- Email: [email protected]
A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas
Biochimie 94 (2012) 1376e1381
Contents lists available at SciVerse ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas Renjie Li a,1, Hui Wang a,1, Yingchun Jiang a, Yang Yu a, Lei Wang a, *, Mei Zhou a, *, Yingqi Zhang b, Tianbao Chen a, Chris Shaw a a b
Natural Drug Discovery Group, School of Pharmacy, Queen’s University, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK Tangshan Gongren Hospital, No.27, Wenhua Road, Tangshan City, Hebei Province 063000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 January 2012 Accepted 8 March 2012 Available online 23 March 2012
The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki ¼ 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequence: ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-Cx(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Amphibian Skin Peptide Protein Trypsin Inhibitor Cloning
1. Introduction Amphibian skin is generally smooth and moist but produces a chemically-complex defensive secretion to deter predators and protect against microbial colonisation [1e4]. The source of this bioactive cocktail are the dermal granular glands and biochemically, its constituents are representative of many molecular classes including biogenic amines, peptides, proteins, alkaloids and heterocyclics [1e4] and as a consequence, extracts of amphibian skin have been used for centuries in folk medicine and witchcraft due to their possession of a wide spectrum of often devastating pharmacological effects [1e4]. Peptides, that are the predominant bioactives in many species, display a wide range of pharmacological activities and may belong to different, structurally-related peptide families [1e4]. They vary in many features that may include primary structure, the presence of disulphide bonds and a plethora of post-translational
* Corresponding authors. Fax: þ44 28 9024 7794. E-mail addresses: [email protected] (L. Wang), [email protected] (M. Zhou). 1 These authors contributed equally to this work. 0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2012.03.009
modifications such as pyroglutamation of N-terminal glutamine residues, amidation of C-terminal residues, hydroxylation of proline, sulphation of tyrosine and racemisation of certain L-amino acids to their D-isomers [1,2]. There is thus enormous scope in the study of these secretions for the discovery of novel structural and functional molecules with possible unique attributes. One class of peptides/proteins that have been isolated from this source are the protease inhibitors, which have been known for some time to be produced by many diverse animals ranging from nematodes to humans and to have an almost ubiquitous distribution in other life forms, such as microorganisms and plants [5,6]. Trypsin inhibitors related to the Ascaris trypsin inhibitor (ATI) have been isolated from ranid frogs and bombinid toads [7,8], a Kunitz-type inhibitor from the tomato frog, Dyscophus guineti, an inhibitor displaying a whey acidic protein (WAP) motif from a ranid frog and Bowman Birk-type inhibitors from ranid frogs [9e11]. Inhibitors for other serine proteases have been found in skin secretions of Phyllomedusa sauvagei (a Kazal inhibitor of post-proline cleaving enzyme) [12] and most recently in that of the African hyperoliid frog, Kassina senegalensis (a Kunitz-type inhibitor of chymotrypsin) [13]. In this study, we describe the structural and functional characterisation of a novel Kazal-type trypsin inhibitory protein from the
R. Li et al. / Biochimie 94 (2012) 1376e1381
defensive skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas. Named A. callidryas Kazal trypsin inhibitor (ACKTI), the full open-reading frame of a cDNA encoding its biosynthetic precursor was deduced following RACE-PCR cloning from a skin cDNA library. Once full structural characterisation had been achieved, the inhibition constant of the protein was determined and bioinformatic analyses were performed.
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Animals were kept under these conditions for at least 3 months prior to acquisition of skin secretion. This was obtained by transdermal electrical stimulation using the method of Tyler et al. [14], washed from the skin with de-ionised water, snapfrozen in liquid nitrogen, lyophilised and stored at 20 C prior to analysis. 2.2. Reverse-phase HPLC
2. Materials and methods Five milligrammes of lyophilised skin secretion were dissolved in 0.5 ml of trifluoroacetic acid (TFA)/water (0.1:99.9; v/v), clarified by centrifugation and the decanted supernatant was subjected to reverse-phase HPLC fractionation using a Cecil Adept Binary HPLC system (Cambridge, UK) fitted with a Jupiter C-5 analytical column (250 4.6 mm). Bound peptides were eluted with a linear gradient formed from 0.05/99.5 (v/v) TFA/water to
2.1. Acquisition of skin secretions Adult red-eyed leaf frogs, A. callidryas, of the Costa Rican strain (both sexes; snout-to-vent length 4e6 cm) were housed in a purpose-designed terrarium under a 12 h/12 h light/dark cycle and were fed multivitamin-loaded crickets three times per week.
A
918
Absorbance [mA]
714
511
307
103 28:59
37:34
46:09
54:43
63:18
71:53
Time [mm:ss]
B
Voyager Spec #1=>BC=>NF0.7[BP = 5894.1, 2513] 5894.09
100
2513.2
90
80
70
% Intensity
60
5875.36
3251.73
50
5913.08
40
30
2947.18
20
3298.10
10
0 2873.0
3636.4
4399.8
5163.2
Mass (m/z)
5926.6
0 6690.0
Fig. 1. (A) Region of reverse-phase HPLC chromatogram of Agalychnis callidryas skin secretion indicating elution position/retention time of the trypsin inhibitor (arrow). This corresponds to fraction number 30. Y-axis represents milli-absorbance units. (B) MALDI-TOF mass spectrum of a sample from fraction number 30. The major singly-charged ion at m/z 5894.09 and its related doubly-charged ion at m/z 2947.18, correspond to an observed parent molecular ion of average mass 5892.73 Da (calculated mass from gene sequence with oxidised Cys residues, 5892.82 Da).
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0.05/19.95/80.0 (v/v/v) TFA/water/acetonitrile in 80 min at a flow rate of 1 ml/min. Fractions (1 ml) were collected at minute intervals and the effluent absorbance was continuously monitored at l214 nm. Samples (200 ml) were removed from each fraction in triplicate, lyophilised and stored at 20 C prior to screening for trypsin inhibition. 2.3. Trypsin inhibition assay Trypsin (10 ml of a 0.1 mM stock solution in 1 mM HCl) was added to the wells of a micro-titre plate containing substrate (Phe-ProArg-NHMec, obtained from Sigma/Aldrich, Poole, Dorset, UK) (50 mM) and individual reconstituted reverse-phase HPLC fractions in 10 mM phosphate buffer, pH 7.4, containing 2.7 mM KCl and 137 mM NaCl (final volume 210 ml). Each determination was carried out in triplicate. The rate of hydrolysis of substrate was monitored continuously at 37 C, by measuring the rate of increase of fluorescence due to production of 7-amino-4-methylcoumarin (NH2Mec) at 460 nm (excitation 360 nm) in a CYTOFLUORÒ multi-well plate reader Series 4000 spectrofluorimeter.
