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Journal of Venomous Animals and Toxins including Tropical Diseases

On-line version ISSN 1678-9199

J. Venom. Anim. Toxins incl. Trop. Dis vol.25  Botucatu  2019  Epub Aug 19, 2019

http://dx.doi.org/10.1590/1678-9199-jvatitd-2019-0029 

Research

Protein identification from the parotoid macrogland secretion of Duttaphrynus melanostictus

Douglas Oscar Ceolin Mariano1 

Marcela Di Giacomo Messias2 

Patrick Jack Spencer2 

Daniel Carvalho Pimenta1  * 
http://orcid.org/0000-0003-2406-0860

1Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, SP, Brazil.

2Biotechnology Center, Nuclear and Energy Research Institute (IPEN), São Paulo, SP, Brazil.

ABSTRACT

Background:

Bufonid parotoid macrogland secretion contains several low molecular mass molecules, such as alkaloids and steroids. Nevertheless, its protein content is poorly understood. Herein, we applied a sample preparation methodology that allows the analysis of viscous matrices in order to examine its proteins.

Methods:

Duttaphrynus melanostictus parotoid macrogland secretion was submitted to ion-exchange batch sample preparation, yielding two fractions: salt-displaced fraction and acid-displaced fraction. Each sample was then fractionated by anionic-exchange chromatography, followed by in-solution proteomic analysis.

Results:

Forty-two proteins could be identified, such as acyl-CoA-binding protein, alcohol dehydrogenase, calmodulin, galectin and histone. Moreover, de novo analyses yielded 153 peptides, whereas BLAST analyses corroborated some of the proteomic-identified proteins. Furthermore, the de novo peptide analyses indicate the presence of proteins related to apoptosis, cellular structure, catalysis and transport processes.

Conclusions:

Proper sample preparation allowed the proteomic and de novo identification of different proteins in the D. melanostictus parotoid macrogland secretion. These results may increase the knowledge about the universe of molecules that compose amphibian skin secretion, as well as to understand their biological/physiological role in the granular gland.

Keywords Amphibian skin secretion; Bufonidae; Duttaphrynus melanostictus; Proteomics; Batch chromatography; Asian common toad

Background

Anuran skin participates in different physiological process and has an important role in chemical defense [1-4]. In these animals, the tegument contains specialized glands, termed granular glands, capable of storing a higher diversity of biological molecules [1, 4-6]. However, some anurans developed glandular accumulation on different body regions. One example is the Bufonidae family, which possess a parotoid macrogland located in the dorsum of the head [7,8].

Bufonid parotoid macrogland secretion contains a wide quantity of alkaloids and steroids. Several studies purified and biologically and/or chemically characterized these molecules on Bufo or Rhinella species [9-12]. In relation to peptides, Rash et al. [13] identified these molecules in low abundance in R. marina. Recently, Huo et al. [14] identified 939 unique peptides in B. gargarizans parotoid macrogland secretion by de novo approach.

Proteins are also present in bufonids. Sciani et al. [15] investigated the protein profile of parotoid macrogland secretion of nine bufonids (Rhinella sp. and Rhaebo sp.). The authors showed the presence of several proteins by electrophoresis. Other two researches identified and characterized baserpin and lysozyme from B. andrewsi parotoid macrogland secretion [16, 17]. Besides that, proteomics studies performed with B. bufo, B. gargarizans and R. schneideri parotoid macrogland secretion identified 13, 8 and 104 proteins, respectively [14, 18, 19].

In bufonids, protein identification and/or characterization from parotoid macrogland secretion may be limited, mainly because this biological sample is highly viscous, sticky and often water insoluble [15, 20]. Recently, our group developed a methodology to analyze viscous secretions [21]. Employing the ion-exchange batch sample preparation methodology, we were able to biochemically characterize the parotoid macrogland secretion of the Asian common toad Duttaphrynus melanostictus, including the retrieval of peptidasic activity, as assessed by zymography [21].

Since our sample preparation step yielded to a protein-rich solution, we performed an in-solution proteomics and de novo peptide sequencing after anion-exchange batch sample processing of D. melanostictus parotoid macrogland secretion.

