 2011) in the environment.

This study has used HPLC coupled with MALDI-TOF mass spectrometry and

automated Edman degradation to analyse the complex array of host-defense peptides in

norepinephrine-stimulated skin secretions of the dodecaploid frog X. ruwenzoriensis. In

common with the diploid X. tropicalis and the other polyploidy species within the Xenopus

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

identified on the basis of structural similarity to orthologs from X. laevis (Gibson et al., 1986;

Giovannini et al., 1987; Zasloff, 1987). Evolutionary pressure to conserve the primary

structures of these peptides during the radiation of the species has not been particularly

strong. A comparison of the amino acid sequences of the peptides isolated from all species to-

date allows derivations of the consensus sequences for each peptide family (Fig. 4). Despite

this variability in sequence, all peptides belonging to these five families are cationic and have

the propensity to adopt an amphipathic α-helical conformation in a membrane-mimetic

solvent (Conlon and Mechkarska, 2014).

Nonfunctionalization (“gene silencing” either by deletion of the duplicated gene or by its

degeneration into a pseudogene by incorporation of premature stop codons or frame-shift

mutations) is the most common fate of duplicated genes following polyploidization events

within the Xenopus genus (Evans 2007; 2008). In the case of X. ruwnzoriensis, elimination of

multiple paralogous genes related to the immune system has been demonstrated (Du

Pasquier et al., 2009) and electrophoresis of serum from this species demonstrate the

11

presence of three albumin bands instead of the predicted six (Graf and Fischberg, 1986).

Consistent with previous data obtained from the analysis of skin secretions of octoploid frogs

(Conlon et al., 2010, 2015b; King et al., 2012; Mechkarska et al., 2014), the extent to which

the genes encoding the host-defense peptides have been silenced in the dodecaploid frog X.

ruwnzoriensis has not been uniform. The six paralogous genes encoding PGLa expected

from three putative allopolyploidization events have been retained and are expressed whereas

three of the genes encoding magainin and three of the genes encoding XPF have been either

been completely silenced or levels of expression are very low. In the case of the CPF and

CPF-RP, unambiguous interpretation is not possible as it is unclear to what extent the

multiplicity of the peptides arose from expression of separate genes or, as in the case of X.

laevis (Richter et al., 1986), from the fact that multiple peptides are encoded by the same

gene.

In addition to the cationic α-helical host-defense peptides, the X. ruwenzoriensis skin

secretions contained caerulein that is identical in structural to the peptide in all other Xenopus

species investigated to-date except for X. borealis (Zahid et al. 2011), X. fischbergi, and X.

clivii (M. Mechkarska, unpublished data) which synthesize the molecular variant caerulein-

B1. The X. ruwenzoriensis secretions also contained two larger peptides that were identified

on the basis of partial amino acid sequencing as members of the TFF trefoil factor family

(Fig. 2). TFF peptides have previously been isolated from skin secretions of X. boumbaensis

and it is speculated that they may play a role in mucosal protection and reconstitution

(Conlon et al., 2015b).

The initial promise of frog skin host-defense peptides as templates for the development

of therapeutically valuable antimicrobial or anticancer agents has not been fulfilled but the

highly variable primary structures of such peptides have proved to be useful as regards

gaining insight into the evolutionary history of species within a particular genus.

12

Traditionally, frogs within the Xenopodinae have been divided into subgroups largely on the

basis of morphological features and advertisement calls (Kobel et al., 1996). These

subgroups comprised the Silurana subgenus (X. tropicalis  and X. epitropicalis),

the laevis subgroup (X. laevis, X. gilli, X. largeni), the muelleri subgroup (X. muelleri, X.

borealis, X. clivii, and the species now referred to as X. fischbergi), the fraseri subgroup (X.

fraseri, X. amieti, X. andrei, X. boumbaensis, X. pygmaeus, X. ruwenzoriensis), the vestitus-

wittei subgroup (X. vestitus, X. wittei) and the longipes sub-group (X. longipes). In the light of

more recent molecular phylogenetic data and discovery of new species, the groupings have

been reformulated (Evans et al., 2015). A more inclusive amieti subgroup of frogs that share

a common evolutionary history has been proposed that includes X. allofraseri, X. amieti, X.

andrei, X. boumbaensis, X. eysoole, X. itombwensis, X. lenduensis, X. parafraseri, X.

pygmaeus, X. ruwenzoriensis, X. vestitus, and X. wittei. The Silurana, laevis and muelleri

subgroups are retained but the taxonomic status of X. fraseri and X. largeni is now unclear.

