Amphibian Skin Secretomics: Application of Parallel Quadrupole Time

May 9, 2007 - Amphibian Skin Secretomics: Application of Parallel Quadrupole. Time-of-Flight Mass Spectrometry and Peptide Precursor cDNA. Cloning to ...
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Amphibian Skin Secretomics: Application of Parallel Quadrupole Time-of-Flight Mass Spectrometry and Peptide Precursor cDNA Cloning to Rapidly Characterize the Skin Secretory Peptidome of Phyllomedusa hypochondrialis azurea: Discovery of a Novel Peptide Family, the Hyposins Alan Hunter Thompson,† Anthony John Bjourson,† David Francis Orr,† Chris Shaw,‡ and Stephen McClean*,† Institute of Biomedical Sciences, University of Ulster, Coleraine, Co Londonderry BT52 1SA, UK, and School of Pharmacy, Queen’s University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK Received May 9, 2007

This study reports the variety of peptides present in the skin secretory peptidome of Phyllomedusa hypochondrialis azurea. Peptide structures, along with post-translational modifications, were elucidated by QTOF MS/MS analysis, cDNA sequencing, or a combination of both. Twenty-two peptides, including 19 novel structures, were identified from six different structural classes, including tryptophyllins, dermorphins, and a novel group of peptides termed hyposins. The study demonstrates the power of this combined approach to mine the rich peptidome compliment of the amphibian defensive skin secretome. Keywords: amphiban skin • secretome • host defense • hyposin • mass spectrometry • de novo sequencing • Phyllomedusa hypochondrialis azurea

Introduction The merits of frog skin secretions have long been extolled, with numerous historical references, having been made to their roles in medicine, folklore, and shamanistic rituals. Even today, the Matses Indians of northern Peru are known to regularly use “Sapo” from tree frogs to improve “luck” when hunting, with beneficial effects including increased strength, heightened senses, and an increased capacity to deal with stressful situations bestowed upon the user.1 Since the 1960s, a concerted effort has been made to isolate and identify many of these pharmacologically valuable agents from amphibian venoms. More than 500 distinct species from six different continents have been studied to date,2 successfully identifying numerous bioactive agents, including bufodienolides, alkaloids, peptides, and proteins.3-5 Recent advances in mass spectrometric instrumentation have led to improved identification of compounds in amphibian secretions, most notably from the peptidome. Aided by cDNA sequencing, peptide, and protein sequences can be quickly elucidated following minimal sample preparation steps. This parallel cDNA sequencing/mass spectrometric approach has been used extensively in recent years, not only to identify * To whom correspondence should be addressed. Dr Stephen McClean, Institute of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine BT52 1SA, Northern Ireland. Tel., + 44 2870 324406; Fax, +44 2870324375; E-mail, [email protected]; Website, http://www. venomics.co.uk. † University of Ulster. ‡ Queen’s University of Belfast.

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proteins and peptides in the defensive products of amphibians,6,7 but also spiders,8 snakes,9 and scorpions,10,11 and is highly favored, owing to its high throughput at a relatively inexpensive cost. The complementary nature of this mode of data acquisition allows simultaneous validation of data derived from both methods, while also catering for identification of post-translational modifications and differentiation of isobaric amino acids not readily achievable by low-energy collisioninduced dissociation. A further advantage bestowed by this approach is the ability to obtain extensive proteomic and genomic information from a single venom sample, without the need to sacrifice the test organism. This has been largely facilitated by the discovery, within our group, that complete mRNA sequences can be isolated directly from a venom secretion,12 leading to construction of cDNA libraries without the need to sacrifice the specimen. By aligning cDNA sequencing with the high sensitivity of QTOF mass spectrometry, extensive information regarding the excreted proteomic content can be acquired while causing minimum discomfort to the test organism. This method of data acquisition has been deemed highly favorable in the modern age, given the current plight of much of the world’s biodiversity. Indeed, this modern methodology bears a stark contrast to older methods that relied on a functional assessment of the peptides prior to their structural elucidation. Such a scheme required the sacrifice of huge numbers of animals in order to attain significant amounts of defensive product to conduct bioactivity assays,13 as well as being timeconsuming, requiring several clean up and preparation steps 10.1021/pr0702666 CCC: $37.00

