Peptide 46 - Institutional repositoryuir.ulster.ac.uk/38739/1/Rhinophrynus dorsalis.docx · Web...
Transcript of Peptide 46 - Institutional repositoryuir.ulster.ac.uk/38739/1/Rhinophrynus dorsalis.docx · Web...
Peptidomic analysis of skin secretions of the Mexican burrowing toad Rhinophrynus
dorsalis (Rhinophrynidae): insight into the origin of host-defense peptides within the
Pipidae and characterization of a proline-arginine-rich peptide
J. Michael Conlona*, Laure Guilhaudisb, Jérôme Leprincec, Laurent Coquetd,
Maria Luisa Mangonie, Samir Attoubf, Thierry Jouenned, Jay D. Kingg
eSAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of
Ulster, Coleraine BT52 1SA, U.K.
b UNIROUEN, INSA Rouen, CNRS, COBRA, Normandy University 76000 Rouen, France
cInserm UU1239, PRIMACEN, Institute for Research and Innovation in Biomedicine (IRIB),
Normandy University, 76000 Rouen, France
dCNRS UMR 6270, PISSARO, Institute for Research and Innovation in Biomedicine (IRIB),
Normandy University, 76000 Rouen, France
eDepartment of Biochemical Sciences Instituto Pasteur-Fondazione Cenci Bolognetti, ,
Sapienza University of Rome, Rome, Italy
fDepartment of Pharmacology, College of Medicine and Health Sciences, United Arab
Emirates University, Al Ain, United Arab Emirates
g Rare Species Conservatory Foundation, St. Louis, MO 63110, U.S.A.
*Corresponding author. [email protected]
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
ABSTRACT
The Mexican burrowing toad Rhinophrynus dorsalis is the sole extant representative of the
Rhinophrynidae. United in the superfamily Pipoidea, the Rhinophrynidae is considered to be
the sister-group to the extant Pipidae which comprises Hymenochirus, Pipa,
Pseudhymenochirus and Xenopus. Cationic, α-helical host-defense peptides of the type found
in Hymenochirus, Pseudhymenochirus, and Xenopus species (hymenochirins,
pseudhymenochirins, magainins, and peptides related to PGLa, XPF, and CPF) were not
detected in norepinephrine-stimulated skin secretions of R. dorsalis. Skin secretions of
representatives of the genus Pipa also do not contain cationic α-helical host-defense peptides
which suggests, as the most parsimonious hypothesis, that the ability to produce such
peptides by frogs within the Pipidae family arose in the common ancestor of (Hymenochirus
+ Pseudhymenochirus) + Xenopus after divergence from the line of evolution leading to
extant Pipa species. Peptidomic analysis of the R. dorsalis secretions led to the isolation of
rhinophrynin-27, a proline-arginine-rich peptide with the primary structure
ELRLPEIARPVPEVLPARLPLPALPRN, together with rhinophrynin-33 containing the C-
terminal extension KMAKNQ. Rhinophrynin-27 shows limited structural similarity to the
porcine multifunctional peptide PR-39 but it lacks antimicrobial and cytotoxic activities. Like
PR-39, the peptide adopts a poly-L-proline helix but some changes in the circular dichroism
spectrum were observed in the presence of anionic sodium dodecylsulfate micelles consistent
with the stabilization of turn structures.
.
Key words: Frog skin; Rhinophrynidae, Pipidae, Host-defense, Antimicrobial, PR-39
2
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
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
3
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
fragment (CPF) and caerulein-precursor fragment-related peptide (CPF-RP) derived from the
post-translational processing of procaeruleins (reviewed in [11,15]). In contrast, cytotoxic
HDPs were not detected in skin secretions of Pipa pipa ([11], Pipa carvalhoi [16]) and Pipa
parva (J.M. Conlon, unpublished data),
The New world frog R. dorsalis is found in coastal lowland regions (from sea-level to
500 m) in a range of habitats, such as tropical dry forests, grasslands, thorn scrub, and
cultivated fields. Although relatively rare in Texas, the species is common and widespread in
Mexico and northern Central America and is listed as Species of Least Concern by the
International Union for Conservation of Nature (IUCN) Red List [17]. The animal is strictly
fossorial, only emerging from its underground burrow to reproduce after the first rains with
the result that it is rarely seen in the wild. The present study uses peptidomic analysis
(reversed-phase HLPC coupled with MALDI-TOF mass spectrometry and automated Edman
degradation) to investigate the occurrence of HDPs in norepinephrine-stimulated skin
secretions from R. dorsalis.