Cladistic analyses based upon the primary stuctures of the dermal host-defense peptides

(Fig. 3) support the placement of X. ruwenzoriensis in the amieti species group. Consistent

with analyses based upon comparisons of the nucleotide sequences of mitochondrial genes

(Evans et al., 2004) and RAG1 and RAG2 nuclear genes (Evans 2007), the data suggest that

X. ruwenzoriensis and the common octoploid ancestor of X. amieti, X. andrei, and X.

boumbaensis share an evolutionary history. Magainin-R2 and -R3 (Fig. 3A), PGLa-R1, -R3

and R-4 (Fig. 3B), XPF-R1, -R2, and R3 (Fig. 3C), and CPF-RP-R1, -R3 and -R3 (Fig. 3E)

segregate in well-defined clades containing the corresponding peptides from these three

species. X. ruwenzoriensis is believed to have arisen from a relatively recent

allopolyploidization event involving such an ancestral octoploid species and the ancestor of a

present-day tetraploid species (Evans et al. 2004; Evans et al., 2015). It has been proposed

that the dodecaploidization of X. ruwenzoriensis was independent from that of all other

13

dodecaploids (Evans et al., 2015). X. ruwenzoriensis is known to be sympatric with the

tetraploid X. pygmaeus over part of its range (Evans et al.  2011, Frost 2015) and nucleotide

sequence analysis of the RAG1 and RAG2 genes (Evans 2007) suggest that these species are

in a close phylogenetic relationship. This proposal is supported by the observations that X.

ruwenzoriensis PGLa-R3, PGLa-R4 and X. pygmaeus PGLa-PG1 segregate in a well-defined

clade (Fig. 3B) and a close phylogenetic relationship between the two species is suggested by

the fact that the primary structures CPF-RP-R2 and CPF-RP-PG1 are identical (Fig 3E). In

contrast, a comparison of the primary structures of the CPF peptides (Fig. 3D) indicates that

an ancestor of X. laevis may have been involved in the allopolyploidization that produced X.

ruwenzoriensis but this is not consistent with phylogenies based upon mitochondrial and

RAG gene sequences. It must be stressed that caution is warranted when interpreting

phylogenetic trees based on the primary structures of relatively small peptides as the number

of informative characters that define the analysis is not very great.

Acknowledgments

The authors thank Peter R. Flatt, Ulster University for providing JMC with laboratory

facilities and Odile Bronchain and Albert Chesneau of the Université Paris-Sud for assisting

in the collection process.

14

REFERENCES

Ali, M.F., Soto, A., Knoop, F.C., Conlon, J.M., 2001. Antimicrobial peptides isolated from

skin secretions of the diploid frog, Xenopus tropicalis (Pipidae). Biochim. Biophys.

Acta 1550, 81-89.

Anastasi, A., Bertaccini, G., Cei, J.M., De Caro, G., Erspamer, V., Impicciatore. M.,

Roseghini, M., 1970. Presence of caerulein in extracts of the skin of Leptodactylus

pentadactylus labyrinthicus and of Xenopus laevis. Br. J. Pharmacol. 38, 1–28.

Conlon, J.M., 2011. The contribution of skin antimicrobial peptides to the system of innate

immunity in anurans. Cell Tissue Res. 343, 201-212.

Conlon, J.M., Mechkarska, M., 2014. Host-defense peptides with therapeutic potential from

skin secretions of frogs from the family Pipidae. Pharmaceuticals (Basel). 7, 58-77.

Conlon, J.M., Al-Ghaferi, N., Ahmed, E., Meetani, M.A., Leprince, J., Nielsen, P.F., 2010.

Orthologs of magainin, PGLa, procaerulein-derived, and proxenopsin-derived peptides

from skin secretions of the octoploid frog Xenopus amieti (Pipidae). Peptides 31, 989-

994.