 2007 American Chemical Society

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to isolate compounds of interest. With the wide-scale introduction of solid-phase peptide synthesis peptides of interest can now be produced on a larger scale, facilitating their examination in a number of assays without the sacrifice of a single animal. Such an approach has been met with quite a degree of success in recent years in identifying the structure and activity of numerous bioactive peptides from amphibian skin secretions.14-17 The present work describes the identification of 22 peptides from the skin secretions of the Tiger Leg leaf frog, Phyllomedusa hypochondrialis azurea. The peptides belong to a number of previously described families, including tryptophyllins and opioid-like peptides, though a novel family of unknown peptides termed hyposins are also described here for the first time. Sequences were either acquired directly by de novo sequencing following QTOF MS/MS or by a combination of de novo sequencing and cDNA sequencing. The current work underlines the effectiveness of these methods in identifying complete peptide structures including post-translational modifications, without the need for specimen sacrifice.

Experimental Section Specimen Biodata and Skin Secretion Acquisition. Specimens of P.hypochondrialis azurea were acquired from a commercial source in Yorkshire, England. They were maintained in purpose-designed terrariums at 20-25 °C under a 12/12 h light/dark cycle and fed multi-vitamin loaded crickets three times per week. Following a 3-month adaptation period, dermal secretions were extracted from the frogs by gently massaging the dorsal region, or exposing the area to a series of mild electrical stimulations as proposed by Tyler et al.18 The resulting foamy secretions were rinsed from the dorsal surface using deionized water into a clean glass beaker. This was then snapfrozen in liquid nitrogen, lyophilised, and stored at -20 °C prior to further analysis. Following secretion acquisition, the animals were returned to their terrariums where they quickly recovered. All experiments were carried out in accordance with the UK Animal (Scientific Procedure) Act 1986. Peptide Purification and Identification. Five milligrams of lyophilised secretion was reconstituted in 1 mL of trifluoroacetic acid (TFA)/water (0.05/99.95 v/v) and cleared of microparticulates by centrifugation. Peptide separation was performed by injecting the crude extract onto a Jupiter C18 reversed-phase chromatographic column (10 mm × 250 mm) (Phenomenex, Macclesfield, Cheshire, UK) on a gradient HPLC system (ThermoFinnigan, San Jose, CA). A gradient was formed from 0.05/99.95 (v/v) TFA/water to 0.05/19.95/80.0 (v/v/v) TFA/ water/acetonitrile over 80 min at a flow rate of 1 mL/min. Effluent from the column was flow split with 10% entering an interfaced LCQ electrospray ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA). Fractions were collected every minute using an automated fraction collector and absorbance was monitored at 214 and 280 nm. Molecular Mass and Sequence Determination. The molecular masses of the peptide content in each fraction were initially determined by MALDI-TOF mass spectrometry using a linear time-of-flight Voyager-DE mass spectrometer (Perseptive Biosystems, MA). One microliter aliquots were allowed to air-dry onto wells of the target plate prior to the addition of 1 µL of matrix (40 mM R-cyano-4-hydroxy cinnamic acid in acetonitrile/water/TFA, 50/49.5/0.5, v/v/v). The samples were analyzed in positive detection mode, and internal mass calibration with known standards established mass accuracy as ( 0.1%. Fifty

Table 1. Primers Targeting Various Regions of cDNA Transcriptsa peptides

Pha-T1

Hyposin-HA1 PRP-HA1

sense primerb

anti-sense primer

5′-CCTGAAGAAATC TCTTTTCCTTGTACTA-3′ 5′-GTTGACCTTGAAG AAATCTCTGTTA-3’ 5′-TIMGICCIGCI GTIATHMGICC-3′ 5′-TYRTACTWTT CCTYGGATTGG-3′