Material and Methods

Reagents

All employed reagents were purchased from Sigma Co. (St. Louis, MO, USA), unless otherwise stated. Amicon Ultra-4 Centrifugal 3 kDa filter and syringe filter (Millex-GV, hydrophobic PVDF 0.22 μm) were purchased from Millipore, USA. pH test strip (pH-Fix 0-14) was obtained from Macherey-Nagel, Germany. QAE-Sephadex A-25 was obtained from Pharmacia Fine Chemicals AB Uppsala, Sweden.

Skin secretion collection

D. melanostictus lyophilized parotoid macrogland secretion was kindly provided by Venom Supplies Pty ltd., Australia.

Anionic-exchange batch sample methodology

Sample preparation

The material was analyzed according to the protocol developed by Mariano et al. [21] as follows:

Step 1 - resin preparation: we resuspended 0.5 g of QAE Sephadex A-25 resin with 12.5 mL of 25 mM ammonium bicarbonate (pH 8.5) for 18h, at room temperature. Then, the tube was centrifuged at 500 g, for 5 min, and the supernatant was discarded. After that, the resin was washed with 12.5 mL 25 mM of ammonium bicarbonate during 30 minutes; after, the tube was centrifuged (500 g, 5 min) and the supernatant was discarded. We repeated this last process twice.

Step 2 - sample preparation: D. melanostictus lyophilized parotoid macrogland secretion (~ 100 mg) was resuspended in 20 mL of 25 mM ammonium bicarbonate (pH 8.5) under constant agitation, followed by sonication. We transferred the solution to a tube containing the anionic resin.

Step 3 - unbound fraction: we maintained the tube under constant homogenization during 1 h, at room temperature. After that, we centrifuged the tube (500 g, during 5 min), collected the supernatant and termed it as ‘anionic unbound fraction’ (A-UBF). Then, we added 20 mL of 25 mM ammonium bicarbonate (pH 8.5) at the tube and left 1h under constant homogenization. The sample was centrifuged and the supernatant was collected and pooled as A-UBF.

Step 4 - salt fraction: following the removal of A-UBF fraction, we added 25 mM ammonium bicarbonate, containing 2 M NaCl (pH 8.5). Here, we conducted this phase as described in step 3: homogenization (1 h) and centrifugation (500 g, during 5 min); however, we collected the supernatant and termed it as ‘anionic salt-displaced fraction’ (A-SDF). This step was repeated twice.

Step 5 - acid fraction: finally, we added 25 mM ammonium bicarbonate (pH ~ 3-4). Again, we repeated this step twice: homogenization (1 h), centrifugation (500 g, during 5 min) and supernatant collection [termed as ‘anionic acid-displaced fraction’ (A-ADF)].

A-SDF and A-ADF were mechanically filtered (.22 μm syringe filters) prior to lyophilization.

Desalting

A-SDF and A-ADF were desalted by a HiPrep 26/10 desalting column (GE Healthcare) coupled to an AKTA avant 25 preparative system (GE Healthcare). We resuspended all samples in 5 mL of 25 mM Tris (pH 8.5) and individually loaded into the system. The column eluted at a constant flow rate of 10 mL/min with 25 mM Tris buffer (pH 8.5) and monitored at 220 nm. We collected each peak corresponding to a protein signal and subsequently all samples were lyophilized.

Chromatographic analysis

Both desalted fractions were concentrated using an Amicon Ultra-4 centrifugal filter (3 kDa), dried, resuspended in 2 mL of 25 mM Tris (pH 8.5) and individually loaded into a Mono Q 5/50 GL column, in a two-buffer system: (1) 25 mM Tris (pH 8.5) and (2) 25 mM Tris, 2 M NaCl (pH 8.5). The column was eluted at a constant flow rate of 1 mL.min-1 under a 0 to 50% gradient of buffer 2, during 20 min. We monitored the eluates at 220 nm and automatically collected one mL fractions during the gradient phase.

Proteomic analysis: in-solution digestion

We dried aliquots of the A-SDF (1-10) and A-ADF (1- 6) fractions and resuspended each fraction in 8 M urea (100 mM Tris-HCL, pH 8.5) and Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 5 mM final concentration, for 1h, at room temperature. After that, we added iodoacetamide (IAA) (dissolved in water) (10 mM final concentration) and incubated all samples for 1h, at room temperature and protected from the light. Next, we added 100 mM Tris-HCl (pH 8.5), for urea dilution (2 M final concentration), and 10 µL trypsin (10 ng.µL-1 in 100 mM Tris-HCl, pH 8.5) and incubated all samples during 18h, at 30°C. Finally, we stopped the enzymatic reaction adding 50% ACN/5% TFA and dried all samples. Prior to analysis in the mass spectrometer, we used a ZipTip® C-18 pipette tips (Millipore) to desalt and for peptide concentration.