Conlon, J.M., Mechkarska, M., Ahmed, E., Leprince, J., Vaudry, H., King, J.D., Takada, K.,

2011. Purification and properties of antimicrobial peptides from skin secretions of the

Eritrea clawed frog Xenopus clivii (Pipidae). Comp. Biochem. Physiol. C Toxicol.

Pharmacol. 153, 350-354.

Conlon, J.M., Mechkarska, M., Prajeep, M., Sonnevend, A., Coquet, L., Leprince, J.,

Jouenne, T., Vaudry, H., King, J.D., 2012. Host-defense peptides in skin secretions of

the tetraploid frog Silurana epitropicalis with potent activity against methicillin-

resistant Staphylococcus aureus (MRSA). Peptides 37, 113-119.

15

Conlon, J.M., Mechkarska, M., Kolodziejek, J., Nowotny, N., Coquet, L., Leprince, J.,

Jouenne, T., Vaudry, H., 2014a. Host-defense peptides from skin secretions of

Fraser’s clawed frog Xenopus fraseri (Pipidae): further insight into the evolutionary

history of the Xenopodinae. Comp. Biochem. Physiol. D Genomics Proteomics 12,

45-52.

Conlon, J.M., Mechkarska, M., Lukic, M.L., Flatt, P.R., 2014b. Potential therapeutic

applications of multifunctional host-defense peptides from frog skin as anti-cancer,

anti-viral, immunomodulatory, and anti-diabetic agents. Peptides 57, 67-77.

Conlon, J.M., Mechkarska, M., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., Measey,

G.J., 2015a. Evidence from peptidomic analysis of skin secretions that allopatric

populations of Xenopus gilli (Anura:Pipidae) constitute distinct lineages. Peptides

63,118-125.

Conlon, J.M., Mechkarska, M., Kolodziejek, J., Leprince, J., Coquet, L., Jouenne, T., Vaudry,

H., Nowotny, N., King, J.D., 2015b. Host-defense and trefoil factor family peptides in

skin secretions of the Mawa clawed frog Xenopus boumbaensis (Pipidae). Peptides

72, 44-49.

de Sá, R.O and Hillis, D.M., 1990. Phylogenetic relationships of the pipid frogs Xenopus and

Silurana: an integration of ribosomal DNA and morphology. Mol. Biol. Evol. 7, 365-

376.

Du Pasquier, L., Wilson, M., Sammut B., 2009. The fate of duplicated immunity genes in the

dodecaploid Xenopus ruwenzoriensis. Front Biosci 14, 177-191.

Evans, B.J., 2007. Ancestry influences the fate of duplicated genes millions of years after

polyploidization of clawed frogs (Xenopus). Genetics 176, 1119-1130.

Evans, B.J., 2008. Genome evolution and speciation genetics of clawed frogs (Xenopus and

Silurana). Front. Biosci. 13, 4687-4706.

16

Evans, B.J., Kelley, D.B., Tinsley, R.C., Melnick D.J., Cannatella, D.C., 2004. A

mitochondrial DNA phylogeny of African clawed frogs: phylogeography and

implications for polyploid evolution. Mol. Phylogenet. Evol. 33, 197-213.

Evans, B.J., Greenbaum, E., Kusamba, C., Carter, T.F., Tobias, M.L., Mendel, S.A., Kelley,

D.B., 2011. Description of a new octoploid frog species (Anura: Pipidae: Xenopus)

from the Democratic Republic of the Congo, with a discussion of the biogeography of

African clawed frogs in the Albertine Rift. J. Zool. 283, 276-290.

Evans, B.J., Carter, T.F., Greenbaum, E., Gvoždík, V., Kelley, D.B., McLaughlin, P.J.,

Pauwels, O.S., Portik, D.M., Stanley, E.L., Tinsley, R.C., Tobias, M.L., Blackburn,

D.C., 2015. Genetics, morphology, advertisement calls, and historical records

distinguish six new polyploid species of African clawed frog (Xenopus, Pipidae) from

west and central Africa. PLoS One 10, e0142823.

Frost, D.R., 2015. Amphibian species of the world: an online reference. Version 6.0

American Museum of Natural History, New York, USA. Electronic database accessible

at http://research.amnh.org/ herpetology/ amphibia /index.php.