5′-CTGATAATTGT GCTCCTCAAGGT-3′

N/A N/A

a Sense primers for PHA-T1 were designed for a highly conserved signal region, whereas the primer for Hyposin-HA1 targeted a specific peptide encoding region. The sense primer for Novel Peptide 3 was intended to identify the dermorphin precursor. The antisense primer for PHA-T1 was designed for a region in the 3′-nontranslated domain. b I ) base pairs with A, G, C or T; R ) A+G; Y ) C+T; M ) A+C; W ) A+T; H ) A+C+T.

laser scans were averaged for each sample, and variable laser intensities were used to ensure the most representative mass spectra for the wells was produced. Following mass determination, peptides were sequenced by MS/MS fragmentation and de novo sequencing experiments on a Q-ToF Ultima API mass spectrometer (Micromass, Manchester, UK) using argon as the collision gas. The instrument was calibrated with a multi-point calibration using selected product ions that resulted from the collision-induced decomposition (CID) of Glu-fibrinogen peptide B (Sigma, UK). Peptide-containing fractions were directly introduced to picospray and nanospray sources using a syringe pump. The mass spectrometer was operated in positive ion mode with a source temperature of 80 °C and a cone gas flow of 0.1-1 µL/min. A voltage of 3.0-4.0 kV was applied to the probe tip. Mass spectra were acquired with the Q-ToF analyzer in the V-mode of operation, the spectra being integrated over 1 second intervals. Peptides of interest were selected using the quadrupole mass analyzer and fragmented in the collision chamber. Collision energy was slowly raised from 10 to 35 eV until the peptide was completely fragmented. Several spectra were integrated at various collision energies for each peptide. Data were processed using MassLynx 3.5 software, which incorporated the MaxEnt3 deconvolution algorithm with sequence analysis by MassSeq and PepSeq tools (Micromass, Manchester, UK). Construction and Sequencing of cDNA Library. Skin secretion was gently swabbed from the dorsal region of the frogs and added directly to 1 mL of lysis/mRNA stabilization buffer from a Dynal mRNA isolation kit (Dynal UK Ltd, Bromborough, UK). Magnetic oligo-dT beads were used to isolate polyadenylated mRNA as described by the manufacturer (Dynal Biotech, Bromborough, UK). The isolated mRNA was subjected to 5′- and 3′-rapid amplification of cDNA ends (RACE) procedures using a SMART-RACE kit (Clontech, Basingstoke, UK) essentially as described by the manufacturer. The 3′-RACE reactions employed a universal primer mix (UPM) primer (supplied with kit) in conjunction with degenerate sense primers designed to target specific coding regions of cDNA. Several sense primers were designed based on highly conserved signal regions of precursors encoding tryptophyllins, dermaseptins and bradykinins in other Phyllomedusid frogs,14,19-21 whereas a degenerate primer was designed to target the N-terminal region of Hyposin-HA1. A further degenerate primer was constructed based on the signal region of previously studied dermorphin precursors.19,22 The primers are listed in Table 1. The 3′-RACE reactions were cleaned using a Wizard SV Gel and PCR Cleanup System (Promega, Southampton, UK) Journal of Proteome Research • Vol. 6, No. 9, 2007 3605

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Figure 1. QTOF MS/MS MaxEnt 3 processed spectra of Tryptophyllin-like peptides. Top spectrum (a) displays Pha-T1, whereas lower spectrum (b) shows [Hyp]3-Pha-T1. The hydroxyproline amino acid is denoted by an isobaric leucine residue. The immonium ions for arginine (R), lysine (K), and tryptophan (W) are clearly marked at the lower end of the spectra.

and cloned using a pGEM-T vector system (Promega, Southampton, UK). All products were sequenced using an ABI 3100 automated DNA sequencer. Sequence data obtained from the 3′-RACE product encoding PHA-T1 was used to design a genespecific antisense primer for a region of the 3′-nontranslated region (Table 1). 5′-RACE was carried out using UPM in conjunction with this primer. Following PCR, all products were cleaned, cloned and sequenced as described above. Analysis and alignment of all cDNA data was performed using the VectorNTI 9.0.0 Suite (InforMax, London, UK).