We analyzed all samples in an electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) (Micromass, UK) equipped with binary ultra-performance liquid chromatography system (UPLC) (Acquity, Waters, MA, USA). Samples (5 μL) were separated on a C18 column, using the following mobile phase: (A) 0.1% formic acid (FA) (1:999, v/v) and (B) 0.1% FA in 90% acetonitrile (ACN) (1:900:99, v/v/v). The gradient condition was: 2% B in 0-5 min; 2-40% B in 5-60 min. The mass spectrometry (MS) was equipped with a locked ESI probe and operated in positive mode (ESI+). The electrospray capillary voltage was 3.1 kV, with a cone voltage of 113 V. The cone and desolvation gas flows were 185 and 600 l h−1, respectively. The desolvation temperature was 150°C. MS scans were acquired at 350-1600 mass charge rate (m/z) and MS/MS scans at 50-2000 m/z. The collision energy of the MS/MS analysis was 10-10.6 eV. The software selected automatically ions with a threshold intensity of ≥ 10 for fragmentation.

Data processing

We loaded and analyzed micromass RAW files by Peaks Studio V7.0 software (BSI, Canada). We adjusted the following parameters for de novo peptide sequencing: error tolerance (MS and MS/MS) was set to 0.2 Da; methionine oxidation and carbamidomethylation as variable and fixed modification, respectively; trypsin as cleavage enzyme; and average local confidence (ALC) ≥ 80 %. We performed a basic local alignment search tool (BLAST) with all de novo peptides, limiting the search for Amphibia class (taxid: 8292). For a deeper analysis, we only consider alignments presenting the higher scores.

For proteomic identification, we set the following parameters on Peaks software: error tolerance (MS and MS/MS) set to 0.2 Da; methionine oxidation and carbamidomethylation as variable and fixed modification, respectively; trypsin as cleavage enzyme; three maximum missed cleavages; three maximum variable PTMs per peptide; one non-specific cleavage; false discovery rate was ≤ 1 %; and we analyzed all data against Amphibia protein database (167530 entries) (built by retrieving all Uniprot entries associated with this taxon).

Results

Chromatographic analysis of D. melanostictus parotoid macrogland secretion after batch sample preparation

After batch processing, we analyzed A-SDF and A-ADF by anionic-exchange chromatography. According to the chromatographic profile and peak distribution, we obtained a total of 10 and 6 fractions from A-SDF and A-ADF, respectively. The resulting profiles are very similar to those previously obtained. Please refer to Figure 3 in Mariano et al. [21].

Mass spectrometry analysis

Proteomic Identification

We identified 24 proteins in A-SDF collected fractions (Table 1), being 18 proteins identified in fractions 3-7, after in solution digestion. Proteins such as acyl-CoA-binding protein homolog, alcohol dehydrogenase, calmodulin 1, diazepam binding inhibitor, galectin-1, histone H2B and prostaglandin reductase 1 were found in more than one fraction. Only in A-SDF fraction 1 no protein was identified.