Gibson, B.W., Poulter, L., Williams, D.H., Maggio, J.E., 1986. Novel peptide fragments originating

from PGLa and the caerulein and xenopsin precursors from Xenopus laevis. J. Biol. Chem. 261,

5341-5349.

Giovannini, M.G., Poulter, L., Gibson, B.W., Williams, D.H., 1987. Biosynthesis and degradation of

peptides derived from Xenopus laevis prohormones. Biochem. J. 243, 113-120.

Graf, J.D., Fischberg, M., 1986. Albumin evolution in polyploid species of the genus Xenopus.

Biochem Genet. 11-12, 821-837.

Greenbaum, E., Kusamba, C., 2010. Geographic distribution: Xenopus ruwenzoriensis. Herpetolog.

Rev. 41, 376.

17

IUCN SSC Amphibian Specialist Group. 2014. Xenopus ruwenzoriensis. The IUCN Red List of

Threatened Species 2014: e.T58180A16942495.

Jackson JA, Tinsley RC. 2003. Parasite infectivity to hybridising host species: a link between hybrid

resistance and allopolyploid speciation? Int J Parasitol. 33, 137-144.

King, J.D., Mechkarska, M., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., Takada, K., Conlon,

J.M., 2012. Host-defense peptides from skin secretions of the tetraploid frogs Xenopus petersii

and Xenopus pygmaeus, and the octoploid frog Xenopus lenduensis (Pipidae). Peptides 33, 35-

43.

King, J.D, Mechkarska, M., Meetani, M.A., Conlon, J.M., 2013. Peptidomic analysis of skin

secretions provides insight into the taxonomic status of the African clawed frogs Xenopus

victorianus and Xenopus laevis sudanensis (Pipidae). Comp Biochem Physiol Part D

Genomics Proteomics 8, 250-254.

Kobel, H.R., 1996. Allopolyploid speciation. In: Tinsley, R.C., Kobel, H.R. (Eds.) The

biology of Xenopus. Clarendon Press, Oxford, pp. 391-401.

Kobel, H.R., Loumont, C., Tinsley, R.C., 1996. The extant species. In: Tinsley, R.C., Kobel,

H.R. (Eds.). The biology of Xenopus. Clarendon Press, Oxford, pp. 9-33.

Mechkarska, M., Ahmed, E., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., King, J.D.,

Conlon, J.M., 2010. Antimicrobial peptides with therapeutic potential from skin

secretions of the Marsabit clawed frog Xenopus borealis (Pipidae). Comp. Biochem.

Physiol. C Toxicol. Pharmacol. 152, 467-472.

Mechkarska, M., Ahmed, E., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., King, J.D.,

Conlon, J.M., 2011a. Peptidomic analysis of skin secretions demonstrates that the

allopatric populations of Xenopus muelleri (Pipidae) are not conspecific. Peptides 32,

1502-1508.

18

Mechkarska, M., Ahmed, E., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., King, J.D.,

Takada, K., Conlon, J.M., 2011b. Genome duplications within the Xenopodinae do

not increase the multiplicity of antimicrobial peptides in Silurana paratropicalis and

Xenopus andrei skin secretions. Comp Biochem Physiol D Genomics Proteomics 6,

206-212.

Mechkarska, M., Meetani, M., Michalak, P., Vaksman, Z., Takada, K., Conlon, J.M., 2012.

Hybridization between the African clawed frogs Xenopus laevis and Xenopus

muelleri (Pipidae) increases the multiplicity of antimicrobial peptides in skin

secretions of female offspring. Comp. Biochem. Physiol. D Genomics Proteomics 7,

285-291.

Mechkarska, M., Prajeep, M., Leprince, J., Vaudry, H., Meetani, M.A., Evans, B.J., Conlon, J.M.,

2013. A comparison of host-defense peptides in skin secretions of female Xenopus laevis ×

Xenopus borealis and X. borealis × X. laevis F1 hybrids. Peptides 45, 1-8.

Mechkarska, M., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., Michalak, K., Michalak,

P., Conlon, J.M., 2014. Host-defense peptides from skin secretions of the octoploid

frogs Xenopus vestitus and Xenopus wittei (Pipidae): insights into evolutionary

relationships. Comp. Biochem. Physiol. D Genomics Proteomics 11, 20-28.

