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First published online August 8, 2008
Journal of Experimental Biology 211, 2707-2711 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.019315
Novel surfactant proteins are involved in the structure and stability of foam nests from the frog Leptodactylus vastus
1 Departamento de Biologia, Universidade Federal do Ceará, Av. Humberto
Monte 2775 Campus Pici, Bloco 909, Fortaleza, Brazil, 60455-000
2 Departamento de Bioquímica e Biologia Molecular, Universidade Federal
do Ceará, Campus Pici, Fortaleza, Brazil
3 Departamento de Engenharia Química, Universidade Federal do Rio Grande
do Norte, Natal, Brazil
* Author for correspondence (e-mail: vmmmelo{at}ufc.br)
Accepted 5 June 2008
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Summary |
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Key words: Amphibia, Leptodactylus vastus, foam nest, surfactants, frog proteins
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Andrade and Abe,
1997; Downie and Smith,
2003). In amphibians the foam nests are associated with their
evolution to terrestrial life (Heyer,
1975) and several functions have been attributed to them, such as
protection against potential predators, pathogens
(Heyer, 1969;
Downie, 1993;
Cooper et al., 2005) and
thermal damage (Gorzula,
1977), a food source for tadpoles
(Tanaka and Nishihira, 1987),
desiccation prevention (Downie,
1988) and respiratory advantages to the embryos
(Seymour, 1994).
The neotropical frogs of the genus Leptodactylus are an example of
a group under transition of reproductive and larval development modes from an
aquatic to a terrestrial habitat (Heyer,
1969) that have benefited from the capacity to produce foam nests.
The species Leptodactylus vastus A. Lutz 1930 is endemic to South
America, occurring specifically in Northeast Brazil
(Heyer, 2005). The female of
this frog releases eggs and a proteinaceous fluid, which is whipped up by the
male using his legs in a rapid motion that simultaneously fertilizes the eggs.
The foam nests are gradually disintegrated and by the end of several weeks
they naturally turn into a dense liquid. Similar behaviour has been observed
in the frogs Physalaemus pustulosus found in Central/South America
and parts of the Caribbean (Cooper et al.,
2005) and Polypedates leucomystax found in Malaysia and
adjacent regions of South East Asia
(McMahon et al., 2006).
In spite of some interesting properties assigned to amphibian foam nests,
little is known about their composition and molecular mechanisms of production
and long-term stability. One of the few works on this subject focussed on the
foam nest of P. pustulosus, which was revealed to be a rich source of
proteins with unusual primary structures and remarkable surfactant activity,
named ranaspumins (Cooper et al.,
2005). More recently, McMahon and colleagues described the crystal
structure of the 13 kDa surfactant protein isolated from foam nests of P.
leucomystax, named ranasmurfin
(McMahon et al., 2006). It has
been hypothesized that it could be a novel protein since searches with the
available partial sequence data did not reveal any match to any known protein
or structure.
Taking into consideration the physicochemical characteristics of foam nest
proteins and the importance of surfactants produced by vertebrates,
particularly due to their biodegradability and biocompatibility, proteins like
ranaspumins and ranasmurfin open up huge possibilities of biomedical and
industrial applications (Kaneko et al.,
1990; Makkar and Rockne,
2003; Cooper et al.,
2005). Keeping these clues in mind, the main objective of the
present work was to study the foam nests of L. vastus in order to add
information regarding their composition and function and to improve the
understanding of ranaspumins, which are probably a novel class of surfactant
proteins.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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), and total carbohydrate by using the phenol/sulphuric acid
method with glucose as the standard
(Dubois et al., 1956).
Absorption spectrum
The ultraviolet-visible spectrum of foam fluid (1.0 mg
ml–1 protein) was determined in 1 cm pathlength quartz
cuvettes using a spectrophotometer (Genesys 10UV, Spectronic Unicam,
Rochester, NY, USA) at a wavelength range of 210 to 700 nm.