2.4. Structural characterisation of the trypsin inhibitor Reverse-phase HPLC fraction #30, which exhibited the highest trypsin inhibitory activity, was used for the structural characterisation of the active moiety. A small sample (2 ml) of this fraction was removed and subjected to mass spectrometric analysis using a MALDI-TOF mass spectrometer (Voyager DE, Perseptive Biosystems, MA, USA) and an additional sample (20 ml) was removed subsequently and subjected to primary structural analysis by automated Edman degradation performed using an Applied Biosystems 491 Procise microsequencer in pulsed-liquid mode. The limit for detection of phenylthiohydantoin (PTH) amino acids was 0.05 pmol. 2.5. Molecular cloning of trypsin inhibitor precursor-encoding cDNA An additional 5 mg sample of lyophilised skin secretion was dissolved in 1 ml of cell lysis/mRNA protection buffer supplied by Dynal Biotec, UK. Polyadenylated mRNA was isolated by the use of
Fig. 2. (A) NCBI-BLASTp analysis of N-terminal Edman sequence of the Agalychnis callidryas skin trypsin inhibitor (ACKTI) incorporating Cys residues in blank cycles 6, 12 and 20. Alignments with regions of the Kajal type serine protease inhibitor from the Pearl oyster (Pinctada fucata), accession number ADC52432.1, and the ovoinhibitor-like protein from the turkey (Meleagris gallopavo), accession number XP003210422.1. Residue numbers are given in parentheses and identical residues are indicated by asterisks. The P1 residue in the active site (R (Arg) or K (Lys), consistent with trypsin inhibition) is indicated by an arrow. (B) Nucleotide and translated open-reading frame amino acid sequence of cloned cDNA encoding the biosynthetic precursor of A. callidryas Kajal trypsin inhibitor (ACKTI). The putative signal peptide is double-underlined, the mature ACKTI is single-underlined and the stop codon is indicated by an asterisk. Site of signal peptide cleavage predicted using the on-line Signal P 4.0 server, technical University of Denmark.
R. Li et al. / Biochimie 94 (2012) 1376e1381
magnetic oligo-dT beads as described by the manufacturer (Dynal Biotec, UK). The isolated mRNA was subjected to 50 and 30 -rapid amplification of cDNA ends (RACE) procedures to obtain full-length trypsin inhibitor precursor nucleic acid sequence data using a SMART-RACE kit (Clontech UK) essentially as described by the manufacturer. Briefly, the 30 -RACE reactions employed a nested universal (NUP) primer (supplied with the kit) and a sense primer (S: 50 - GCIACIAARCCIMGITGYCARTA-30 ) that was complementary to the N-terminal amino acid sequence of the trypsin inhibitor, ATKPRCQY-, determined by automated Edman degradation (see Results section 3.1). The 30 -RACE reactions were purified and cloned using a pGEM-T vector system (Promega Corporation) and sequenced using an ABI 3100 automated capillary DNA sequencer. The sequence data obtained from these 30 -RACE products was used to design a specific antisense primer (AS: 50 -CCATGAA GACTCTGAAACTCTCCAA-30 ) to a conserved site within the 30 -nontranslated region of the trypsin inhibitor precursor-encoding cDNA. 50 -RACE was carried out using this specific primer in conjunction with the NUP RACE primer and resultant products were purified, cloned and sequenced. 3. Results 3.1. Identification, isolation and partial structural characterisation of the trypsin inhibitor peptide Trypsin inhibitory activity was identified following reversephase HPLC fractionation of skin secretion with peak activity being resolved into fraction #30 (Fig. 1A). MALDI-TOF mass spectrometer analysis of this fraction indicated a major component with a singly-charged ion of m/z of 5894.09 and a doubly-charged ion of m/z of 2947.18 (Fig. 1B). A contaminant with m/z of 3251.73 was an antimicrobial peptide, dermaseptin, which represents a class of peptides that are devoid of trypsin inhibitory activity. Partial automated Edman degradation of this fraction established the sequence of the first twenty amino acid residues of the major peptide as: ATKPR-QYIVL-PRILRPV-GT. Blank cycles were assumed to represent cysteinyl residues as these are the most common cause of such effects during repeated cycles of Edman degradation. An
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NCBI-BLAST search using the sequence obtained incorporating the blank cycles, produced no relevant hits but incorporation of cysteinyl residues at these sites produced significant hits with Kazal inhibitor proteins from molluscs and with avian ovomucoids (Fig. 2A). 3.2. Molecular cloning of trypsin inhibitor precursor-encoding cDNA By means of the molecular cloning strategy described, that used the partial N-terminal amino acid sequence data generated to synthesise a degenerate primer pool for 30 -RACE-PCR, a single transcript that encoded the precursor of a Kunitz protein was repeatedly cloned and was of identical nucleotide sequence in at least 25 different clones. The nucleotide and translated openreading frame amino acid sequence of these clones is shown in Fig. 2B. The open-reading frame of the precursor consisted of 78 amino acid residues with the first 26 constituting the putative signal peptide (predicted using the SignalP 4.0 server, Technical University of Denmark), and the remaining 52 residues constituting the mature Kazal inhibitor protein. This novel Kazal protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). The nucleotide sequence of the skin secretion-derived Kazal trypsin inhibitor has been deposited in the EMBL Nucleotide Sequence Database under the accession code HE653907. 3.3. Additional bioinformatic analyses performed on ACKTI NCBI-BLASTp analysis of the signal peptide sequence produced no relevant hits with any amphibian skin peptide precursor but the analysis of the mature peptide sequence revealed that ACKTI displayed the highest sequence identity (22 residues) with Kazal inhibitors of the bony fishes, Salmo salar (Atlantic salmon) and Danio rerio (Zebrafish) (Fig. 3A). Alignment of the primary structure of ACKTI with those of PSKP-1 and PSKP-2 from the skin of P. sauvagei [12], two Kazal-type inhibitors of post-proline cleaving enzyme, revealed a similar shared sequence identity (20 residues) (Fig. 3B). Secondary structure prediction analysis of ACKTI using the SWISS-MODEL workspace [15], and with porcine pancreatic
A
B
Fig. 3. (A) NCBI-BLASTp analysis of the cloned cDNA open-reading frame-deduced sequence of the mature Agalychnis callidryas skin trypsin inhibitor (ACKTI). ACKTI e A. callidryas Kazal skin trypsin inhibitor; SSKTI - Salmo salar (Atlantic salmon) Kazal trypsin inhibitor e accession number NP_001140094; DRKTI-1 - Danio rerio (zebrafish) Kazal trypsin inhibitor-1 e accession number XP_003199918; DRKTI-2- D. rerio (Zebrafish) Kazal trypsin inhibitor 2 e accession number XP_003199919. Gaps have been included to maximise alignments. Conserved residues are indicated by asterisks and the canonical Kazal inhibitor motif is shown below. The common P1 residue (R- Arg, consistent with trypsin inhibition), is indicated by an arrow. (B) Alignment of ACKTI with Phyllomedusa sauvagei skin-derived post-proline cleaving enzyme Kazal inhibitors, PSKP-1 and PSKP-2 (accession numbers P83578 and P83579, respectively). Gaps have been included to maximise alignments. Conserved residues are indicated by asterisks. Note that the P1 residue (arrow) in both PSKP-1 and PSKP-2 is P (Pro), consistent with their post-proline enzyme inhibitory activity and lack of trypsin inhibitory activity.