Otto, S.P., Whitton, J., 2000. Polyploid incidence and evolution. Annu. Rev. Genet. 34, 401–

437.

Richter, K., Egger, R., Kreil, G., 1986. Sequence of preprocaerulein cDNAs cloned from skin

of Xenopus laevis. A small family of precursors containing one, three, or four copies

of the final product. J. Biol. Chem. 261, 3676-3680.

Roelants, K., Fry, B.G., Ye, L., Stijlemans, B., Brys, L., Kok, P., Clynen, E., Schoofs, L.,

Cornelis, P., Bossuyt, F., 2013. Origin and functional diversification of an amphibian

defense peptide arsenal. PLoS Genet. 9, e1003662

19

Salamone, L. 2006. Phylogénie moléculaire et biogéographie de la sous-famille des

Xenopodinae avec la description de deux nouvelles espèces. L’Hermine. Bulletin de la

Société Zoologique de Genève 147, 2.

Schmid, M., Evans, B.J., Bogart, J.P., 2015. Polyploidy in Amphibia. Cytogenet. Genome

Res. 14, 315-330.

Schwarz, R., Dayhoff, M., 1979. Matrices for detecting distant relationships. In: Dayhoff, M.

(Ed.), Atlas of protein sequences. National Biomedical Research Foundation:

Washington, DC, pp. 353-358.

Selmecki, A.M., Maruvka, Y.E., Richmond, P.A., Guillet, M., Shoresh, N., Sorenson, A.L,

De, S., Kishony, R., Michor, F., Dowell, R., Pellman, D.,2015. Polyploidy can drive

rapid adaptation in yeast. Nature 519, 349-352.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular

Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729.

Tymowska, J., Fischberg, M., 1980. The karyotype of the hexaploid species Xenopus ruwenzoriensis

Fischberg and Kobel (Anura: Pipidae). Cytogene. Cell Genet. 27: 39–44.

Tymowska, J., Fischberg, M. 1982, A comparison of the karyotype, constitutive

heterochromatin, and nucleolar organizer regions of the new tetraploid species

Xenopus epitropicalis Fischberg and Picard with those of Xenopus tropicalis Gray

(Anura, Pipidae). Cytogene. Cell Genet. 34, 49-157.

Yang, Z.,1994. Maximum likelihood phylogenetic estimation from DNA sequences with

variable rates over sites: approximate methods. J. Mol .Evol. 39, 306-314.

Zahid, O.K., Mechkarska, M., Ojo, O.O., Abdel-Wahab, Y.H., Flatt. P.R., Meetani, M.A.,

Conlon, J.M., 2011. Caerulein-and xenopsin-related peptides with insulin-releasing

activities from skin secretions of the clawed frogs, Xenopus borealis and Xenopus

amieti (Pipidae). Gen. Comp. Endocrinol. 172, 314-320.

20

Zasloff, M., 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation,

characterization of two active forms and partial cDNA sequence of a precursor. Proc.

Natl. Acad. Sci. USA 84, 5449-5453.

Xu, X., Lai, R., 2015. The chemistry and biological activities of peptides from amphibian

skin secretions. Chem. Rev. 115, 1760-1846.

21

Legend to Figures

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

X. ruwenzoriensis after partial purification on Sep-Pak cartridges. The peaks designated 1 -

17 contained host-defense peptides that were purified to near homogeneity by further

chromatography. The peak designated A contained caerulein and Peak B contained desulfated

caerulein. The dashed line shows the concentration of acetonitrile in the eluting solvent.

Fig. 2. Amino acid sequences, observed molecular masses (Mr obs), and calculated molecular

masses (Mr calc) of the antimicrobial peptides isolated from skin secretions of X.

ruwenzoriensis. Peak number refers to the chromatogram shown in Fig. 1. <E denotes

pyroglutamate. a indicates that the peptide is C-terminally α-amidated. + indicates that the

peptide was identified as the Na+ adduct of the desulfated form.

Fig. 3. Phylogenetic trees, generated using the maximum likelihood method, that are derived

from the amino acid sequences of the (A) magainin, (B) PGLa, (C) XPF, (D) CPF, and (E)

CPF-RP peptides from frogs belonging to the genus Xenopus.. The sequence of the peptides

from X. tropicalis are used as outgroup to polarize the ingroup taxa.