2. Experimental
2.1. Collection of skin secretions
All experiments with live animals were approved by the Gladys Porter Zoo Scientific
and Research Committee and were carried out by authorized investigators. Four adult R.
dorsalis frogs (1 male, 16 g body weight; 3 female, 20, 20, and 30 g body weight) were
collected in the grounds of Rio Grande City High School, Rio Grande City, TX and were
housed in a vivarium at the Gladys Porter Zoo, Brownsville, TX.
4
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
Each frog was injected via the dorsal lymph sac with norepinephrine hydrochloride
(40 nmol/g body weight) and placed in a solution (100 ml) of distilled water for 15 min. The
injection did not produce the kind of copious, milky skin secretions often seen when Xenopus
species are stimulated with norepinephrine but the collection solution became more turbid
and foamy. The frog was removed and the collection solution was acidified by addition of
trifluoroacetic acid (TFA) (1 ml) and immediately frozen for shipment to Ulster University.
The solutions containing the secretions from each frog were pooled and passed at a flow rate
of 2 ml/min through 6 Sep-Pak C-18 cartridges (Waters Associates, Milford, MA) connected
in series. Bound material was eluted with acetonitrile/ water/TFA (70.0:29.9:0.1, v/v/v) and
freeze-dried. The material was redissolved in 0.1% (v/v) TFA/water (2 ml).
2.2. Peptide purification
The pooled skin secretions from R. dorsalis, after partial purification on Sep-Pak
cartridges, were injected onto a semipreparative (1 cm x 25 cm) Vydac 218TP510 (C-18)
reversed-phase HPLC column (Grace, Deerfield, IL) equilibrated with 0.1% (v/v) TFA/water
at a flow rate of 2.0 ml/min. The concentration of acetonitrile in the eluting solvent was
raised to 21% (v/v) over 10 min and to 63% (v/v) over 60 min using linear gradients.
Absorbance was monitored at 214 nm and peak fractions were collected by hand. The
peptides within the peaks that were present in major abundance were subjected to further
purification. These components were purified to near homogeneity, as assessed by a
symmetrical peak shape and mass spectrometry, by chromatography on (1.0 cm x 25 cm)
Vydac 214TP510 (C-4) and (1.0 cm x 25 cm) Vydac 219TP510 (phenyl) columns. The
concentration of acetonitrile in the eluting solvent was raised from 7% to 35% over 50 min
5
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
for the more hydrophilic components and from 21% to 49% over 50 min for the more
hydrophobic components. The flow rate was 2.0 ml/min.
2.3. Structural characterization
MALDI-TOF mass spectrometry was carried out using a Voyager DE-PRO
instrument (Applied Biosystems, Foster City, CA) that was operated in reflector mode with
delayed extraction and the accelerating voltage in the ion source was 20 kV. The instrument
was calibrated with peptides of known molecular mass in the 2 - 4 kDa range. The accuracy
of mass determinations was 0.02%. Spectra were recorded using both -cyano-4-
hydroxycinnamic acid and sinapinic acid as matrix solutions. The complete primary
structures of peptides in the mass range 1 - 4 kDa were determined by automated Edman
degradation using a model 494 Procise sequenator (Applied Biosystems). For larger
peptides/proteins (molecular mass > 4kDa), only the amino acid sequence at N-terminus
(residues 1-10) were determined in order to permit identification. Amino acid composition
analyses were performed by the University of Nebraska Medical Center Protein Structure
Core Facility (Omaha, NE).
2.4. Peptide synthesis
Rhinophrynin-27 (ELRLPEIARPVPEVLPARLPLPALPRN) was supplied at a purity
> 95% by Synpeptide Co., Ltd (Shanghai, China). Its identity and purity were confirmed by
electrospray mass spectrometry.