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secretory Kazal-type 1 trypsin inhibitor as a reference, showed unequivocally that in terms of putative secondary structure, ACKTI was very similar to this related protein from a mammalian source (Fig. 4A). 3.4. Determination of the inhibition constant of ACKTI for trypsin ACKTI was incubated with substrate and trypsin at a range of concentrations and a Morrison plot of initial rates (vi) was used to calculate the Ki of 1.9 nM (Fig. 4B). 4. Discussion Amphibian skin secretions are an intriguing resource for the discovery of novel natural bioactive peptides with a wide spectrum of pharmacological actions and putative physiological functions [1e4]. While low molecular mass peptides of short chain length can
be readily sequenced using Edman degradation, higher molecular mass polypeptides and proteins present the investigator with some technical problems that are often solved by use of protease digestion resulting in generation of a mixture of shorter oligopeptides from the parent molecule. The procedure essentially progresses through fractionation of the digest, sequencing of individual peptides and finally piecing together the structure of the parent polypeptide/protein [16,17]. Problems however can arise if the polypeptide/protein being studied is a protease inhibitor, especially one that is active against trypsin e the protease that is most often employed for this purpose [7,9]. Trypsin inhibitors are also the commonest group of protease inhibitors that have been identified in amphibian skin secretions [7e11]. However, acquiring the molecular mass of the polypeptide/protein of interest and also a short N-terminal amino acid sequence, can provide sufficient information for degenerate primer design to initiate precursorencoding cDNA cloning studies [18,19]. Previously, the presence in
Fig. 4. Secondary structure prediction analysis of (A) porcine pancreatic secretory Kazal-type 1 trypsin inhibitor (accession number P00998) and (B) ACKTI, using the SWISS-MODEL workspace. Note the high degree of secondary structural similarity between mammal and amphibian proteins especially in the C-terminal region with sequential a-helix (H), random coil (C) and extended beta sheet (E). (C) A Morrison plot of trypsin inhibitory activity of ACKTI at different concentrations indicating a Ki value of 1.9 nM.
R. Li et al. / Biochimie 94 (2012) 1376e1381
lyophilised amphibian skin secretions of PCR amenable polyadenylated mRNA transcripts encoding constituent peptides/ proteins was established [20]. Thus, parallel peptide/protein sequencing, molecular mass determination and precursorencoding cDNA cloning as employed in the present study, can provide rapid and unequivocal structural determinations of novel molecules. Peptidic inhibitors of proteases are ubiquitous in Nature and are found in multiple forms in animals, plants and microorganisms [5,6]. They are grouped primarily following establishment of the presence of well-defined structural motifs that are invariably intimately related to their functions [5,6,21]. Such groups include the Kunitz-, Bowman Birk- and Kazal-type inhibitors of serine proteases [5]. These inhibitors function by binding to their cognate enzyme in a substrate-like manner, forming a stable complex [5]. Representatives of all three types have been isolated from amphibian skin although the Kazal inhibitors thus far appear to be the least common with only two, closely-related inhibitors of postproline cleaving enzyme, PSKP-1 and PSKP-2, having been isolated from the skin of the waxy monkey frog, P. sauvagei [12]. The subfamily of South and Central American leaf frogs to which this species belongs, the Phyllomedusinae, contains about 50 species in several genera [22]. The largest genus, Phyllomedusa, contains species with skin secretions that have been described as a “huge factory and store-house of a variety of (biologically) active peptides” [22]. This was a major reason to focus our efforts on the skin secretions of another member of this subfamily, the red-eyed leaf frog, A. callidryas, in the search for novel inhibitors of trypsin. The single endogenous inhibitor of trypsin identified in the skin of this species was of Kazal-type. While there have been two Kazal proteins previously identified in phyllomedusine frog skin secretion (PSKP-1 and PSKP-2), neither was found to be an inhibitor of trypsin. However, changing the Pro (P) residue in the P1 position to Arg (R), virtually abolished the original post-prolineecleavage activity and produced a potent trypsin inhibitor [12]. The crucial role played by the nature of the residue in the P1 position of the active site of Kazal inhibitors was thus elegantly demonstrated by these data. Bioinformatic analysis of ACKTI produced interesting results. Database interrogation with the N-terminal 22-residue sequence obtained by Edman degradation of the intact protein, incorporating the blank cycles, resulted in no appropriate hits. However, incorporation of the putative Cys residues at these sites, resulted in a clear indication of relatedness to reactive centre domains of Kazal trypsin inhibitors from organisms as diverse as molluscs and birds (Fig. 2A). Three of the Cys residues incorporated represent fundamental sites of the canonical Kazal motif (Fig. 3A) and the presence of such residues at these sites was unequivocally confirmed following successful cloning of ACKTI precursorencoding cDNA (Fig. 2B). Database interrogation using the full primary structure of mature ACKTI revealed the presence of a full canonical Kazal inhibitor motif and a significant degree of relatedness to bony fish analogues (salmon and zebrafish) (Fig. 3A) that was similar to that exhibited between all three amphibian skin analogues (ACKTI, PSKP-1 and PSKP-2) (Fig. 3B). Although primary structural identity per se between various Kazal inhibitors was relatively low (approx. 40%), secondary structural prediction comparison between ACKTI and porcine pancreatic Kazal trypsin inhibitor-1 (Fig. 4A) revealed a greater degree of similarity. Thus primary structure per se outside of defined functional motifs or domains, appears not to be a major determining factor in the function of this class of protease inhibitor. The Ki determined for ACKTI in this study, was in the low nanomolar range (1.9 nM),
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which is consistent with that determined for many other Kazaltype trypsin inhibitors from many different sources [5]. The data presented in this report substantiates the widely-held view that amphibian skin secretions, in this case those of a phyllomedusine frog, are “huge factories and store-houses of biologicallyactive peptides” [22], and that this assertion could be extended to include polypeptides and proteins. Further systematic searches of this resource would thus be highly-likely to result in the discovery of novel biologically-active molecules with applications as pharmacological tools or in the more general fields of biotechnology and biomedical sciences.