Table 1. Proteins identified in fractions 1-10 of A-SDF 

Fraction Entry name Identified protein Organism 10lgP Score Peptide Molecular mass (Da) PTM Top BLAST hit
2 ACBP_PELRI Acyl-CoA-binding protein homolog Pelophylax ridibundus TKPTDDELKELYGLYK 9808
SPQADFDKAAGD(+43.99)VK(+42.01)K Carboxylation (DKW); Acetylation
GLG(sub S)KEDAMSAYVSK Mutation
A4K520_BUFGR Diazepam binding inhibitor Bufo gargarizans 30.66 GMSKEDAMSAYVSK 9905
3 C1C3M2_LITCT NADP-dependent leukotriene B4 12-hydroxydehydrogenase Lithobates catesbeiana 95.25 ASPEGYDC(+57.02)*YFENVGGK 35412 Prostaglandin reductase 1-like [Nanorana parkeri]
IGFDEAFNYK
ACBP_PELRI Acyl-CoA-binding protein homolog Pelophylax ridibundus 51.94 TKPTDDELKELYGLYK 9808
F7A8C0_XENTR Uncharacterized protein (Fragment) Xenopus tropicalis 50.66 IGFDEAFNYK 32004 Prostaglandin reductase 1 [Xenopus tropicalis]
QLLQWVIEGK
LEG1_RHIAE Galectin-1 Rhinella arenarum 35.36 LNLKPGHC(+57.02)*VEIK 14711
NLNLKPGHC(+57.02)*VEIK
AK1A1_XENLA Alcohol dehydrogenase [NADP(+)] Xenopus laevis 34.37 MPLIGLGTWK 37100
4 LEG1_RHIAE Galectin-1 Rhinella arenarum 53.51 LNLKPGHC(+57.02)*VEIK 14711
NLNLKPGHC(+57.02)*VEIK
GFAVNLGEDASNL(sub F)LLHL(sub F)NAR Mutation
ACBP_PELRI Acyl-CoA-binding protein homolog Pelophylax ridibundus 46.76 TKPTDDELKELYGLYK 9808
AK1A1_XENLA Alcohol dehydrogenase [NADP(+)] Xenopus laevis 44 MPLIGLGTWK 37100
A4K520_BUFGR Diazepam binding inhibitor Bufo gargarizans 21.03 GMSKEDAMSAYVSK 9905
5 A4K520_BUFGR Diazepam binding inhibitor Bufo gargarizans 156.1 GMSKEDAMSAYVSK 9905
KGMSKEDAMSAYVSK
SPQADFDKAAED(+14.02)VKK Methyl ester
ANELIEKH(sub Y)GL Mutation
QSTVGDINIDC(+57.02)*PGMLDLK
ACBP_PELRI Acyl-CoA-binding protein homolog Pelophylax ridibundus 74.97 TKPTDDELKELYGLYK 9808
Q4KLC5_XENLA MGC116485 protein Xenopus laevis 29.86 WEAWNSKK 8374 Acyl-CoA-binding protein homolog [Xenopus laevis]
AKWEAWNSKK
6 C1C3M2_LITCT NADP-dependent leukotriene B4 12-hydroxydehydrogenase Lithobates catesbeiana 54.04 ASPEGYDC(+57.02)*YFENVGGK 35412
IGFDEAFNYK
LEG1_RHIAE Galectin-1 Rhinella arenarum 56.72 NLNLKPGHC(+57.02)*VEIK 14711
GFAVNLGEDASNL(sub F)LLH Mutation
GSIPPDC(+57.02)*KGFAVNLGEDASNL(sub F)LLHL(sub F)NAR Mutation
PGHC(+57.02)*VEIK
F7A8C0_XENTR Uncharacterized protein (Fragment) Xenopus tropicalis 40.44 IGFDEAFNYK 32004 Prostaglandin reductase 1 [Xenopus tropicalis]
QLLQWVIEGK
7 Q641J7_XENTR Calmodulin 1 Xenopus tropicalis 57.87 VFDKDGNGYISAAELR 16838
LEG1_RHIAE Galectin-1 Rhinella arenarum 41.85 NLNLKPGHC(+57.02)*VEIK 14711
GSIPPDC(+57.02)*KGFAVNLGEDASNL(sub F)LLHL(sub F)NAR Mutation
ACBP_PELRI Acyl-CoA-binding protein homolog Pelophylax ridibundus 28.9 TKPTDDELKELYGLYK 9808
8 C1C4P2_LITCT Calmodulin Lithobates catesbeiana 54.52 SLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMAR 16838
VFDKDGNGYISAAELR
LEG1_RHIAE Galectin-1 Rhinella arenarum 51.47 SGDQFSFPVR 14711
IVC(+57.02)*NSKEADAWGSEQRE
NLNLKPGHC(+57.02)*VEIK
9 A0A1L8G795_XENLA Histone H2B Xenopus laevis 34.09 NSFVNDIFER 13935
10 ACBP_PELRI Acyl-CoA-binding protein homolog Pelophylax ridibundus 40.54 TKPTDDELKELYGLYK 9808

*Cysteine carbamidomethylation.

Furthermore, we identified 18 proteins in A-ADF collected fractions (Table 2), being six of them already identified in A-SDF. In the acid fraction, we can highlight the presence of the protein ATP synthase (subunit alpha and beta) and also hemoglobin (subunit beta).