2.5. Antimicrobial and cytotoxicity assays
6
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
Reference strains of microorganisms were purchased from the American Type Culture
Collection (Rockville, MD, USA). Minimum inhibitory concentrations (MIC) of
rhinophrynin-27 against reference strains of Staphylococcus epidermidis (ATCC 12228),
Bacillus megaterium (Bm11), Escherichia coli (ATCC 25922) and Candida parapsilosis
(ATCC 22019) were measured by standard microdilution methods [18,19] as previously
described [20]. Hemolyic activity was determined by incubation of washed erythrocytes (2 x
107 cells) from male NIH male Swiss mice (Harlan Ltd, Bicester, UK) with rhinophrynin-27 (
31.3 - 500 μM ) for 60 min at 37 oC as previously described [8]. Cytotoxicity of
rhinophrynin-27 against human non-small cell lung adenocarcinoma A549 cells was
measured as previously described [21]. The effects of the peptide (1 - 100 μM) on cell
viability were determined by measurement of ATP concentrations using a CellTiter-Glo
Luminescent Cell Viability assay (Promega Corporation, Madison, WI, USA). All animal
experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act
1986 and EU Directive 2010/63EU for animal experiments.
2.6. CD spectra
Spectra were obtained using a MOS-500 Circular Dichroism Spectrometer (Bio-Logic,
Claix, France). Data points were collected from 260 nm to 185 nm, with an integration time
of 2 s per point and a step size of 1 nm, using a 1.0 mm path length rectangular quartz cell.
Measurements were carried out at room temperature, 20°C and 5.5°C. Rhinophrynin-27 was
dissolved in water, in 2,2,2-trifluoroethanol (TFE)-water (25% and 50%, v/v), in 20 mM
sodium dodecyl sulfate (SDS) aqueous solution, and in 20 mM dodecylphosphocholine
(DPC) aqueous solution at a final concentration of 0.18 - 0.21 mg/ml. The concentration of
20 mM for detergents was chosen to ensure micelle formation. For each spectrum, three scans
7
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
were accumulated and then averaged. The baseline was obtained by recording a spectrum of
the solvent, and the mean residue molar ellipticity ([θ]MRE), deg cm2 dmol−1, was calculated
from the observed ellipticity after baseline correction. The -helical content was estimated by
using the Forood formula [22].
3. Results
3.1. Purification of the peptides
The pooled skin secretions from R. dorsalis, after partial purification on Sep-Pak C-18
cartridges, were chromatographed on a Vydac C-18 semipreparative reversed-phase HPLC
column (Fig. 1). The prominent peaks designated 1 - 19 were collected by hand and subjected
to further purification. The major components present in each peak were purified to near
homogeneity, as assessed by a symmetrical peak shape and mass spectrometry, by further
chromatography on semipreparative Vydac C-4 and Vydac phenyl columns. The
methodology is illustrated by the purification of rhinophrynin-27 (Fig. 2).
3.2. Structural characterization
The molecular masses of the components purified from R. dorsalis skin secretions are
shown in Table 1. The compounds from peaks 1-4, 7-9, and 15 with masses < 600 kDa were
shown by Edman degradation and amino acid composition analysis not to be peptides. The
nature of these substances, which are present in relatively high concentrations in the
secretions, remains to be determined. The complete primary structures of the peptides present
in peaks 5, 6, 12, and 13 were determined by Edman degradation. A BLAST search (National
8
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
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.
9
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
3.3. Antimicrobial and cytotoxic activities
Rhinophrynin-27 produced < 5% hemolysis during a 60 min incubation with freshly-
prepared mouse erythrocytes at concentrations up to 500 μM and showed <5% cytolytic
activity against human non-small cell lung adenocarcinoma A549 cells at concentrations up
to 100 μM (data not shown). Rhinophrynin-27 did not significantly inhibit the growth of the
Gram-negative bacterium E. coli, the Gram-positive bacteria S. epidermidis and B.
megaterium, and the opportunist yeast pathogen C. parapsilosis at concentrations up to 128
μM.
3.4. Conformational analysis
In water, the CD spectrum of rhinophrynin-27 exhibited a strong negative band at 198 nm
with a slight shoulder around 225 nm (Fig. 3). This spectrum resembles those of other proline
rich peptides that have been reported to adopt a left handed polyproline type II (PPII) helical
structure [23-27]. The essential features of this structure are a strong negative band in the
vicinity of 200 nm and a weak positive band around 220 nm. In contrast, disordered
structures tend to exhibit a negative peak around 195 nm, and a positive peak around 220 nm,
is absent. Although a positive peak around 220 nm was not observed in the spectrum of
rhinophrynin-27, the strong intensity of the negative band as well as its high proline content
(26%) suggest the presence of a PPII conformation in the peptide. Similar conclusions were
drawn from CD analyses of several Pro-rich peptides, for which it was reported that the
absence of the weak positive peak was attributable to a relative small number of proline
residues or to deviation from the ideal PP-II structure [27-29].
