Background

Amphibians inhabit a diverse range of environments worldwide [1, 2]. As amphibiotic organisms they require water reservoirs to complete their larval development. After metamorphosis most species, like bufonid toads, are usually terrestrial [1]. The migration from an aquatic to a more hybrid (semiaquatic and terrestrial) environment led to many unique traits in amphibian evolution. For instance, amphibian skin plays a crucial role in their adaptation to the terrestrial environment, being responsible for respiration, water balance regulation, excretion, temperature control, osmotic changes, reproduction, and also defence [3,4,5]. The tegument contains two types of specialized glands, termed mucous and granular (or poison) glands. The latter form in bufonids large parotoid macroglands located in the postorbital region (Fig. 1A) [3, 4] and are involved in chemical defence [6,7,8].

Fig. 1
figure 1

A Female common toad Bufo bufo exhibiting the left parotoid macrogland (dashed line) localized at the postorbital region. Black triangles point to the glandular pores. B Longitudinal section of parotoid bottle-shaped glands. Abbreviations: cl—chromatophore layer; ct—connective tissue; ep—epidermis; mg—mucous glands; pg—poison glands; pl—epithelial plug. Photos by Anna Kowalczewska (A) and Krzysztof Kowalski (B)

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Poison secreted from parotoids is used to protect against fungi, microorganisms and predators [9,10,11,12,13,14], as it may be irritating and even lethal for the latter [6, 13]. The chemical compounds contained in parotoid secretions have been classified into four main categories: (i) biogenic amines, such as adrenaline, noradrenaline, bradykinin, or histamine; (ii) bufadienolides; (iii) alkaloids, such as batrachotoxin or tetrodotoxin, and (iv) peptides and proteins [4, 5, 11,12,13, 15]. Bufadienolides are the major components of the bufonid parotoid secretion responsible for its toxicity [8, 11, 15]. According to Steyn and Heerden [16], the toad gland secretion may contain up to 86 types of bufadienolides, including arenobufagin, bufalin, bufogenin, bufotalin, cinobufagin, cinobufotalin, gamabufotalin, marinobufagin, and telocinobufagin [8, 11, 17].

Recently, some peptides and proteins have been identified in the toad parotoid secretions [12, 13, 18,19,20,21,22,23,24], but their abundance seems to be much lower than those of bufadienolides and biogenic amines [4, 12]. For instance, Sousa-Filho et al. [20] obtained 104 proteins, including actin, beta-actin, ribosomal proteins, catalase, galectin, and uncharacterised proteins, in the Rhinella schneideri parotoid secretion by proteomic analysis. No peptides were found. Huo et al. [25] identified 939 unique peptides by de novo approach in parotoid macrogland secretion of Bufo gargarizans. Mariano et al. [23] identified 42 proteins and sequenced de novo 153 peptides in the Duttaphrynus melanostictus skin secretion. The most common proteins were acyl-CoA-binding protein, alcohol dehydrogenase, calmodulin, catalase, galectin, proteasome subunit alpha- and beta-type, and histone. It is assumed that bufonid parotoid gland secretions do not contain bioactive peptides similar to those from the skin of many frog species [4, 12]. It has been also suggested that peptides from parotoid secretion of Rhinella marina could be breakdown products of larger proteins that ensure the proper gland functioning [12, 23]. However, our previous results indicate that some proteins from parotoid secretion of Bufo bufo may participate in the toad chemical defence [13].

Most studies of bufonid chemical defence focused on the biochemistry and pharmacology of parotoid gland secretions. Little is known about the functioning of parotoid macroglands and processes that regulate toxin biosynthesis, poison production and excretion. Therefore, to understand these processes, in this work we aimed to investigate protein content in the extract from parotoid macroglands. As a model species we used the common toad, Bufo bufo, which is known for producing potent poison in its parotoids [13, 26]. Some proteins and peptides, such as muscle creatine kinase, proteasome subunit α type, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, serine/threonine-protein kinase, so far have successfully been identified in this species [13].