Microbiota associated with foam nests
The colonization of the foam nests by bacteria was evaluated by cell viable
counting. For the assay, 1.0 g of foam nests collected aseptically was
immediately dissolved in 9.0 ml of sterile saline and the suspension was
homogenized by vortexing for 2 min. The resultant solution was serially
diluted (10–1, 10–2, up to
10–6) and 0.1 ml of each dilution was spread onto
Müller–Hinton Agar (Difco Laboratories, Detroit, MI, USA) in
duplicate. Simultaneously, samples of pond water from the same environment as
the foam nests were analysed. The plates were incubated at 30°C and
observed for development of colonies over 48h. The test was repeated twice.
The macromorphology of colonies was analysed and cell morphology studied in
slide preparations stained by the Gram procedure.
Antimicrobial activity
The antibacterial susceptibility test was determined by the disc diffusion
method on Müller–Hinton agar, as described by Bauer and colleagues
(Bauer et al., 1966) against
the following bacteria from American Type Culture Collection (ATCC):
Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa
(ATCC 25319), Salmonella choleraesuis (ATCC 10708), Bacillus
subtilis (ATCC 6633), Chromobacterium violaceum (ATCC 12472),
Klebsiella pneumoniae (ATCC 10031) and Enterobacter
aerogenes (ATCC 13048). The antifungal activity test was done as
described by Bormann and colleagues
(Bormann et al., 1999).
Briefly, an agar plug containing mycelia of the test fungus was placed in the
centre of a Petri dish containing 15 ml of Sabouraud agar (Difco) and
incubated until the colony reached a diameter of 3–4 cm. A 6 mm sterile
paper disc that had absorbed 15 µl of foam fluid was placed at the growing
front of the hyphae, and the test plates were incubated at 30°C and
monitored for inhibition of hyphal growth. The tests were done against the
fungi Trichoderma viride, Penicillium herguei, Colletotrichum
lindemuthianum, Fusarium solani, Aspergillus fumigatus, Pythium oligandrum,
Phomopsis sp., Rhizoctonia solani, Mucor sp. and
Neurospora sp. The tests were done in duplicate and repeated twice.
All microorganisms were obtained from the culture collections of the Microbial
Ecology and Biotechnology Laboratory, Biology Department of the Federal
University of Ceara, Brazil.
Haemagglutinating activity
Haemagglutinating activity of the foam fluid was carried out essentially as
described by Moreira and Perrone (Moreira
and Perrone, 1977). Serial 1:2 dilutions of the foam fluid were
mixed in small glass tubes with untreated or trypsin-treated rabbit, rat and
mouse erythrocytes (2% suspension prepared in 0.15 moll–1
NaCl). The enzyme-treated cells were obtained by incubation of trypsin (0.1
mg) with 25 ml of the 2% suspension for 60 min at 4°C. After washing six
times, a 2% suspension was prepared in 0.15 moll–1 NaCl. The
degree of agglutination was monitored visually after the tubes had been left
to stand at 37°C for 30 min and at room temperature for an additional 30
min. The haemagglutination titre was defined as the minimal amount (µg) of
protein per millilitre able to induce visible erythrocyte agglutination. The
assay was performed in duplicate and repeated twice.
Haemolytic activity
The assay for haemolytic activity of the foam fluid was done using rabbit
erythrocytes according to a previous publication
(Nakajima et al., 2003).
Erythrocytes were isolated from heparinized blood by centrifugation at 1000
g for 5 min and washed three times with phosphate-buffered
saline (PBS), pH 7.4. A 1000 µl sample of cell suspension adjusted to
1x109 cells ml–1 in PBS, pH 7.4, was
incubated at 37°C for 30 min with an equal volume of foam fluid. The
incubation media were centrifuged at 1000 g for 5 min and
aliquots (200 µl) of supernatant were diluted in water (2000 µl). The
absorbance of the diluted solution was measured at 420 nm. The absorbance
values obtained after treating erythrocytes with PBS and 2% SDS were taken as
0% and 100%, respectively. The test was performed in duplicate and repeated
twice.