Results Peptide Purification and Primary Structure Analysis. RPHPLC fractionation of the crude secretion resulted in a complex chromatogram, with over thirty peaks identified on both the UV absorbance trace and the accompanying total ion count 3606

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(TIC) trace from the ion-trap mass spectrometer (data not shown). Each of the fractions displaying UV absorbance peaks were sequentially analyzed using MALDI-TOF MS. Peptide masses were noted and compared to those of existing sequenced peptides from previously studied amphibian secretions including bradykinins and phylloseptins. A number of peptides were also noted to have masses co-incident with those of [Hyp]6-dermorphin and several tryptophyllin-like peptides. All fractions containing peptides were subjected to collision induced dissocation and de novo sequencing analysis using QTOF MS/MS and were postacquisition processed using the MaxEnt3 algorithm. Sequence viabilities were determined by comparing the predicted hydrophobicities of the peptides with their retention times. Thirteen short tryptophan-containing peptides resembling tryptophyllins recently categorized by Chen et al.14 were identified in several of the fractionated

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Amphibian Skin Secretomics Table 2. Tryptophyllin-Like Peptides Identified in the Skin Secretions of P. hypochondrialis azureaa peptide name

amino acid sequence

experimental mass (Da)

theoretical mass (Da)

accession number

979.60 995.64

979.56 995.59

P84945 (see above)

691.28 673.38 657.39 675.31 680.40 810.46 826.43 842.47 814.40

691.34 673.32 657.36 675.32 680.40 810.52 826.48 842.44 814.43

P84952 P84943 P84942 P84948 P84941 P84946 P84947 P84953 P84944

1613.91 1597.83

1613.80 1597.76

P84949 P84950

T-1 peptides Pha-T1 [Hyp]3 -Pha-T1

Arg-Pro-Pro-Ser-Trp-Ile-Pro-Lys* Arg-Pro-Hyp-Ser-Trp-Ile-Pro-Lys*

Pha-T2-1 Pha-T2-2 Pha-T2-3 Pha-T2-4 Pha-T2-5 Pha-T2-6 Pha-T2-7 Pha-T2-8 Pha-T2-9

Phe-Pro-Pro-Trp-Phe-NH2* Phe-Pro-Pro-Trp-Glu-NH2* Phe-Pro-Pro-Trp-Leu-NH2 Phe-Pro-Pro-Trp-Met-NH2 Leu-Pro-Pro-Trp-Ile-Gly-NH2* Lys-Pro-Hyp-Trp-Arg-Leu-NH2* Lys-Pro-Trp-Glu-Arg-Leu-NH2* Lys-Pro-Trp-Glu-Arg-Glu-NH2* Val-Pro-Pro-Ile-Gly-Trp-Phe*

Pha-T3-1 Pha-T3-2

T-3 peptides pGlu-Asp-Lys-Pro-Phe-Trp-Pro-Pro-Pro-Ile-Tyr-Ile-Met* pGlu-Asp-Lys-Pro-Phe-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met*

T-2 peptides

a Peptides were classified into three structural groups based on their structural similarities, as previously suggested by Chen et al.12 Novel peptides are indicated by asterisks.