10
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
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.
11
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
267
268
269
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
12
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
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
13
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
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.
14
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
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.
15
344
345
346
347
348
349
350
351
REFERENCES
[1] D.R. Frost, 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, 2017.
[2] R.O. de Sá, D.M. Hillis, Phylogenetic relationships of the pipid frogs Xenopus and
Silurana: an integration of ribosomal DNA and morphology, Mol. Biol. Evol. 7 (1990)
365-376.
[3] B.J. Evans, Genome evolution and speciation genetics of clawed frogs (Xenopus and
Silurana), Front. Biosci. 13 (2008) 4687-4706.
[4] B.J. Evans, T.F. Carter, E. Greenbaum, V. Gvoždík, D.B. Kelley, P.J. McLaughlin, ,et
al., 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 (2015) e0142823.
[5] S. McLoughlin, The breakup history of Gondwana and its impact on preCenzoic
floristic provincialism, Aust. J. Bot. 49 (2001) 271-300.
[6] A.J. Bewick, F.J. Chain, J. Heled, B.J. Evans, The pipid root, Syst. Biol. 61 (2012) 913-
926.
[7] D.R. Frost, T. Grant, J. Faivovich, R.H. Bain, A. Haas, C.F.B. Haddad, et al., The
amphibian tree of life, Bull. Am. Mus. Nat. His. 297 (2012) 1-370.
[8] M. Mechkarska, M. Prajeep, L. Coquet, J. Leprince, T. Jouenne, H. Vaudry et al., The
hymenochirins: a family of host-defense peptides from the Congo dwarf clawed frog,
Hymenochirus boettgeri (Pipidae). Peptides 35 (2012) 269-275.
16
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
[9] S. Matthijs, L. Ye, B. Stijlemans, P. Cornelis, F. Bossuyt, K. Roelants, Low structural
variation in the host-defense peptide repertoire of the dwarf clawed frog Hymenochirus
boettgeri (Pipidae), PLoS One 9 (2014) e86339.
[10] J.M. Conlon, M. Prajeep, M. Mechkarska, L. Coquet, J. Leprince, T. Jouenne, et al.,
Characterization of the host-defense peptides from skin secretions of Merlin's clawed
frog Pseudhymenochirus merlini: insights into phylogenetic relationships among the
Pipidae, Comp. Biochem. Physiol. Part D Genomics Proteomics 8 (2013) 352-357.
[11] J.M. Conlon, M. Mechkarska, Host-defense peptides with therapeutic potential from
skin secretions of frogs from the family Pipidae, Pharmaceuticals (Basel) 7 (2014) 58-
77.
[12] L. Coquet, J. Kolodziejek, T. Jouenne, N. Nowotny, J.D. King, J.M. Conlon,
Peptidomic analysis of the extensive array of host-defense peptides in skin secretions
of the dodecaploid frog Xenopus ruwenzoriensis (Pipidae), Comp. Biochem. Physiol.
Part D Genomics Proteomics 19 (2016) 18-24.
[13] J.M. Conlon, M. Mechkarska, M.L. Lukic, P.R. Flatt, Potential therapeutic
applications of multifunctional host-defense peptides from frog skin as anti-cancer,
anti-viral, immunomodulatory, and anti-diabetic agents, Peptides 57 (2014) 67-77.
[14] X. Xu, R. Lai, The chemistry and biological activities of peptides from amphibian
skin secretions, Chem. Rev. 115 (2015) 1760-1846.
[15] K. Roelants, B.G. Fry, L. Ye, B. Stijlemans, B. L. Brys, P. Kok, P. et al., Origin and
functional diversification of an amphibian defense peptide arsenal, PLoS Genet. 9
(2013e1003662.
17
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
[16] D.O. Mariano, L.F. Yamaguchi, C. Jared, M.M. Antoniazzi, J.M. Sciani, M. J. Kato, et al.,
Pipa carvalhoi skin secretion profiling: absence of peptides and identification of kynurenic
acid as the major constitutive component, Comp. Biochem. Physiol. C Toxicol. Pharmacol.
167 (2015) 1-6.
[17] G. Santos-Barrera, G. Hammerson, F. Bolaños, F., Chaves G, Wilson, L.D., Savage,
J., et al., Rhinophrynus dorsalis, The IUCN Red List of Threatened Species 2010:
e.T59040A11873951.