Table 2 Proteins identified in fractions 1-6 of A-ADF 

Fraction Entry name Identified protein Organism 10lgP Peptide Molecular mass (Da) PTM Top BLAST hit
1 LEG1_RHIAE Galectin-1 Rhinella arenarum 29.27 NLNLKPGHC(+57.02)*VEIK 14711
2 A4K520_BUFGR Diazepam binding inhibitor Bufo gargarizans 117.39 QSTVGDINIDC(+57.02)*PGMLDLK 9905
SPQADFDKAAED(+14.02)VKK Methyl ester
ANELIEKH(sub Y)GL Mutation
AKWEAWNS(sub L)KK Mutation
GMSKEDAM(+15.99)#SAYVSK
KGMSKEDAMSAYVSK
ACBP_PELRI Acyl-CoA-binding protein homolog Pelophylax ridibundus 44.78 TKPTDDELKELYGLYK 9808
3 A0A1L8H8W7_XENLA Uncharacterized protein Xenopus laevis 27.03 LVAM(+15.99)#GIPESIR 128290 TBC1 domain family member 8-like isoform X2 [Xenopus laevis]
F6U3Y7_XENTR Myosin IH Xenopus tropicalis 23.29 INSSLANK 120196
A0A1L8ESG2_XENLA Protein Wnt Xenopus laevis 23.27 SSRFSPGTAGRTC(+57.02)*SR 39675
4 Q7ZWR6_XENLA ATP synthase subunit beta Xenopus laevis 112.11 866. TVLIMELINNVAK 56374
SLQDIIAILGMDELSEEDKLTVSR
LVLEVAQHLGES(sub N)TVR Mutation
SLQDIIAILGM(+15.99)#DELSEEDKLTVSR
KGSITSVQAIYVPAN(sub D)DLTDPAPATTFAHLDATTVLSR Mutation
A0A1L8G6S1_XENLA Histone H4 Xenopus laevis 78.17 AMGIMNSFVNDIFER 26318
LEG1_RHIAE Galectin-1 Rhinella arenarum 41.11 GFAVNLGEDASNL(sub F)LLHL(sub F)NAR 14711 Mutation
SGDQFSFPVRK
Q3KPP1_XENLA MGC52881 protein Xenopus laevis 35.47 LFIGGLSFETTEESLR 36486 Heterogeneous nuclear ribonucleoprotein A2 homolog 2 isoform X3 [Xenopus laevis]
Q6DD58_XENLA Tubulin alpha chain Xenopus laevis 33.45 AVFVDLEPTVIDEVR 49887
6 Q7ZWR6_XENLA ATP synthase subunit beta Xenopus laevis 134.99 DQEGQDVLLFIDNIFR 56374
TVLIMELINNVAK
IGLFGGAGVGK
FTQAGSEVSALLGR
VVDLLAPYAK
SLQDIIAILGM(+15.99)#DELSEEDKLTVSR
Q9I9P5_LITCT Inner-ear cytokeratin Lithobates catesbeiana 108.48 SLDLDSIIAEVK 56619
FLEQQNKVLETK(-.98) Amidation
TP(sub L)NNKFASFIDKVR Mutation
F7CIH4_XENTR ATP synthase subunit alpha Xenopus laevis 91.29 1059. VLSIGDGIAR 57548
EVAAFAQFGSDLDAATQQLLS(sub N)R Mutation
TGAIVDVPVGD(+14.02)ELLGR Methyl ester
TSIAIDTIINQK
AVDSLVPIGR
G1FF50_9SALA Beta-actin (Fragment) Eurycea cirrigera 75.81 SYELPDGQVITIGNER 23707
TTGIVMDSGDGVTHTVPIYEGYALPHAILR
GYSFTTTAER
G5DYL7_9PIPI Tubulin alpha chain (Fragment) Hymenochirus curtipes 58.22 AVFVDLEPTVIDEVR 10921
HBB_PELES Hemoglobin subunit beta Pelophylax esculentus 50.95 LLVVYPWTQR 15424
LEG1_RHIAE Galectin-1 Rhinella arenarum 25.59 GFAVNLGEDASNL(sub F)LLHL(sub F)NAR 14711 Mutation

# Methionine oxidation; *Cysteine carbamidomethylation.

Based on the Gene Ontology (GO) project [22], employing the ‘molecular function’ identifier, we observed that proteins present in A-SDF were associated to binding (four proteins) and/or catalytic activities (seven proteins). While in A-ADF, besides binding activity (12 proteins), we also found proteins classified into nucleoside-triphosphatase activity (five proteins), oxygen carrier activity (one protein), structural molecule activity (three proteins) and transmembrane transporter activity (two proteins).

de novo peptides

This analysis led us to the identification of 102 and 41 different de novo peptides in A-SDF and A-ADF collected fractions, respectively (Additional files 1 and 2). Among them, only ten de novo peptides were present in both fractions.