Larvicidal activity
The larvae toxicity assay was run against Aedes aegypti according
to Thangam and Kathiresan (Thangam and
Kathiresan, 1991), with some modifications. Third stage larvae
were collected with a Pasteur pipette placed on filter paper for removal of
excess water and transferred (10 per test) with a tiny brush into test tubes
containing 5 ml of 20% foam fluid (v/v). Larvae were exposed to the samples at
room temperature and mortality/survival registered after 24h. Each test was
run in triplicate and distilled water was used as the control.
Toxicity activity
Foam fluid toxicity was assessed in mice (Swiss strain from the animal
facilities of the Federal University of Ceara, Brazil) by intraperitoneal
injection of fluid (30 mg protein kg–1 body weight) according
to Litchfield and Wilcoxon (Litchfield and
Wilcoxon, 1949). The animals were kept under observation until 48h
after inoculation for description of symptoms or death quantification.
Surfactant activity
The surfactant activity was evaluated by the emulsification activity
(Iqbal et al., 1995). Briefly,
the foam fluid (1.0 mg ml–1) or the purified protein (0.1 mg
ml–1) was combined with the same volume of kerosene in a 20
ml screw-capped tube and was homogenized using a vortex for 2 min and left to
stand for 24h. The emulsification index (EI, %) was calculated using the
equation EI=(height of emulsion layer/height of oil plus emulsion
layer)x100. The capacity of the foam fluid to reduce the water surface
tension was also measured according to the Du Nouy ring method (ASTM D971,
ASTM International Standards Worldwide;
http://www.astm.org/)
using pure water at 30°C for calibration. Both the surface tension and EI
measurements were performed in triplicate and each experiment was repeated
twice.
Isolation and characterization of the surfactant protein of the foam fluid
The surfactant protein of the foam fluid was purified by CM-Sepharose
chromatography. Briefly, 16mg of lyophilized foam fluid was dissolved in 2.0
ml of 0.05 moll–1 Tris-HCl buffer pH 7.0 and applied to a
CM-Sepharose fast flow column equilibrated with the same buffer. After elution
of non-retained proteins with 0.05 moll–1 Tris-HCl buffer pH
7.0, the adsorbed proteins were eluted with the same buffer containing 0.5
moll–1 NaCl. The chromatography was monitored at 280 nm and
the surfactant activity followed through an emulsification activity test. The
protein fractions were subjected to Tricine-SDS-PAGE according to
Schägger and von Jagow (Schägger
and von Jagow, 1987). Protein bands were stained with 0.05%
Coomassie Brilliant Blue R-250.
N-terminal sequence analysis
The N-terminal amino acid sequence of the surfactant protein was determined
on a Shimadzu PPSQ-10 automated protein sequencer (Kyoto, Japan) performing
Edman degradation. The sequence was determined from protein blotted on
polyvinylidene fluoride (PVDF) after Tricine-SDS-PAGE. Phenylthiohydantoin
(PTH) amino acids were detected at 269 nm after separation on a reversed phase
C18 column (4.6 mmx2.5 mm) under isocratic conditions,
according to the manufacturer's instructions. The sequence was compared with
available amino acid sequence databases. The sequence was subjected to
automatic alignment, which was performed using the NCBI-BLAST search system
(Altschul et al., 1997).
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RESULTS |
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Biological activity
The foam fluid did not show antimicrobial activity, acute toxicity to mice
or larvicidal action. As for haemagglutinating activity, the foam fluid showed
only traces of agglutination in trypsin-treated rat erythrocytes but it was
negative for rabbit and mouse erythrocytes. In addition, trypsin-treated rat
and rabbit erythrocytes showed only slight haemolysis when in contact with
foam fluid.