References [1] V. Erspamer, Bioactive secretions of the integument, in: H. Heatwole, G.T. Barthalmus (Eds.), The Integument, Amphibian Biology, vol. 1, Surrey Beatty & Sons, Chipping Norton, 1994 (Chapter 9). [2] B.L. Bevins, M. Zasloff, Peptides from frog skin, Ann. Rev. Biochem. 59 (1990) 395e414. [3] L.H. Lazarus, M. Attila, The toad, ugly and venomous, wears yet a precious jewel in his skin, Prog. Neurobiol. 41 (1993) 473e507. [4] B.T. Clarke, The natural history of amphibian skin secretions, their normal functioning and potential medical applications, Biol. Rev. Camb. Philos. Soc. 72 (1997) 365e379. [5] M. Laskowski, I. Kato, Protein inhibitors of proteinases, Ann. Rev. Biochem. 49 (1980) 593e626. [6] W. Bode, R. Huber, Natural protein proteinase inhibitor and their interaction with proteinases, Eur. J. Biochem. 204 (1992) 433e451. [7] M.F. Ali, K.R. Lips, F.C. Knoop, B. Fritzsch, C. Miller, J.M. Conlon, Antimicrobial peptides and protease inhibitors in the skin secretions of the crawfish frog, Rana areolata, Biochim. Biophys. Acta 160 (2002) 55e63. [8] T. Chen, C. Shaw, Identification and molecular cloning of novel trypsin inhibitor from the dermal venom of the Oriental fire-bellied toad (Bombina orientalis) and the European yellow-bellied toad (Bombina variegata), Peptides 24 (2003) 873e880. [9] J.M. Conlon, J.B. Kim, A protease inhibitor of the Kunitz family from skin secretions of the tomato frog, Dyscophus guineti (Microhylidae), Biochem. Biophys. Res. Commun. 279 (2000) 961e964. [10] G. Song, M. Zhou, W. Chen, T. Chen, B. Walker, C. Shaw, HV-BBIea novel amphibian skin Bowman-Birk-like trypsin inhibitor, Biochem. Biophys. Res. Commun. 372 (2008) 191e196. [11] M. Wang, L. Wang, T. Chen, B. Walker, M. Zhou, D. Sui, J.M. Colon, C. Shaw, Identification and molecular cloning of a novel amphibian Bowman-Birk-like trypsin inhibitor from the skin of the Hejiang Odorous Frog, Odorrana Hejiangensis, Peptides 33 (2012) 245e250. [12] L.G. Gebhard, F.U. Carrizo, A.L. Stern, N.I. Burgardt, J. Faivovich, E. Lavilla, M.R. Ermácora, A Kazal prolyl endopeptidase inhibitor isolated from the skin of Phyllomedusa sauvagei, Eur. J. Biochem. 271 (2004) 2117e2126. [13] H. Wang, L. Wang, M. Zhou, M. Yang, C. Ma, T. Chen, Y. Zhang, M. Zeller, M. Hornshaw, C. Shaw, Functional peptidomics of amphibian skin secretion: a novel Kunitz-type chymotrypsin inhibitor from the African hyperoliid frog, Kassina Senegalensis, Biochimie 94 (2012) 891e899. [14] M.J. Tyler, D.J.M. Stone, J.H. Bowie, A novel method for the release and collection of dermal, glandular secretions from the skin of frogs, J. Pharmacol. Toxicol. Methods 28 (1992) 199e200. [15] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling, Bioinformatics 22 (2006) 195e201. [16] R. Deutzmann, Structural characterization of proteins and peptides, Methods Mol. Med. 94 (2004) 269e297. [17] V.C. Pham, W.J. Henzel, J.R. Lill, Rapid on-membrane proteolytic cleavage for Edman sequencing and mass spectrometric identification of proteins, Electrophoresis 26 (2005) 4243e4251. [18] T. Chen, Y. Xue, M. Zhou, C. Shaw, Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8ea novel prokineticin from Bombina orientalis, Peptides 26 (2005) 377e383. [19] L. Odum, J.R. Bundgaard, A.H. Johnsen, A Kazal-type trypsin inhibitor from the protochordate Ciona intestinalis, Eur. J. Biochem. 259 (1999) 872e876. [20] T. Chen, S. Farragher, A.J. Bjourson, D.F. Orr, P. Rao, C. Shaw, Granular gland transcriptomes in stimulated amphibian skin secretions, Biochem. J. 371 (2003) 125e130. [21] R.M. Roberts, N. Mathialagan, J.Y. Duffy, G.W. Smith, Regulation and regulatory role of proteinase inhibitors, Crit. Rev. Eukaryot. Gene Exp. 5 (1995) 385e436. [22] V. Erspamer, P. Melchiorri, G. Falconieri Erspamer, P.C. Montecucchi, R. de Castiglione, Phyllomedusa skin: a huge factory and store-house of a variety of active peptides, Peptides 6 (1985) 7e12.