BLAST alignment showed that several de novo peptides aligned with proteins already identified in our proteomic study, like acyl-CoA-binding protein, alcohol dehydrogenase, galectin and prostaglandin reductase 1 (Additional files 1 and 2, Blast E-value < 0.01, color code: light red). The remaining de novo peptides suggest proteins related to: apoptosis (apoptosis regulator Bcl-2-like), binding activity (spectrin beta chain, non-erythrocytic 4; FYN-binding protein-like; FRAS1-related extracellular matrix protein 3), cytoskeleton (keratin; neurofilament light polypeptide), fertilization (zona pellucida sperm-binding protein 4-like), enzymatic activity (hexokinase-2-like; protein ABHD14B-like; serine/threonine-protein kinase akt-1-like; UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 9) and transport (golgi to ER traffic protein; large neutral amino acids transporter; apolipoprotein B-100) (Blast E-value < 1, color code: light blue).

It is also important to mention that de novo BLAST alignment suggest the presence of the following proteins (Additional files 1 and 2) (Blast E-value > 1, color code: light green): antimicrobial peptide precursor, catechol O-methyltransferase-like, cytochrome P450, estradiol 17-beta-dehydrogenase 12-B-like S, integumentary mucin B.1, metalloproteinase ADAM10-like protein, NADH dehydrogenase subunit 6, N-acetylneuraminate lyase, pappalysin-1, phospholipid-transporting ATPase IC-like, phospholipase A2 crotoxin basic subunit CBb-like, proteasome 26S subunit, protein kinase C delta type-like, protein-tyrosine kinase 2-beta, proteoglycan 4, squalene synthase-like, trans-1,2-dihydrobenzene-1,2-diol dehydrogenase-like and trefoil factor 2-like. We also found uncharacterized proteins in our analysis (Additional files 1 and 2).

Discussion

Bufonidae is a worldwide amphibian taxon popularly known as the true toads. These anurans store a huge arsenal of bioactive molecules in their parotoid macroglands, such as alkaloids and steroids [5, 10, 12, 23].

However, only a few studies focus on the biochemical and/or biological characterization/identification of proteins in bufonids. One explanation is that the parotoid macrogland secretion exhibit a viscous aspect, which difficult its solubilization. Another point to highlight is the higher quantity of low molecular mass molecules, which makes necessary to prepare properly the sample prior to the protein study.

Huo et al. [14] submitted B. gargarizans parotoid macrogland secretion to a cut-off filter (10 kDa). Based on de novo sequencing, the authors obtained a < 10 kDa fraction rich in peptides. This fraction exhibited anti-proliferative activity on SMMC-7721 cells under different concentrations. However, the authors do not comment about the presence of low weight molecular mass molecules in < 10 kDa fraction; neither if these molecules were removed nor how they did it.

In another study, Rash et al. [13] submitted R. marina parotoid macrogland secretion to two sample preparation steps: dialysis (to remove molecules below 1 kDa) and subsequently, cut-off membrane filter (to remove molecules over 10 kDa and insoluble material). When the authors analyzed the < 10 kDa fraction (termed peptide-enriched sample), they found that the peptide abundance in this material was very low (the authors sequenced only 14 de novo peptides). Furthermore, even after dialysis and membrane filter, this material contained high quantities of low molecular mass molecules (< 900 Da).

Utilizing a different approach, Mariano et al. [21] observed a similar result as obtained by Rash et al. [13]. After analyzing D. melanostictus parotoid macrogland secretion by batch sample preparation, the authors obtained soluble protein fractions. However, in these soluble fractions they also observed the presence of low molecular mass molecules, however, in low abundance.

The presence of non-protein compounds (alkaloids, biogenic amines, mucus and steroids) interfere in many protein quantification assays [24]. Employing different chromatography strategies (gel filtration, ion exchange and high-performance liquid chromatography), βγ-CAT (a complex of non-lens βγ-crystallin and trefoil factor) [25], lysozyme [17] and KPHTI (a trypsin inhibitor) [26] were purified from Bombina maxima, B. andrewsi and Kaloula pulchra hainana skin secretion, respectively. Using a similar chromatography steps, Anjolette et al. [24] obtained active protein fractions active on the complement system [24].