Materials and methods

Toads trapping and housing

Trapping of toads was performed from March to April in 2016. Adult female toads Bufo bufo were captured (using 10-L pitfalls and drift fences) in gardens and parks in Poznań (western Poland), placed into transporters and carried to the laboratory. Then, they were placed into large (46 × 30 × 28 cm, 39 l) aqua-terraria equipped with bedding (a mixture of peat and sand). The terraria were regularly irrigated to maintain the adequate humidity. Each terrarium contained a shelter (flowerpot) and a water tank to allow the toads to submerge in water. Food (mealworms and crickets) and water were provided ad libitum. The toads were kept in the breeding room under standard laboratory conditions (temperature: 21 °C; humidity: 65–70%; artificial photoperiod: 12L:12D).

Extraction of toad parotoid macroglands and sample preparation

Two toads were decapitated and their parotoid glands were carefully isolated (to not squeeze them and not release the poison) and transferred into 600 µl of methanol. Tissues were next homogenized and samples were centrifuged at 10,000 × g and 4 °C for 30 min. The supernatants were collected, and the protein content was determined using a Direct Detect spectrometer (MERCK Millipore, Warsaw, Poland). Then, the supernatants were used for peptide analysis by reverse phase high-performance liquid chromatography (RP-HPLC). Separation was performed using a Dionex Ultimate 3000 chromatographic system comprising a dual pump programmable solvent module. Supernatants were analysed using a BioBasic-18 analytical column (5 µm, 150 × 4.6 mm; Thermo Scientific). The samples were eluted with a gradient of 5–60% acetonitrile (ACN)/0.1% TFA with a flow rate 0.5 ml/min for 55 min. The eluent was monitored at 214 nm and fractions were collected into 1.5-ml tubes.

Protein identification and analysis of their biological functions

Peptides and proteins from the methanolic extract of the toad parotoid glands were analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Nano-Acquity LC system (Waters, Milford, Massachusetts, USA) and an OrbitrapVelos mass spectrometer (Thermo Electron Corp., San Jose, CA). Before performing the analysis, the proteins were subjected to an ion-solution digestion procedure. Proteins were: (1) reduced with 50 mM TCEP for 30 min at 60 °C, (2) alkylated with 200 mM MMTA for 30 min at room temperature and (3) digested overnight with trypsin (sequencing Grade Modified Trypsin—Promega V5111). Next, the samples were applied to an RP-18 precolumn (nanoACQUITY Symmetry® C18—Waters 186,003,514) using water containing 0.1% TFA as a mobile phase and were transferred to a nano-HPLC RP-18 column (nanoACQUITY BEH C18—Waters 186,003,545). The samples were eluted with a gradient of 0–35% acetonitrile in the presence of 0.05% formic acid with a flow rate of 250 nl/min for 180 min. The column was directly coupled to the ion source of the spectrometer working within data dependent on the MS to MS/MS switch. To ensure a lack of cross contamination from previous samples, each analysis was preceded by a blank run.

The proteins were identified by a Mascot Search (Matrix Science, London, UK) against the SwissProt database. The search parameters were as follows: type of search: MS/MS Ion Search; enzyme specificity: trypsin; fixed methylthio modification of cysteine; variable modifications: methionine oxidation; mass values: monoisotopic; protein mass: unrestricted; peptide mass tolerance: 20 ppm; fragment mass tolerance: 0.1 D; number of missed cleavage sites allowed: 1; instrument type: HCD. Peptides with Mascot scores exceeding the threshold value of p < 0.05 were considered positively identified. The protein content was calculated based on the Exponentially Modified Protein Abundance Index (emPAI) [27].

The biological functions of the identified proteins were determined by searching the UniProt database. All functions were then classified into 11 categories (Table 1). The percentage of proteins displaying particular functions from each category was calculated. To understand the cellular processes in toad’s parotoids, protein-protein interaction networks were built using STRING database [28]. Because molecular data on bufonid toads are scarce, predicted interactions between identified in this work proteins were analysed based on homology to the western clawed frog Xenopus tropicalis, a common anuran model. Then, Gene ontology (GO) enrichment analysis was performed to predict which metabolic processes the identified proteins are involved in, and what are their molecular functions (GO molecular function analysis). KEGG analysis was carried out to predict which signalling pathways the identified proteins participate in. Finally, GO component analysis was performed to predict the localisation of particular proteins within the cell.