Table 3. Peptides Identified in the Skin Secretions of P. hypochondrialis azurea, Including a Novel Group Termed Hyposins, [Hyp]6-Dermorphin, and Three Structurally Novel Peptidesa peptide name

sequence

experimental mass (Da)

theoretical mass (Da)

Hyposin-HA1 Hyposin-HA2 Hyposin-HA3 Hyposin-HA4 Hyposin-HA5 [Hyp]6-Dermorphin TRP-HA1 GRP-HA1 PRP-HA1

LRPAVI-RPKGK-NH2 LRPAFI-RPKGK-NH2 LRPAVIVRTKGK-NH2 FRPALIVRTKGTRL-NH2 LGPALITRKPLKGKP YAFGYPS-NH2 VMYYSLPRPV-NH2 pEQGEGGPYGGLSPLRFS FLFFAFPHPL-NH2

1232.89 1280.87 1335.87 1626.12 1514.01 818.38 1222.72 1731.86 1233.77

1232.82 1280.82 1335.88 1626.02 1513.92 818.39 1222.65 1731.82 1233.67

accession number

P84954 P84955 P84956 P84957 P84958 (N/A) P84951 P84959 P84960

a Conserved residues are in bold, whereas gaps (-) have been introduced to maximize alignment. Amidated C-terminals are denoted by (-NH2). Hydroxyproline and pyroglutamate are represented by P and pE, respectively.

samples. Typical sequence determination of the peptides is demonstrated in the de novo sequenced mass spectra in Figure 1. The immonium ions for arginine (R), lysine (K), and tryptophan (W) are clearly marked at the lower end of the spectra and assisted in the assignment of amino acid sequences. The peptides were organized into three discrete structural groups (T1, T2, and T3) in Table 2 based on the classification suggested by Chen et al.14 Peptides were named according to their structural class and species of origin and submitted to the UniProt Knowledgebase under the accession numbers listed in Table 2. Many of the peptides were found to share a high degree of homogeneity with existing tryptophyllins. Indeed, PHA-T2-3 and PHA-T2-4 were identical to two peptides previously identified in P. rohdei by Montecucchi.23 Comparison with existing structures assisted in the identification of post-translational modifications in the current crop. Pha-T1 and Pha-T2-6 were understood to have undergone hydroxyprolination in a similar manner to PdT-1 from P. dacnicolor,14 whereas many of the T-2 peptides were assumed to feature amidation of the C-terminus in accordance with previously described sequences from P. rohdei.23 Likewise, both Pha-T3-1 and Pha-T3-2 appeared to feature a pyroglutamate modification at the N-terminus in common with those from P. rohdei, P. bicolor, and P. sauvagei.14 A novel family of peptides termed Hyposins were also identified in the current study. Sequence determination of the five peptides was easily elucidated by Q-TOF MS/MS analysis as shown in the selected de novo sequenced mass spectra in

Figure 2, though leucine and isoleucine could not be differentiated under these low-energy collision conditions owing to their isobaric masses. Sequencing of the peptides was aided by the high conservation of their N-termini, as demonstrated in Table 3. The five peptides were 11-15 residues in length and rich in basic (Lys and Arg) amino acids. The majority of the peptides appeared to feature an amidation modification at the Cterminus. Extensive trawling of online databases failed to find any sequences with relevant homology. [Hyp]6-dermorphin (Figure 3a), previously isolated from P. rohdei and P. sauvagei24,25 and three novel peptides were also identified following mass spectrometric analysis. TRP-HA1 (tyrosine-rich peptide HA1) closely resembled the peptide Leu-Met-Tyr-Tyr-Thr-LeuPro-Arg-Pro-Val-NH2 previously identified in P. sauvagei,13 differing only by conserved substitutions at positions 1 (Val for Leu) and 5 (Ser for Thr). Comparison with this sequence appeared to confirm the presence of a C-terminal amide. GRPHA1 (Glycine-rich peptide HA1) displayed an excellent fragmentation profile exemplified by the relative abundance of peaks corresponding to b- and y-series ions though difficulty in sequencing the N-terminus appeared to indicate a pyroglutamate modification. Indeed, comparisons with Pha-T3-1 and Pha-T3-2 showed that these peptides appeared to fragment in a similar manner, with the N-terminal proving particularly resilient to collision induced decomposition. Sequencing of Novel peptide 3 revealed a structure very rich in hydrophobic phenylalanine residues a motif which was justified by its relatively late retention time during HPLC analysis. The peptide Journal of Proteome Research • Vol. 6, No. 9, 2007 3607