Surfactant activity
The whole foam fluid (1.0±0.2mg proteinml–1) was
able to emulsify kerosene and this activity was quite stable, persisting for
over a month, even when the tubes containing the fluid and the hydrocarbon
were placed upside down (data not shown). The emulsification index was 62%.
The foam fluid also reduced the surface tension of the water from 74 to 52 mN
m–1.
Purification and characterization of a surfactant protein
The foam fluid was separated into two fractions by ion-exchange
chromatography. The non-retained fraction eluted with 0.05
moll–1 Tris-HCl pH 7.0 showed a well-defined and dense
protein band of approximately 20 kDa, whereas the retained fraction, eluted
with 0.5 moll–1 NaCl in the same buffer, was characterized by
exclusion of the 20 kDa band (Fig.
2). Both fractions showed surfactant activity and the 20 kDa
protein at 0.1 mg ml–1 presented an emulsification index of
57%. The N-terminal amino acid sequence of the 20 kDa protein had the
following composition: FLEGFLVPKVVPGPTAALLKKALDD.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). The
foam nests also make it possible for L. vastus to lay the eggs at the
beginning of the rainy season, even before the complete formation of the water
ponds in the margins of which the foam nests are produced, as the tadpoles can
remain in the foam nests until the water volume increases and washes the
tadpoles to the pond.
Investigation of the role of foam nests in chemical defence revealed no
significant biological activity that could be associated with some recognized
defence compounds such as lectins, enzymes, haemolysins, antimicrobial
compounds or toxins, commonly detected in secretions released by vertebrates
(Bols et al., 1986;
Conceição et al.,
2007; Che et al.,
2008) and invertebrates
(Canicatti et al., 1992;
Becerril et al., 1996;
Melo et al., 2000;
Derby, 2007).
The foam nests of L. vastus seem to be involved in the physical
protection of eggs and tadpoles. This protection is ensured by its unusual
stability, which allows the eggs to be suspended in the water or to develop on
land, giving good access to moisture, oxygen and a food source, the latter
being the foam itself or the non-fecundated eggs
(Vinton, 1951;
Muedeking and Heyer, 1976).
Foam nests may float on the surface of warm tropical pools, providing access
to atmospheric oxygen for the newly hatched tadpoles
(Seymour and Loveridge, 1994)
and preventing the eggs from sinking to areas with lower oxygen content
(Seymour and Roberts, 1991).
Terrestrial incubation of typically aquatic eggs provides increased access to
oxygen but also increased risk of desiccation and sun damage
(Martin and Strathmann, 1999).
In this particular case, the UV absorbance of the foam nests may be
fundamental to the prevention of desiccation and sun damage since the foams
absorb maximally at 280 nm, protecting eggs and embryos against UV injury. As
a matter of fact, this was one of the first hypothesized functions for foam
nests (Gorzula, 1977;
Downie, 1988). The UV
absorbance of foam nests is particularly important since the decline in
amphibian populations has been widely reported in recent times and several
studies have suggested that UV radiation may be one of the contributing
factors (Blaustein and Belden,
2003).
The foam nest of the frog L. vastus is characterized by long-term stability and surfactant activity. These properties probably provide desiccation resistance either by slowing evaporative water loss or by drawing water towards the developing eggs.
Cooper and colleagues have already described the foam nest properties of
the common frog from Central/South America and parts of the Caribbean, P.
pustulosus (Cooper et al.,
2005). Those authors have also reported some physicochemical
properties of the crude foam fluid of this frog, and have named the mixture of
proteins in the 10–40 kDa mass range ranaspumin. They presented evidence
of the extraordinary surfactant activity of ranaspumin and its involvement in
foam nest stabilization. Nevertheless, the authors did not associate any
protein in particular with the surfactant activity. In the present work, the
foam fluid of L. vastus showed a different profile comprising several
proteins with molecular masses in the range 14 to over 97 kDa.