Contents lists available at SciVerse ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas Renjie Li a,1, Hui Wang a,1, Yingchun Jiang a, Yang Yu a, Lei Wang a, *, Mei Zhou a, *, Yingqi Zhang b, Tianbao Chen a, Chris Shaw a a b
Natural Drug Discovery Group, School of Pharmacy, Queen’s University, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK Tangshan Gongren Hospital, No.27, Wenhua Road, Tangshan City, Hebei Province 063000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 January 2012 Accepted 8 March 2012 Available online 23 March 2012
The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki ¼ 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequence: ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-Cx(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Amphibian Skin Peptide Protein Trypsin Inhibitor Cloning
1. Introduction Amphibian skin is generally smooth and moist but produces a chemically-complex defensive secretion to deter predators and protect against microbial colonisation [1e4]. The source of this bioactive cocktail are the dermal granular glands and biochemically, its constituents are representative of many molecular classes including biogenic amines, peptides, proteins, alkaloids and heterocyclics [1e4] and as a consequence, extracts of amphibian skin have been used for centuries in folk medicine and witchcraft due to their possession of a wide spectrum of often devastating pharmacological effects [1e4]. Peptides, that are the predominant bioactives in many species, display a wide range of pharmacological activities and may belong to different, structurally-related peptide families [1e4]. They vary in many features that may include primary structure, the presence of disulphide bonds and a plethora of post-translational
* Corresponding authors. Fax: þ44 28 9024 7794. E-mail addresses: [email protected] (L. Wang), [email protected] (M. Zhou). 1 These authors contributed equally to this work. 0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2012.03.009
modifications such as pyroglutamation of N-terminal glutamine residues, amidation of C-terminal residues, hydroxylation of proline, sulphation of tyrosine and racemisation of certain L-amino acids to their D-isomers [1,2]. There is thus enormous scope in the study of these secretions for the discovery of novel structural and functional molecules with possible unique attributes. One class of peptides/proteins that have been isolated from this source are the protease inhibitors, which have been known for some time to be produced by many diverse animals ranging from nematodes to humans and to have an almost ubiquitous distribution in other life forms, such as microorganisms and plants [5,6]. Trypsin inhibitors related to the Ascaris trypsin inhibitor (ATI) have been isolated from ranid frogs and bombinid toads [7,8], a Kunitz-type inhibitor from the tomato frog, Dyscophus guineti, an inhibitor displaying a whey acidic protein (WAP) motif from a ranid frog and Bowman Birk-type inhibitors from ranid frogs [9e11]. Inhibitors for other serine proteases have been found in skin secretions of Phyllomedusa sauvagei (a Kazal inhibitor of post-proline cleaving enzyme) [12] and most recently in that of the African hyperoliid frog, Kassina senegalensis (a Kunitz-type inhibitor of chymotrypsin) [13]. In this study, we describe the structural and functional characterisation of a novel Kazal-type trypsin inhibitory protein from the
R. Li et al. / Biochimie 94 (2012) 1376e1381
defensive skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas. Named A. callidryas Kazal trypsin inhibitor (ACKTI), the full open-reading frame of a cDNA encoding its biosynthetic precursor was deduced following RACE-PCR cloning from a skin cDNA library. Once full structural characterisation had been achieved, the inhibition constant of the protein was determined and bioinformatic analyses were performed.
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Animals were kept under these conditions for at least 3 months prior to acquisition of skin secretion. This was obtained by transdermal electrical stimulation using the method of Tyler et al. [14], washed from the skin with de-ionised water, snapfrozen in liquid nitrogen, lyophilised and stored at 20 C prior to analysis. 2.2. Reverse-phase HPLC
2. Materials and methods Five milligrammes of lyophilised skin secretion were dissolved in 0.5 ml of trifluoroacetic acid (TFA)/water (0.1:99.9; v/v), clarified by centrifugation and the decanted supernatant was subjected to reverse-phase HPLC fractionation using a Cecil Adept Binary HPLC system (Cambridge, UK) fitted with a Jupiter C-5 analytical column (250 4.6 mm). Bound peptides were eluted with a linear gradient formed from 0.05/99.5 (v/v) TFA/water to
2.1. Acquisition of skin secretions Adult red-eyed leaf frogs, A. callidryas, of the Costa Rican strain (both sexes; snout-to-vent length 4e6 cm) were housed in a purpose-designed terrarium under a 12 h/12 h light/dark cycle and were fed multivitamin-loaded crickets three times per week.
A
918
Absorbance [mA]
714
511
307
103 28:59
37:34
46:09
54:43
63:18
71:53
Time [mm:ss]
B
Voyager Spec #1=>BC=>NF0.7[BP = 5894.1, 2513] 5894.09
100
2513.2
90
80
70
% Intensity
60
5875.36
3251.73
50
5913.08
40
30
2947.18
20
3298.10
10
0 2873.0
3636.4
4399.8
5163.2
Mass (m/z)
5926.6
0 6690.0
Fig. 1. (A) Region of reverse-phase HPLC chromatogram of Agalychnis callidryas skin secretion indicating elution position/retention time of the trypsin inhibitor (arrow). This corresponds to fraction number 30. Y-axis represents milli-absorbance units. (B) MALDI-TOF mass spectrum of a sample from fraction number 30. The major singly-charged ion at m/z 5894.09 and its related doubly-charged ion at m/z 2947.18, correspond to an observed parent molecular ion of average mass 5892.73 Da (calculated mass from gene sequence with oxidised Cys residues, 5892.82 Da).
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0.05/19.95/80.0 (v/v/v) TFA/water/acetonitrile in 80 min at a flow rate of 1 ml/min. Fractions (1 ml) were collected at minute intervals and the effluent absorbance was continuously monitored at l214 nm. Samples (200 ml) were removed from each fraction in triplicate, lyophilised and stored at 20 C prior to screening for trypsin inhibition. 2.3. Trypsin inhibition assay Trypsin (10 ml of a 0.1 mM stock solution in 1 mM HCl) was added to the wells of a micro-titre plate containing substrate (Phe-ProArg-NHMec, obtained from Sigma/Aldrich, Poole, Dorset, UK) (50 mM) and individual reconstituted reverse-phase HPLC fractions in 10 mM phosphate buffer, pH 7.4, containing 2.7 mM KCl and 137 mM NaCl (final volume 210 ml). Each determination was carried out in triplicate. The rate of hydrolysis of substrate was monitored continuously at 37 C, by measuring the rate of increase of fluorescence due to production of 7-amino-4-methylcoumarin (NH2Mec) at 460 nm (excitation 360 nm) in a CYTOFLUORÒ multi-well plate reader Series 4000 spectrofluorimeter.