Table 1 Categories of biological functions and number of proteins identified in the extract from parotoids of the common toad Bufo bufo displaying particular functions
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Results and discussion

Bufonids are worldwide distributed amphibians [1, 2] that store potent chemical weapon in their parotoid macroglands [6,7,8, 13]. Most of the studies dealing with toad parotoid secretions focus on biological activities of bufadienolides, biogenic amines and alkaloids [8, 11, 15]. Therefore, these molecules and their toxic and pharmacological effects are particularly well characterised [8]. Little is known about the protein and peptide content in the bufonid parotoids, as well as their toxic properties. Also, the processes related to poison production and excretion remain unknown.

Here, we identified 162 proteins in the extract from parotoid macroglands of B. bufo (Table 2; Additional file 1: Table A1). The most abundant (with the highest emPAI) were proteins related to the cell structure maintenance and proteins with enzymatic activities involved in metabolic pathways (see Additional file 1: Table A1). For each identified protein at least one biological function was assigned by searching UniProt database (Table 2). Then, all functions were assigned to one of 11 categories (Tables 1 and 2).

Table 2 Proteins identified in the extract from parotoids of the common toad Bufo bufo based on tandem mass spectrometry analysis
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STRING protein-protein interaction network predicted for proteins identified in the extract from B. bufo parotoids confirms the results obtained by searching the UniProt database (Additional file 2: Fig. A1). Most proteins are related to the metabolic processes with the strongest interactions between proteins involved in glycolysis, including phosphoglucomutase (pgm1), fructose-bisphosphate aldolase (aldoa), triosephosphate isomerase (tpi1), phosphoglycerate kinase 1 (pgk1) and transaldolase (taldo1). Strong interactions occur also between proteins, such as transitional endoplasmic reticulum ATPase (vcp), S-phase kinase-associated protein 1 (skp1), NEDD8 (nedd8), small ubiquitin-related modifier 2 (sumo2) and UV excision repair protein (rad23b), that participate in stress response and DNA repair and replication. This finding indicates that proteins present in the toad parotoids may effectively protect skin cells from DNA damage and thus minimize the harmful effects of UV radiation. Similarly, the results of GO enrichment and KEGG analyses are consistent with previous findings. Most predicted genes code intracellular proteins involved in metabolic processes and stress response (see Additional file 2: Figs A2–A5).

Regarding the number of identified proteins our results seem to be similar to those obtained recently by other researchers [e.g., 20, 23, 25] (see Introduction for details). One-third (34.6%) of the identified in this work proteins are involved in cell metabolism (Fig. 2 and Additional file 2: Figs A1–A4) with many of them being related to biosynthesis, regulation of transcription and translation, and gland functioning (Table 2 and Additional file 2: Figs A1–A4). This finding is consistent with the results of protein abundance analysis. Similarly, Sousa-Filho et al. [20] found numerous proteins related to cell metabolism and cell matrix in the R. schneideri parotoid secretion. Here, presence of two proteins, phosphomevalonate kinase (PMVK) and isopentenyl-diphosphate delta-isomerase 1 (IDI1; Table 2), is particularly noteworthy. These molecules are involved in the mevalonate arm of the cholesterol biosynthesis pathway [29, 30]. PMVK catalyses formation of mevalonate 5-diphosphate from mevalonate 5-phosphate, an essential step in the mevalonate pathway, while IDI1 performs isomerisation of isopentenyl diphosphate into dimethylallyl diphosphate (Fig. 3) [29, 30]. Cholesterol has been confirmed to be a precursor for synthesis of various steroids including bufadienolides [31,32,33], and it has been assumed that it is synthesised in the liver and then secreted and transferred through the bloodstream to the parotoid glands [31, 32]. Recent studies showed that genes coding PMVK and IDI1 are expressed in 27 human tissues including liver, kidney, spleen, adrenal glands and brain [34]. Genes coding IDI1 have been also proven to express in the liver of X. tropicalis frog [35]. PMVK has not been reported in any amphibian organs and tissues so far. Thus, confirming the presence of PMVK and IDI1 in the toad parotoid extract indicates that cholesterol necessary for toxin production may be also synthesised in parotoids, major glands that store bufadienolides.