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Figure 2. Selected QTOF MS/MS MaxEnt 3 processed spectra of novel Hyposins. (a) and (b) Hyposins-HA1 and HA2, respectively.

was named Phenylalanine rich peptide or PRP-HA1 accordingly. Once again, structural allocation appeared to favor the presence of an amidated C-terminus. Database searches on both these peptides failed to reveal any sequences with significant homology. Cloning of Peptide Precursor Transcripts. 3′-RACE reactions utilizing primers specific to the conserved signal region consistently generated two Pha-T1-encoding precursors (see EMBL accession number AM292542). Both precursors contained a single copy of the mature peptide and differed by seven nucleotide substitutions confined to the signal region. Subsequent 5′-RACE reactions using an antisense primer specifically tailored for the 5′-untranslated region consistently yielded one full length precursor. The Pha-T1 open reading frame was extremely short, comprising 61 amino acids, and featured a tripartite structure that included a hydrophobic 22-residue signal region, a highly acidic 28-residue spacer, and a single mature peptide. A di-basic propeptide convertase cleavage site was identified immediately prior to the mature peptide. Unusually 3608

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the encoded peptide featured an N-terminal aspartic acid residue which was not present in the final product. A similar motif was identified in the precursors of PdT-1 from Pachymedusa dachnicolor14 and [Thr]6-phyllokinin from Phyllomedusa sauvagei,26 with both reading frames encoding N-terminal acidic residues which were absent following post-translational processing. None of the tryptophyllin-like peptides from the T2 and T3 classes were identified using the aforementioned primers, suggesting that their signal domains are composed differently. Efforts were also made to deduce the precursor structure of the novel Hyposins. 3′-RACE reactions using a degenerate primer which targeted the N-terminus of Hyposin-HA1 succeeded in cloning multiple copies of the precursor. The absence of multiple bands following gel electrophoresis confirmed that each precursor only contained a single copy of the mature peptide. cDNA sequencing validated the de novo derived structure as well as confirming the amidation modification of the final product, with the penultimate glycyl residue of the

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Figure 3. QTOF MS/MS MaxEnt 3 processed spectra of (a) [Hyp]6-Dermorphin and (b) TRP-HA1. The hydroxyproline in the dermorphin peptide is represented by an isobaric leucine residue.

precursor product (LRPAVIRPKGKGK) appropriately positioned to act as an amide donor. A concerted effort was also made to identify the [Hyp]6-Dermorphin precursor by constructing a degenerate primer homologous to the signal region of dermorphin precursors identified in other Phyllomedusid frogs. Although this ultimately proved to be unsuccessful (probably owing to the low expression of the peptide), it did succeed in identifying the precursor for a novel phenylalanine rich peptide, PRP-HA1. Once again, the cDNA sequence confirmed the mass spectrometric derived amino acid sequence of the mature peptide, including the C-terminal amidation motif. This posttranslational modification appears to occur in a similar manner to that of the aforementioned Hyposin-HA1, with the removal of the precursor’s C-terminal -Gly-Arg- residues from the final product. Again, the precursor is organized into a tripartite structure consisting of a highly homologous signal domain, an

acidic spacer and a single copy of the peptide. The mature peptide is immediately preceded by a common di-basic (-KR-) cleavage site. Unsurprisingly, the combined signal and acidic domains displayed very high levels of conservation with dermorphin precursors from P. sauvagei (85% identity)27 and deltorphins from P. bicolor (86% identity).28