2.4. Structural characterisation of the trypsin inhibitor Reverse-phase HPLC fraction #30, which exhibited the highest trypsin inhibitory activity, was used for the structural characterisation of the active moiety. A small sample (2 ml) of this fraction was removed and subjected to mass spectrometric analysis using a MALDI-TOF mass spectrometer (Voyager DE, Perseptive Biosystems, MA, USA) and an additional sample (20 ml) was removed subsequently and subjected to primary structural analysis by automated Edman degradation performed using an Applied Biosystems 491 Procise microsequencer in pulsed-liquid mode. The limit for detection of phenylthiohydantoin (PTH) amino acids was 0.05 pmol. 2.5. Molecular cloning of trypsin inhibitor precursor-encoding cDNA An additional 5 mg sample of lyophilised skin secretion was dissolved in 1 ml of cell lysis/mRNA protection buffer supplied by Dynal Biotec, UK. Polyadenylated mRNA was isolated by the use of
Fig. 2. (A) NCBI-BLASTp analysis of N-terminal Edman sequence of the Agalychnis callidryas skin trypsin inhibitor (ACKTI) incorporating Cys residues in blank cycles 6, 12 and 20. Alignments with regions of the Kajal type serine protease inhibitor from the Pearl oyster (Pinctada fucata), accession number ADC52432.1, and the ovoinhibitor-like protein from the turkey (Meleagris gallopavo), accession number XP003210422.1. Residue numbers are given in parentheses and identical residues are indicated by asterisks. The P1 residue in the active site (R (Arg) or K (Lys), consistent with trypsin inhibition) is indicated by an arrow. (B) Nucleotide and translated open-reading frame amino acid sequence of cloned cDNA encoding the biosynthetic precursor of A. callidryas Kajal trypsin inhibitor (ACKTI). The putative signal peptide is double-underlined, the mature ACKTI is single-underlined and the stop codon is indicated by an asterisk. Site of signal peptide cleavage predicted using the on-line Signal P 4.0 server, technical University of Denmark.
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magnetic oligo-dT beads as described by the manufacturer (Dynal Biotec, UK). The isolated mRNA was subjected to 50 and 30 -rapid amplification of cDNA ends (RACE) procedures to obtain full-length trypsin inhibitor precursor nucleic acid sequence data using a SMART-RACE kit (Clontech UK) essentially as described by the manufacturer. Briefly, the 30 -RACE reactions employed a nested universal (NUP) primer (supplied with the kit) and a sense primer (S: 50 - GCIACIAARCCIMGITGYCARTA-30 ) that was complementary to the N-terminal amino acid sequence of the trypsin inhibitor, ATKPRCQY-, determined by automated Edman degradation (see Results section 3.1). The 30 -RACE reactions were purified and cloned using a pGEM-T vector system (Promega Corporation) and sequenced using an ABI 3100 automated capillary DNA sequencer. The sequence data obtained from these 30 -RACE products was used to design a specific antisense primer (AS: 50 -CCATGAA GACTCTGAAACTCTCCAA-30 ) to a conserved site within the 30 -nontranslated region of the trypsin inhibitor precursor-encoding cDNA. 50 -RACE was carried out using this specific primer in conjunction with the NUP RACE primer and resultant products were purified, cloned and sequenced. 3. Results 3.1. Identification, isolation and partial structural characterisation of the trypsin inhibitor peptide Trypsin inhibitory activity was identified following reversephase HPLC fractionation of skin secretion with peak activity being resolved into fraction #30 (Fig. 1A). MALDI-TOF mass spectrometer analysis of this fraction indicated a major component with a singly-charged ion of m/z of 5894.09 and a doubly-charged ion of m/z of 2947.18 (Fig. 1B). A contaminant with m/z of 3251.73 was an antimicrobial peptide, dermaseptin, which represents a class of peptides that are devoid of trypsin inhibitory activity. Partial automated Edman degradation of this fraction established the sequence of the first twenty amino acid residues of the major peptide as: ATKPR-QYIVL-PRILRPV-GT. Blank cycles were assumed to represent cysteinyl residues as these are the most common cause of such effects during repeated cycles of Edman degradation. An
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NCBI-BLAST search using the sequence obtained incorporating the blank cycles, produced no relevant hits but incorporation of cysteinyl residues at these sites produced significant hits with Kazal inhibitor proteins from molluscs and with avian ovomucoids (Fig. 2A). 3.2. Molecular cloning of trypsin inhibitor precursor-encoding cDNA By means of the molecular cloning strategy described, that used the partial N-terminal amino acid sequence data generated to synthesise a degenerate primer pool for 30 -RACE-PCR, a single transcript that encoded the precursor of a Kunitz protein was repeatedly cloned and was of identical nucleotide sequence in at least 25 different clones. The nucleotide and translated openreading frame amino acid sequence of these clones is shown in Fig. 2B. The open-reading frame of the precursor consisted of 78 amino acid residues with the first 26 constituting the putative signal peptide (predicted using the SignalP 4.0 server, Technical University of Denmark), and the remaining 52 residues constituting the mature Kazal inhibitor protein. This novel Kazal protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). The nucleotide sequence of the skin secretion-derived Kazal trypsin inhibitor has been deposited in the EMBL Nucleotide Sequence Database under the accession code HE653907. 3.3. Additional bioinformatic analyses performed on ACKTI NCBI-BLASTp analysis of the signal peptide sequence produced no relevant hits with any amphibian skin peptide precursor but the analysis of the mature peptide sequence revealed that ACKTI displayed the highest sequence identity (22 residues) with Kazal inhibitors of the bony fishes, Salmo salar (Atlantic salmon) and Danio rerio (Zebrafish) (Fig. 3A). Alignment of the primary structure of ACKTI with those of PSKP-1 and PSKP-2 from the skin of P. sauvagei [12], two Kazal-type inhibitors of post-proline cleaving enzyme, revealed a similar shared sequence identity (20 residues) (Fig. 3B). Secondary structure prediction analysis of ACKTI using the SWISS-MODEL workspace [15], and with porcine pancreatic
A
B
Fig. 3. (A) NCBI-BLASTp analysis of the cloned cDNA open-reading frame-deduced sequence of the mature Agalychnis callidryas skin trypsin inhibitor (ACKTI). ACKTI e A. callidryas Kazal skin trypsin inhibitor; SSKTI - Salmo salar (Atlantic salmon) Kazal trypsin inhibitor e accession number NP_001140094; DRKTI-1 - Danio rerio (zebrafish) Kazal trypsin inhibitor-1 e accession number XP_003199918; DRKTI-2- D. rerio (Zebrafish) Kazal trypsin inhibitor 2 e accession number XP_003199919. Gaps have been included to maximise alignments. Conserved residues are indicated by asterisks and the canonical Kazal inhibitor motif is shown below. The common P1 residue (R- Arg, consistent with trypsin inhibition), is indicated by an arrow. (B) Alignment of ACKTI with Phyllomedusa sauvagei skin-derived post-proline cleaving enzyme Kazal inhibitors, PSKP-1 and PSKP-2 (accession numbers P83578 and P83579, respectively). Gaps have been included to maximise alignments. Conserved residues are indicated by asterisks. Note that the P1 residue (arrow) in both PSKP-1 and PSKP-2 is P (Pro), consistent with their post-proline enzyme inhibitory activity and lack of trypsin inhibitory activity.