Fig. 2
figure 2

Biological functions of proteins identified in the extract from parotoids of the common toad Bufo bufo. Values in parentheses show percentage of proteins classified into each function category. See Table 2 for detailed functions of particular proteins

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Fig. 3
figure 3

The mevalonate pathway in the cholesterol biosynthesis (according to Mazein et al. [27], modified). Blue arrows indicate synthesis processes, while purple arrows catalysis accelerated by enzymes. Question marks indicate uncertain processes. Enzymes (PMVK and IDI1) identified in this work in the extract from B. bufo parotoids are shown in bold

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Parotoids are holocrine glands [6, 36,37,38] in which poison excretion is accompanied by changes in the gland structure (Fig. 1B) [7]. Therefore, the complete poison replenishment is considered to be metabolically costly and time-consuming for toads [7], and may affect their growth and behaviour [39]. According to Jared et al. [7], the Cururu toad Rhinella icterica requires more than 3 months to restore the poison secreted from parotoids after mechanical compression of glands and the parotoid content seems to not return to the state preceding poison extraction. Rapid poison replenishment is necessary to ensure chemical protection against predators and microorganisms. Therefore, for toads, a reduced or depleted poison supply can be life-threatening because of diminished defence capabilities. In this work, we found various proteins involved in cell division and cell cycle regulation (12.0%), as well as cell aging and apoptosis (7.3%; Fig. 2). Together with many proteins that participate in molecule biosynthesis and transport (8.4%) as well as maintenance of cell structure (8.4%; Fig. 2), it may be indicative of a high epithelial cell turnover in parotoids. Such an intense cell turnover in the holocrine gland should require well developed protein machinery that regulates cell cycle and cell divisions to enable quick and effective replenishment of toxins and thus increase the chance of survival during encounter with a predator.

Some proteins found in this study are likely to reinforce toxic activity of parotoid secretion. For instance, gamma-enolase has neurotropic properties [40, 41] and thus its presence in parotoid secretion may increase paralytic activity of toad poison. Antithrombin-III regulates the blood coagulation cascade (Table 2) and may block blood clot formation [42, 43] and thus cause bleeding. Few proteins, such as puromycin-sensitive aminopeptidase-like protein, cytosolic non-specific dipeptidase or proteasome subunit alpha type-1, cause proteolysis [44,45,46,47] and thus may reinforce proteolytic action of toad poison. Previously, we also described in B. bufo parotoid secretion some peptides that may display physiological effects [13]. Mariano et al. [23] found proteasomes in D. melanostictus skin secretions. It is therefore likely that some molecules obtained in this work also participate in the toad chemical defence. However, further studies are required to confirm their biological activity and describe their action modes.

Tropomyosin alpha-3 chain, myosin light polypeptide 6 and calponin-3 are involved in regulation of muscle contraction (Table 2) [48,49,50]. Calmodulin is a Ca+2 binding protein that regulates apoptosis, inflammation and smooth muscle contraction [51,52,53]. Therefore, these proteins may promote poison excretion from parotoids. Also, 78 kDa glucose regulated protein, which is related to toxin transport [54], may participate in poison secretion from toad parotoids. Myosin light polypeptide 6 and calmodulin have already been reported in the D. melanostictus parotoid secretion [22, 23]. Calmodulin has been also found in the toad urinary bladder, oviduct and retina photoreceptors [55,56,57], while myosin and tropomyosin in smooth muscles within the carotid labyrinth of Rh. marina [58] and skeletal muscles of X. tropicalis and the Japanese tree frog Hyla japonica [59].