Demesa-Balderrama et al. [27] and Cavalcante et al. [28] conducted proteomics studies with Lithobates spectabilis and Dermatonotus muelleri skin secretion after electrophoresis and classical in-gel digestion. However, such approaches are purely analytical and rely on protein separation by electrophoresis, not allowing (typically) protein recovery and/or subsequent biological/biochemical assays. Moreover, the scarce available omics databases impair proper proteomic identification.

We employed the ion-exchange batch processing protocol [21] to obtain soluble protein fractions from D. melanostictus parotoid macrogland secretion. Following this methodology, we identified by proteomics proteins already described in other studies, such as alcohol dehydrogenase [NADP(+)], calmodulin, galectin, histone H2B and prostaglandin reductase 1 [18, 27-29].

Alcohol dehydrogenase [NADP(+)] is an enzyme belonging to the protein superfamily aldo-keto-reductases (AKRs), responsible for the reduction of aldehydes and ketones to primary and secondary alcohols [30]. Calmodulin is a Ca+2-receptor protein involved in signaling pathways, such as growth, metabolic homeostasis, osmotic control, proliferation or reproductive process, through interaction with multiple target proteins [31]. Galectins are a phylogenetically conserved family of lectins involved in different cell signaling pathways, in the immune system, and also in the adult and embryonic tissue development and differentiation [32]. Histones are proteins responsible for the nucleosome structure in eukaryotic cells [33]. Another identified enzyme, prostaglandin reductase 1, is responsible for the irreversible degradation of prostaglandin E and F, leukotriene B4 and lipoxin A4, all endogenous lipid mediators involved in immune response and inflammation, for example [34].

In this work, we also identified proteins related to: energy metabolism (ATP synthase subunit alpha and beta), oxygen transport (hemoglobin subunit beta) or structural activity (beta-actin, inner-ear cytokeratin, myosin and tubulin). Furthermore, proteomic analysis revealed the presence of acyl-CoA-binding protein homolog (ACBP) and diazepam binding inhibitor (DBI). Previously, Deng et al. (deposited sequence with no paper associated) found acyl-CoA-binding protein homolog mRNA in B. garzarians venom (GenBank: DQ437101.1; ABD75368.1); recently, Huo et al. [14] sequenced de novo peptides related to this protein in the aqueous extract of B. gargarizans parotoid macrogland secretion.

Studies showed that ACBP and DBI were the same protein [35, 36]. ACBP is highly conserved among different organisms. The literature reports some biological activities of this protein: capacity to displace diazepam from the γ-aminobutyric acid (GABA) receptor in rat brain; to affect the cell growth; to bind to long-chain acyl-CoA esters; to stimulate steroidogenesis in isolated adrenal mitochondria; and to inhibit glucose inducing insulin secretion from pancreas [35, 37]. In addition, after inducing ACBP depletion in a mouse model, animals displayed reduced water content in the intercellular lipid membranes, which led to an elevated transepidermal water loss [38]. In this way, ACBP may act/help on physiological process that happens in anuran skin, once this organ is involved in gas-exchange, ionic and osmotic regulation, protection and thermoregulation [1-4]. The presence of steroid molecules in bufonid parotoid macrogland secretion [9-12, 15] may suggest the involvement of ACBP in the synthesis or transport of such molecules.

Moreover, we identified proteins involved in housekeeping function. Sousa-Filho et al. [18] found a similar protein profile in R. schneideri parotoid macrogland secretion (proteins related to carbohydrate metabolism, cell matrix, lipid metabolism, protein metabolism or uncharacterized proteins). Kowalski et al. [19] identified proteins involved in the antioxidant system (phospholipid hydroperoxide glutathione peroxidase), apoptosis (serine-threonine kinase), energy metabolism (muscle creatine kinase) or protein recycling (proteasome subunit α type-7-A) from B. bufo parotoid macrogland extract. The authors also identified a serine peptidase (snake venom serine protease homolog) that may exert toxic activity [19]. Rash et al. [13] suggested that the de novo peptides obtained from R. marina parotoid macrogland secretion were breakdown products of proteins involved in cell maintenance.