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secretory Kazal-type 1 trypsin inhibitor as a reference, showed unequivocally that in terms of putative secondary structure, ACKTI was very similar to this related protein from a mammalian source (Fig. 4A). 3.4. Determination of the inhibition constant of ACKTI for trypsin ACKTI was incubated with substrate and trypsin at a range of concentrations and a Morrison plot of initial rates (vi) was used to calculate the Ki of 1.9 nM (Fig. 4B). 4. Discussion Amphibian skin secretions are an intriguing resource for the discovery of novel natural bioactive peptides with a wide spectrum of pharmacological actions and putative physiological functions [1e4]. While low molecular mass peptides of short chain length can
be readily sequenced using Edman degradation, higher molecular mass polypeptides and proteins present the investigator with some technical problems that are often solved by use of protease digestion resulting in generation of a mixture of shorter oligopeptides from the parent molecule. The procedure essentially progresses through fractionation of the digest, sequencing of individual peptides and finally piecing together the structure of the parent polypeptide/protein [16,17]. Problems however can arise if the polypeptide/protein being studied is a protease inhibitor, especially one that is active against trypsin e the protease that is most often employed for this purpose [7,9]. Trypsin inhibitors are also the commonest group of protease inhibitors that have been identified in amphibian skin secretions [7e11]. However, acquiring the molecular mass of the polypeptide/protein of interest and also a short N-terminal amino acid sequence, can provide sufficient information for degenerate primer design to initiate precursorencoding cDNA cloning studies [18,19]. Previously, the presence in
Fig. 4. Secondary structure prediction analysis of (A) porcine pancreatic secretory Kazal-type 1 trypsin inhibitor (accession number P00998) and (B) ACKTI, using the SWISS-MODEL workspace. Note the high degree of secondary structural similarity between mammal and amphibian proteins especially in the C-terminal region with sequential a-helix (H), random coil (C) and extended beta sheet (E). (C) A Morrison plot of trypsin inhibitory activity of ACKTI at different concentrations indicating a Ki value of 1.9 nM.
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lyophilised amphibian skin secretions of PCR amenable polyadenylated mRNA transcripts encoding constituent peptides/ proteins was established [20]. Thus, parallel peptide/protein sequencing, molecular mass determination and precursorencoding cDNA cloning as employed in the present study, can provide rapid and unequivocal structural determinations of novel molecules. Peptidic inhibitors of proteases are ubiquitous in Nature and are found in multiple forms in animals, plants and microorganisms [5,6]. They are grouped primarily following establishment of the presence of well-defined structural motifs that are invariably intimately related to their functions [5,6,21]. Such groups include the Kunitz-, Bowman Birk- and Kazal-type inhibitors of serine proteases [5]. These inhibitors function by binding to their cognate enzyme in a substrate-like manner, forming a stable complex [5]. Representatives of all three types have been isolated from amphibian skin although the Kazal inhibitors thus far appear to be the least common with only two, closely-related inhibitors of postproline cleaving enzyme, PSKP-1 and PSKP-2, having been isolated from the skin of the waxy monkey frog, P. sauvagei [12]. The subfamily of South and Central American leaf frogs to which this species belongs, the Phyllomedusinae, contains about 50 species in several genera [22]. The largest genus, Phyllomedusa, contains species with skin secretions that have been described as a “huge factory and store-house of a variety of (biologically) active peptides” [22]. This was a major reason to focus our efforts on the skin secretions of another member of this subfamily, the red-eyed leaf frog, A. callidryas, in the search for novel inhibitors of trypsin. The single endogenous inhibitor of trypsin identified in the skin of this species was of Kazal-type. While there have been two Kazal proteins previously identified in phyllomedusine frog skin secretion (PSKP-1 and PSKP-2), neither was found to be an inhibitor of trypsin. However, changing the Pro (P) residue in the P1 position to Arg (R), virtually abolished the original post-prolineecleavage activity and produced a potent trypsin inhibitor [12]. The crucial role played by the nature of the residue in the P1 position of the active site of Kazal inhibitors was thus elegantly demonstrated by these data. Bioinformatic analysis of ACKTI produced interesting results. Database interrogation with the N-terminal 22-residue sequence obtained by Edman degradation of the intact protein, incorporating the blank cycles, resulted in no appropriate hits. However, incorporation of the putative Cys residues at these sites, resulted in a clear indication of relatedness to reactive centre domains of Kazal trypsin inhibitors from organisms as diverse as molluscs and birds (Fig. 2A). Three of the Cys residues incorporated represent fundamental sites of the canonical Kazal motif (Fig. 3A) and the presence of such residues at these sites was unequivocally confirmed following successful cloning of ACKTI precursorencoding cDNA (Fig. 2B). Database interrogation using the full primary structure of mature ACKTI revealed the presence of a full canonical Kazal inhibitor motif and a significant degree of relatedness to bony fish analogues (salmon and zebrafish) (Fig. 3A) that was similar to that exhibited between all three amphibian skin analogues (ACKTI, PSKP-1 and PSKP-2) (Fig. 3B). Although primary structural identity per se between various Kazal inhibitors was relatively low (approx. 40%), secondary structural prediction comparison between ACKTI and porcine pancreatic Kazal trypsin inhibitor-1 (Fig. 4A) revealed a greater degree of similarity. Thus primary structure per se outside of defined functional motifs or domains, appears not to be a major determining factor in the function of this class of protease inhibitor. The Ki determined for ACKTI in this study, was in the low nanomolar range (1.9 nM),
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which is consistent with that determined for many other Kazaltype trypsin inhibitors from many different sources [5]. The data presented in this report substantiates the widely-held view that amphibian skin secretions, in this case those of a phyllomedusine frog, are “huge factories and store-houses of biologicallyactive peptides” [22], and that this assertion could be extended to include polypeptides and proteins. Further systematic searches of this resource would thus be highly-likely to result in the discovery of novel biologically-active molecules with applications as pharmacological tools or in the more general fields of biotechnology and biomedical sciences.