Mariano et al. [23] identified 62 binding proteins in the skin secretions of D. melanostictus. These proteins might bind to different molecules and play important role in amphibian skin secretion. In this work, we found acyl-CoA-binding domain-containing protein 7 and annexins in the B. bufo parotoid extract. The latter have been suggested to regulate diverse cellular and physiological processes, such as endo- and exocytosis [60, 61], and thus promote toxin excretion from poison glands. Both, acyl-CoA-binding proteins (ACBPs) and annexins, have recently been reported in D. melanostictus skin secretions [22, 23]. ACBPs have also been found in the frog’s brain [62], while annexins in the skin of the large-webbed bell toad Bombina maxima, the Dybovsky’s frog Rana dybowskii, and the Chinese giant salamander Andrias davidianus [63,64,65]. It seems that the latter are unique to the skin and because of their antimicrobial activity related to the immune response of amphibian skin secretions.

It is well known that amphibian skin plays an important role in antimicrobial defence [66, 67]. Proteins related to immune defence have already been reported in anuran skin secretions [18, 66,67,68]. Also, in this work we found proteins involved in innate and adaptive immune response, and antimicrobial, allergic and inflammatory response (7.0%; Table 2, Fig. 2). Thus, our results confirm antimicrobial functions of bufonid skin secretions. Also, proteins related to stress response are quite abundant (6.3%) in the extract from B. bufo parotoids (Fig. 2 and Additional file 2: Figs A1–A3). Here, we identified catalase, peroxiredoxin 6 and superoxide dismutase that are related to the anti-oxidant system [69]. Similar proteins (catalase, glutathione peroxidase 3, peroxiredoxin 6, superoxide dismutase and thioredoxin like 1) have previously been reported in parotoid secretion of R. schneideri [20]. Superoxide dismutase has also been identified in skin secretions of R. dybowskii [64], while catalase in lungs, heart, liver and kidney of the painted frog Discoglossus pictus [70].

Amphibian populations are in decline, which has been exacerbated in the last few decades and caused by several factors [71]. Ultraviolet (UV) radiation is especially dangerous to amphibians because of their thin and poorly cornified integument [72], and may lead to protein denaturation, cell damage and death, and/or mutagenesis in amphibian skin [73, 74]. Amphibians can cope with the harmful effects of UV radiation in many ways which include behavioural and molecular mechanisms. Here, we report 6 proteins in the extract from toad parotoids (Table 2, Fig. 2 and Additional file 2: Fig. A1) that are involved in DNA repair. Also, heat shock proteins (HSPs) may be synthesised under environmental or physiological stress [75]. They interact with denatured proteins and help them to refold and reassemble, turning back their active forms [23, 76]. Therefore, all these molecules may represent molecular mechanisms that may effectively protect toad skin cells from DNA damage and thus minimize the harmful effects of UV radiation. Such proteins involved in protection against UV radiation damage and protein recycling have been recently found in the skin secretion of D. melanostictus [23]. Related to stress response HSPs have been reported for many anuran organs, such as skin, liver, heart, kidneys, lungs and brain [77, 78].

Conclusions

We report here housekeeping proteins in the extract from B. bufo parotoid macroglands. Applying a proteomic approach enabled us to obtain new findings on the molecules that are involved in poison production and secretion, and may contribute to the gland functioning. Most identified proteins are involved in metabolic processes and cell structure maintenance. Our results indicate that in toads cholesterol may be synthesized in parotoids, and not only in the liver from which is then transferred through the bloodstream to the parotoids. Presence of proteins that protect skin cells from DNA damage may help to minimize the harmful effects of UV radiation. It opens up new perspectives for studying biological activity of molecules from parotoid secretions and their role in the toad chemical defence system. Applying de novo peptide sequencing coupled with analyses of various databases will provide new protein dataset in anuran skin secretions and extend our knowledge on the functioning of bufonid parotoids.