Each gland that composes the parotoid macrogland is a syncytial cell filled by cytoplasm. Several nuclei are present on the periphery of the gland, as well as organelles (Golgi stacks, mitochondria and rough endoplasmic reticulum). In a central position, we found several granules [7, 8]. Upon mechanical pressure, the secretion expels/ejects much like a champagne cork. Therefore, cellular components can be expelled together with the secretion, like the cell machinery, cytoplasm, whole organelles and eventually nuclei [39]. Therefore, it was not unusual to identify house-keeping proteins in bufonid parotoid macrogland secretion.

De novo peptide sequencing relies on the determination of a peptide sequence directly from the mass spectrometry data, without the aid of a protein database [40]. Using this rationale, we deduced circa 150 de novo peptides from SDF and ADF fractions. After performing a BLAST search against Amphibia database, we proposed another dataset of proteins related to: binding, enzymatic activity or molecular transport (Additional files 1 and 2).

Furthermore, there were other interesting protein possibilities derived from that approach. However, due to the poor aligned E-value (but not a low ALC score) we will not discuss deeply these results, avoiding too much speculation. The only protein that we would call the attention is the antimicrobial peptide precursor (Additional file 1: fractions 3 and 4; Additional file 2: fraction 5). Recently, Shibao et al. [41] identified several classes of antimicrobial peptides in Rhinella schneideri skin glands by transcriptomic analysis. However, complementary studies are still necessary to confirm the actual existence of them.

Demesa-Balderrama et al. [27] performed an in-gel digestion and de novo peptide sequencing to study proteins from Lithobates spectabilis skin secretion. The authors found 111 de novo peptides, identifying 15 proteins (E-value < 0.077). Nonetheless, their research discuss about the aspects that we must considerer when performing protein identification based on de novo peptides. One of them is the decreased number of amphibian skin proteins deposited in public data banks. Such problematic was also observed by Souza-Filho et al. [18] and in the present study, supporting the need for transcriptome studies with amphibian granular gland.

The IEX batch sample preparation led to the identification of 42 proteins in D. melanostictus parotoid macrogland secretion. These results may help to increase the knowledge on the amphibian skin secretion composition, as well as to infer possible biological activities exerted by these proteins. De novo peptide sequencing and subsequent BLAST alignment suggest a complementary protein dataset in D. melanostictus parotoid macrogland secretion.

>Abbreviations

SDF: salt-displaced fraction; ADF: acid-displaced fraction; A-UBF: anionic unbound fraction; A-SDF: anionic salt-displaced fraction; A-ADF: anionic acid-displaced fraction; TCEP: Tris(2-carboxyethyl)phosphine hydrochloride; IAA: iodoacetamide; ESI-Q-TOF: electrospray-quadrupole-time of flight; UPLC: ultra-performance liquid chromatography system; FA: formic acid; CAN: acetonitrile; MS: mass spectrometry; ALC: average local confidence; AKR: aldo-keto-reductase; ACBP: acyl-CoA-binding protein homolog; DBI: diazepam binding inhibitor; GABA: γ-aminobutyric acid

Acknowledgments

The authors would like to thank Ismael Feitosa Lima, from the Center for Research on Toxins, Immune-Response and Cell Signaling (CeTICS) of Butantan Institute, for the technical assistance.

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Availability of data and materialsAll data generated or analyzed during this study are included in this published article (and its supplementary information files).

FundingThis work was supported by the Coordination for the Improvement of Higher Education Personnel (CAPES - grant n. 969130 to DOCM), the São Paulo Research Foundation (FAPESP) and the National Council for Scientific and Technological Development (CNPq - grant n. 406385/2018-1). DCP is a CNPq fellow researcher (grant n. 303792/2016-7). CeTICS is supported by FAPESP (grant n. 2013/07467-1). A FAPESP grant (n. 2013/07467-1) was used for acquisition of the Q-TOF mass spectrometer. JVATiTD publication is supported by the National Council for Scientific and Technological Development (CNPq) through Programa Editorial CNPq/CAPES (chamada n. 26/2017, processo n. 440954/2017-7). Therefore, all articles published by the journal are in part funded by this grant.

Ethics approvalNot applicable.

Consent for publicationNot applicable.

Received: May 06, 2019; Accepted: July 11, 2019

* Correspondence: dcpimenta@butantan.gov.br

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

All the authors contributed equally in this work. All authors read and approved the final manuscript.

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