References [1] V. Erspamer, Bioactive secretions of the integument, in: H. Heatwole, G.T. Barthalmus (Eds.), The Integument, Amphibian Biology, vol. 1, Surrey Beatty & Sons, Chipping Norton, 1994 (Chapter 9). [2] B.L. Bevins, M. Zasloff, Peptides from frog skin, Ann. Rev. Biochem. 59 (1990) 395e414. [3] L.H. Lazarus, M. Attila, The toad, ugly and venomous, wears yet a precious jewel in his skin, Prog. Neurobiol. 41 (1993) 473e507. [4] B.T. Clarke, The natural history of amphibian skin secretions, their normal functioning and potential medical applications, Biol. Rev. Camb. Philos. Soc. 72 (1997) 365e379. [5] M. Laskowski, I. Kato, Protein inhibitors of proteinases, Ann. Rev. Biochem. 49 (1980) 593e626. [6] W. Bode, R. Huber, Natural protein proteinase inhibitor and their interaction with proteinases, Eur. J. Biochem. 204 (1992) 433e451. [7] M.F. Ali, K.R. Lips, F.C. Knoop, B. Fritzsch, C. Miller, J.M. Conlon, Antimicrobial peptides and protease inhibitors in the skin secretions of the crawfish frog, Rana areolata, Biochim. Biophys. Acta 160 (2002) 55e63. [8] T. Chen, C. Shaw, Identification and molecular cloning of novel trypsin inhibitor from the dermal venom of the Oriental fire-bellied toad (Bombina orientalis) and the European yellow-bellied toad (Bombina variegata), Peptides 24 (2003) 873e880. [9] J.M. Conlon, J.B. Kim, A protease inhibitor of the Kunitz family from skin secretions of the tomato frog, Dyscophus guineti (Microhylidae), Biochem. Biophys. Res. Commun. 279 (2000) 961e964. [10] G. Song, M. Zhou, W. Chen, T. Chen, B. Walker, C. Shaw, HV-BBIea novel amphibian skin Bowman-Birk-like trypsin inhibitor, Biochem. Biophys. Res. Commun. 372 (2008) 191e196. [11] M. Wang, L. Wang, T. Chen, B. Walker, M. Zhou, D. Sui, J.M. Colon, C. Shaw, Identification and molecular cloning of a novel amphibian Bowman-Birk-like trypsin inhibitor from the skin of the Hejiang Odorous Frog, Odorrana Hejiangensis, Peptides 33 (2012) 245e250. [12] L.G. Gebhard, F.U. Carrizo, A.L. Stern, N.I. Burgardt, J. Faivovich, E. Lavilla, M.R. Ermácora, A Kazal prolyl endopeptidase inhibitor isolated from the skin of Phyllomedusa sauvagei, Eur. J. Biochem. 271 (2004) 2117e2126. [13] H. Wang, L. Wang, M. Zhou, M. Yang, C. Ma, T. Chen, Y. Zhang, M. Zeller, M. Hornshaw, C. Shaw, Functional peptidomics of amphibian skin secretion: a novel Kunitz-type chymotrypsin inhibitor from the African hyperoliid frog, Kassina Senegalensis, Biochimie 94 (2012) 891e899. [14] M.J. Tyler, D.J.M. Stone, J.H. Bowie, A novel method for the release and collection of dermal, glandular secretions from the skin of frogs, J. Pharmacol. Toxicol. Methods 28 (1992) 199e200. [15] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling, Bioinformatics 22 (2006) 195e201. [16] R. Deutzmann, Structural characterization of proteins and peptides, Methods Mol. Med. 94 (2004) 269e297. [17] V.C. Pham, W.J. Henzel, J.R. Lill, Rapid on-membrane proteolytic cleavage for Edman sequencing and mass spectrometric identification of proteins, Electrophoresis 26 (2005) 4243e4251. [18] T. Chen, Y. Xue, M. Zhou, C. Shaw, Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8ea novel prokineticin from Bombina orientalis, Peptides 26 (2005) 377e383. [19] L. Odum, J.R. Bundgaard, A.H. Johnsen, A Kazal-type trypsin inhibitor from the protochordate Ciona intestinalis, Eur. J. Biochem. 259 (1999) 872e876. [20] T. Chen, S. Farragher, A.J. Bjourson, D.F. Orr, P. Rao, C. Shaw, Granular gland transcriptomes in stimulated amphibian skin secretions, Biochem. J. 371 (2003) 125e130. [21] R.M. Roberts, N. Mathialagan, J.Y. Duffy, G.W. Smith, Regulation and regulatory role of proteinase inhibitors, Crit. Rev. Eukaryot. Gene Exp. 5 (1995) 385e436. [22] V. Erspamer, P. Melchiorri, G. Falconieri Erspamer, P.C. Montecucchi, R. de Castiglione, Phyllomedusa skin: a huge factory and store-house of a variety of active peptides, Peptides 6 (1985) 7e12.