[18] Clinical Laboratory and Standards Institute. Methods for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically. Approved Standard M07-A8.
CLSI, Wayne, PA, 2008.
[19] Clinical Laboratory and Standards Institute. Reference method for broth dilution
antifungal susceptibility testing of yeast. Approved Standard M27-A3. CLS1, Wayne,
PA, 2008.
[20] M.L. Mangoni, A. Carotenuto, L. Auriemma, M.R. Saviello, P. Campiglia, I.
Gomez-Monterrey, et. al., Structure-activity relationship, conformational and
biological studies of temporin L analogues. J. Med. Chem. 54 (2011) 1298-1307.
[21] S. Attoub, H. Arafat, M. Mechkarska, J.M. Conlon, Anti-tumor activities of the host-
defense peptide hymenochirin-1B, Regul. Pept. 187 (2013) 51-56.
[22] B. Farood, E.J. Filiciano, K.P. Niambiar, Stabilization of α-helical structures in short
peptides via end capping, Proc. Natl. Acad. Sci. USA 90 (1993) 838-842
[23] M.L. Tiffany, S. Krimm, S., Circular dichroism of poly-L-proline in an unordered
conformation, Biopolymers 6 (1968) 1767-1770.
[24] M.L. Tiffany, S. Krimm, Effect of temperature on the circular dichroism spectra of
polypeptides in the extended state, Biopolymers 11 (1972) 2309-2316.
18
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
[25] E.W. Ronish, S. Krimm, The calculated circular dichroism of polyproline II in the
polarizability approximation. Biopolymers 13 (1974) 1635-1651.
[26] A.L. Rucker, C.T. Pager, M.N. Campbell, J.E. Qualls, T.P. Creamer, Host-guest scale
of left-handed polyproline II helix formation, Proteins 53 (2003) 68-75.
[27] J.L. Lopes, A.J. Miles, L. Whitmore, B.A. Wallace, Distinct circular dichroism
spectroscopic signatures of polyproline II and unordered secondary structures:
applications in secondary structure analyses, Protein Sci. 23 (2014) 1765-1772.
[28] P.A. Raj, M. Edgerton, Functional domain and poly-L-proline II conformation for
candidacidal activity of bactenecin 5, FEBS Lett. 368, (1995) 526-530.
[29] T. Niidome, H. Mihara, M. Oka, T. Hayashi, T. Saiki, K. Yoshida, et al., Structure and
property of model peptides of proline/arginine-rich region in bactenecin 5, J. Peptide
Res. 51 (1998) 337-345.
[30] W.A. Elam, T.P. Schrank, A.J. Campagnolo, V.J. Hilser, Temperature and urea have
opposing impacts on polyproline II conformational bias, Biochemistry 52 (2013) 949-
958
[31] K. Roelants, F. Bossuyt, Archaeobatrachian paraphyly and pangaean diversification of
crown-group frogs. Syst. Biol. 54 (2005) 111-126.
[32] K. Roelants, D.J. Gower, M. Wilkinson, S.P. Loader, S.D. Biju, K. Guillaume, et al.,
Global patterns of diversification in the history of modern amphibians, Proc. Natl.
Acad. Sci. USA 104 (2007) 887-892.
[33] I. Irisarri, M. Vences, D. San Mauro, F. Glaw, R. Zardoya, Reversal to air-driven sound
production revealed by a molecular phylogeny of tongueless frogs, family Pipidae, BMC
Evol. Biol. 11 (2011) 114.
[34] D.C. Cannatella, L. Trueb. Evolution of pipoid frogs: intergeneric relationships of the
aquatic frog family Pipidae (Anura). Zool. J. Linnean Soc. 94 (1988) 1-38.
19
409
410
411
412
413
414
415
416
417
418
419
420
421
[35] D.C. Cannatella, L. Trueb. Evolution of pipoid frogs. Morphology and phylogenetic
relationships of Pseudhymenochirus. J. Herpetol. 22 (1988) 439-456.
[36] M. Scocchi, A. Tossi, R. Gennaro, Proline-rich antimicrobial peptides: converging to
a non-lytic mechanism of action, Cell. Mol. Life Sci. 68 (2011) 2317-2330.