The foam fluid (1.0±0.2 mg protein ml–1) of L.
vastus showed strong and stable emulsification and was particularly
effective in reducing the water surface tension, which dropped from 74 to 52
mN m–1. Cooper and colleagues have obtained similar results
for water surface tension with foam fluid from P. pustulosus
(Cooper et al., 2005),
distinguishing the superiority of this property when compared with those of
common globular proteins such as bovine serum albumin (BSA), which at the same
concentration, 1.0mgml–1, reduced the water surface tension
to 65 mN m–1. This is a significant finding considering that
surfactants produced by vertebrates are of great interest due to their
biodegradability and biocompatibility, with a huge potential for biomedical
and industrial utilization (Kaneko et al.,
1990; Makkar and Rockne,
2003; Rodrigues et al.,
2006).
Chemicals with surface-active properties are synthesized by an amazing
variety of living bodies, from plants (e.g. saponins) and microorganisms (e.g.
glycolipids, lipopeptides/lipoproteins, phospholipids, neutral lipids,
substituted fatty acids, lipopolysaccharides and hydrophobins) to higher
complexity animals (e.g. bile acids) for intra- and extracellular activities,
which may range from emulsification of food for the transport of material
across cell membranes to recognition of cells and defence
(Ivshina et al., 1998;
Hofmann, 1999;
Wang et al., 2003). Thus, frog
foam nests appear to be new sources of surface-active compounds that need to
be explored for better characterization, understanding of their biological
role and potential use.
As an attempt to identify and better characterize the proteins responsible for the surfactant activity, the foam fluid of L. vastus was subjected to ion-exchange chromatography, which allowed us to separate its proteins into two fractions. A well-defined and dense protein band of approximately 20 kDa appeared in the non-retained fraction, which showed a strong emulsification activity (EI 57%) at a concentration of 0.1 mg ml–1 similar to that shown by the whole foam fluid tested at 1.0 mg ml–1 (EI 62%). The mixture of proteins in the retained fraction also showed surfactant activity, confirming the participation of more than one protein with this function in the foam of L. vastus. Thus, all these proteins presumably have a role in the construction of the foam nests and it may be hypothesized that the 20 kDa protein isolated in the present work represents a major function in foam nest stabilization.
Partial sequencing of the 20 kDa band revealed an N-terminal amino acid sequence consisting of 25 amino acid residues in the following order: FLEGFLVPKVVPGPTAALLKKALDD. Using the NCBI-BLAST search system program, no matches in current databases were observed, indicating it to be a novel protein. The protein sequence data reported in this work will appear in the UniProt Knowledgebase under the accession number P85507. These data are of paramount importance since they associate the surfactant activity with a component of protein nature originating from a vertebrate. This protein will hereafter simply be referred to as Lv-ranaspumin (ranaspumin from L. vastus).
Lv-ranaspumin seems to be different from ranasmurfin, another
surfactant protein, which has been isolated from the foam nests of P.
leucomystax (McMahon et al.,
2006). These proteins differ in their molecular masses – 20
and 13 kDa for Lv-ranaspumin and ranasmurfin, respectively. Moreover,
ranasmurfin can occur in dimeric form while Lv-ranaspumin occurs only
as a monomer. In addition, the 26 kDa dimer of P. leucomystax is
strongly blue due to a chromogen in its structure whereas
Lv-ranaspumin did not show any colour. Coincidently, neither protein
revealed any match to any known protein or structure in the databases. This
reinforces the notion that Lv-ranaspumin as well as ranasmurfin are
novel proteins and therefore may show different and interesting properties, to
which further studies must be dedicated.
In conclusion, the foam nests of L. vastus are rich sources of surfactant proteins and Lv-ranaspumin is the major surfactant component of the foam. This 20kDa protein did not show any match to any known protein or structure, which suggests that it belongs to a new class of surfactant protein.
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Acknowledgments |
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-lactalbumin (14.2 kDa).