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: [email protected] (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
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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
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(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.
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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
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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