[37] B. Agerberth, J.Y. Lee, T. Bergman, M. Carlquist, H.G. Boman, V. Mutt, et al., Amino
acid sequence of PR-39. Isolation from pig intestine of a new member of the family of
proline-arginine-rich antibacterial peptides, Eur. J. Biochem. 202 (1991) 849-854.
[38] H. Wu, G. Zhang, C.R. Ross, F. Blecha, Cathelicidin gene expression in porcine
tissues: roles in ontogeny and tissue specificity, Infect. Immun. 67 (1999) 439-442.
[39] R. Gennaro, B. Skerlavaj, D. Romeo, Purification, composition, and activity of
two bactenecins, antibacterial peptides of bovine neutrophils, Infect. Immun. 57
(1989) 3142-3146.
[40] J.A. Rees, M. Moniatte, P. Bulet, Novel antibacterial peptides isolated from a
European bumblebee, Bombus pascuorum (Hymenoptera, Apoidea), Insect Biochem.
Mol. Biol. 27 (1997) 413-422.
[41] H.G. Boman, B. Agerberth, A. Boman, Mechanisms of action on Escherichia coli of
cecropin P1 and PR-39, two antibacterial peptides from pig intestine, Infect. Immun.
61 (1993) 2978-2984.
[42] Y.R. Chan, M. Zanetti, R. Gennaro, R.L. Gallo, Antimicrobial activity and cell
binding are controlled by sequence determinants in the anti-microbial peptide PR-39,
J. Invest. Dermatol. 116 (2001) 230–235.
[43] R. Holani, C. Shah, Q. Haji, G.D. Inglis, R.R. Uwiera, E.R. Cobo, Proline-
arginine rich (PR-39) cathelicidin: Structure, expression and functional
implication in intestinal health, Comp. Immunol. Microbiol. Infect. Dis. 49
(2016) 95-101.
20
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
[44] V. Cabiaux, B. Agerberth, J. Johansson, F. Homblé, E. Goormaghtigh, J.M.,
Ruysschaert, Secondary structure and membrane interaction of PR-39, a Pro+Arg-rich
antibacterial peptide, Eur. J. Biochem. 224 (1994) 1019-1027.
21
443
444
445
446
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.
22
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
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.
23
472
473
474
475
476
477
478
479
480
Fig. 1
24
481
482
483
Fig.2
25
484
485
486
487
488
489
Fig. 3.
26
490
491
492
493
494
Fig. 4
27
495
496
497
498
Fig. 5
28
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
Rhinophrynin-33 ELRLPEIARPVPEVL*PARLPLPALPRNKMAKNQ
Rhinophrynin-27 ELRLPEIARPVPEVL*PARLPLPALPRN
PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP
Bac5 RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLRFP
Abaecin PYNPPRPGQSKPFPTFPGHGPFNPKIQWPYPLPNPGH
Fig. 6
29
517
518
519
520
521
522
523
Table 1. Complete or partial amino acid sequences, observed molecular masses ([Mr+H]obs),
and calculated molecular masses ([Mr+H]calc) of components isolated from skin secretions of
R. dorsalis
Peak no. [M + H]+obs [M + H]+
calc Amino acid sequence Putative assignment
1 279.2 Non-peptide
2 321.2 Non-peptide3 414.3 Non-peptide4 430.2 Non-peptide5 1411.8 1411.8 IPHEHRPRIQE Laminin α-chain fragment6 1510.9 1510.8 VIPHEHRPRIQE Laminin α-chain fragment7 398.2 Non-peptide7 444.1 Non-peptide8 383.2 Non-peptide8 397.2 Non-peptide9 412.3 Non-peptide9 428.3 Non-peptide10 5548.3 VIVPPNHKDA….. Unknown11 5663.6 LVIVPPNHKDA….. unknown12 3727.4 3727.2 ELRLPEIARPVPEVLPARL
PLPALPRNKMAKNQRhinophrynin-33
12 7348.2 LKCNYCKNGRSF….. Zinc finger MYM-type protein 2 isoform X1 fragment
13 3027.2 3026.8 ELRLPEIARPVPEVLPARLPLPALPRN
Rhinophrynin-27
14 563.4 Non-peptide15 7266.2 Not determined16 19,148 Not determined17 18,903 Not-determined18 19,017 YRTVYRCSTA,…. Unknown19 5876.6 Non-determined
Peak No. refers to the chromatogram shown in Figure 1.
30
524
525
526
527
528
529
530