Fig.4. Consensus sequences shown in bold of the magainin, PGLa, XPF, CPF, and CPF-RP

peptides from frogs of the genus Xenopus. Amino acid substitutions are arranged vertically

in order of their observed frequency.

22

Fig,1

23

Peak Peptide Primary structure M+H]obs [M+H]calc

No.

A Caerulein <EQDY(S03)TGWMDFa 1272.4+ 1272.5

B Caerulein <EQDYTGWMDFa 1272.4+ 1272.5

1 Magainin-R1 GIGKFLHSAKKFGKAFVGEIMNS 2466.2 2466.3

2 Magainin-R2 GIKEFAHSLGKFGKAFVGGILNQ 2418.1 2418.3

3 Magainin-R3 GVSKILHSAGKFGKAFLGEIMKS 2405.3 2405.3

4 PGLa-R1 GMASKAGSVLGKVAKVALKAALa 2069.5 2069.2

5 PGLa-R2 GMASKAGAIAGKIAKVALKALa 1968.4 1968.2

5 XPF-R1 GWASKIGQTLGKMAKVGLHELIQPK 2690.1 2690.5

6 TFF-R1 YSTIYRCTSQNPSGRQ.... 15,259 unknown

7 TFF-R2 YSTIYRCTSQNPSGRQ.... 14,505 unknown

8 PGLa-R3 GMASKAGTIVGKIAKVALNALa 2012.2 2012.2

8 PGLa-R4 GMATKAGTIVGKIAKVALNALa 2026.2 2026.2

8 XPF-R2 GWASKIGQTLGKMAKVGLQELIQPK 2681.5 2681.5

9 CPF-RP-R1 GFGSVLGKALKIGANLLa 1657.0 1657.0

10 CPF-RP-R2 GFGSLLGKALKIGTNLLa 1701.0 1701.0

11 PGLa-R5 GMASTAGSVLGKLAKVAIGALa 1914.2 1914.1

12 CPF-R1 GFGSFLGKALKAGLKLGANLLGGAPQQ 2613.5 2613.5

12 CPF-R2 GFGSLLGKALKAGLKLGANLLGGAPQQ 2579.5 2579.6

13 CPF-RP-R3 GIGSALAKAAKLVAGIVa 1538.0 1538.0

13 CPF-R3 GLASLLGKALKAGLKIGTHFLGGAPQQ 2646.6 2646.6

13 CPF-R4 GFGSFLGKALKAALKIGANALGGSPQQ 2601.5 2601.4

13 XPF-R3 GWASKIAQTLGKMAKVGLQELIQPK 2695.7 2695.5

14 PGLa-R6 GMASTAGSVLGKLAKTAIGILa 1958.2 1958.2

14 CPF-R5 GFGSFLGKALKAALKIGANALGGAPQQ 2585.5 2585.5

15 CPF-R6 GLGSVLGKILKMGANLLGGAPKQ 2222.4 2222.3

16 CPF-R7 GLASFLGKALKAGLKIGAHLLGGAPQQ 2616.5 2616.5

17 CPF-R8 GFASFLGKALKAALKIGANMLGGAPQQ 2659.6 2659.5

Fig. 2

24

25

26

27

28

Fig. 3

29

Magainin GIGKFLHSAGKFGKAFVGEIMKS VSEIA ALK AQGLLSGVLNQ LKQV T IT LTGG M M A

PGLa GMASKAGSVLGKLAKVALKGAL TTV TIA IT TVIGAIV A AAV V A A N A QTF I

XPF GWASKIGQTLGKMAKVGLQELIQPK LWQTVHSG K FG AFMKAFVNS VKTDAAGA I G AEQILK FFLFLLEQ V VHDVME V M L GG A L A T F N

CPF GFGSFLGKALKAALKIGANLLGGAPRQQ LAGV L LFLFGV LVGHMMA PSKEE L P I AKIP VIPKAI S IGA M A TLTDV T K T SSF VQ

CPF-RP GIGSLLGKALKLGANLL FAGVVANGARIVSGIV L TA K V VATKM AF FIE S P

Fig. 4.

30