Discussion The current study successfully identified 22 peptides from the skin secretome of P. hypochondrialis azurea, including tryptophyllin-like peptides, opioid-like peptides, and a novel family of peptides termed hyposins. Each of these groups is discussed separately below. Tryptophyllins. The tryptophyllins constituted the largest group reported here, featuring 13 peptides including 11 previJournal of Proteome Research • Vol. 6, No. 9, 2007 3609

research articles ously unreported structural isoforms. Members of this peptide family have previously been identified in the skin secretions of various other hylid frogs, including P. rohdei, P. dacnicolor, and L. rubella4,14,29 though their classification as a family has proved somewhat misleading, owing to the high degree of heterogeneity between resident peptides. Indeed, in a recent report, Chen et al.14 re-classified the peptides into three distinct groups based on their structural characteristics. These included T-1 peptides which are 7-8 residues in length and feature conserved lysine, proline, and tryptophan residues; T-2 peptides which are 4-7 residues in length feature a conserved ProTrp motif; and T-3 peptides which are composed of 13 amino acids and characterized by an N-terminal pyroglutamic acid. All of the peptides identified in the current study were allocated to one of these structural groups, as displayed in Table 2 (PhaT2-9 did not feature the Pro-Trp motif required of T-2 peptides, but was included due to its overall homology with other peptides in this group). Until recently, these peptides were thought to be limited to hylid frogs. However, the recent discovery of two tryptophyllin-like peptides in the most primitive extant frog, Ascaphus truei appears to suggest that these peptides have not emerged recently, but rather arose much earlier in the development of anurans.30 The tryptophyllins described in the current study were largely identified by de novo sequencing following Q-TOF MS/MS. Structural determination was greatly eased by the small size of the peptides, which generated uncomplicated fragmentation profiles, whereas the presence of immonium ions in the lower m/z end of the spectra highlighted the existence of certain amino acids in the sequence (Figure 1). Comparison with previously deduced tryptophyllins contributed to the assignment of post-translational modifications, with 11 out of the 13 peptides featuring a C-terminal amidation, N-terminal pyroglutamate, or hydroxyproline modification. Post-translational modifications are common in peptides derived from defensive secretions and are understood to increase the stability of peptides against enzymatic degradation.31,32 Furthermore, the modification of peptides expands their structural repertoire, by introducing conformational changes that can optimize the binding specificity of peptides to particular receptors.31 The T-1 peptide, Pha-T1-1, existed in both an unmodified and hydroxyprolinated form, suggesting that modification of the peptide was mediated in a concentration-dependent manner. The modification affected the second proline (or third residue) of the sequence, in a similar fashion to that of previously studied T-1 peptides (KPHypAWVP-NH2, KPHypSWIP-NH2).14,24 Indeed, this same modification pattern was noted in bradykinins from P. hypochondrialis33 and P. sauvagei26 (RPHypGFTPFR, RPHypGFSPFR). Although the circumstances surrounding hydroxyproline modification remain poorly understood, the enzymes involved in modifying amphibian secretory peptides appear to be specific to regions comprising a basic residue followed by a proline doublet. Indeed, several of the T-2 peptides reported here (Pha-T2-1-Pha-T2-5), despite containing a di-proline motif, did not feature hydroxyprolination, emphasizing the importance of the basic residue for enzymatic recognition. Molecular cloning successfully sequenced two cDNA sequences encoding Pha-T1, confirming the isoleucine and hydroxyprolination feature in the mature peptide. Although both sequences encoded identical mature peptides, the precursor transcripts differed by seven nucleotide bases, indicating that these peptides were encoded by at least two genes. Alignment of the cDNA and translated precursor sequences 3610

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with those encoding PdT-1 from P. dacnicolor14 revealed remarkably high levels of conservation (almost 95% homology), emphasising the close relationship between these two species (Figures 4 and 5). High levels of conservation were also observed within the signal peptide and acidic spacer regions of precursors encoding structurally diverse mature peptide products, including caerins in Litoria caerulea (84%) and dermaseptins in Agalychnis annae (75%) (data not shown). This high level of conservation among geographically distant Hylids highlights the importance of this region for successful peptide maturation, regardless of the peptide’s final function. An unusual feature noted in the Pha-T1 precursor was the presence of an aspartic acid residue, which immediately followed the common N-terminal di-basic cleavage site, but was not present in the mature peptide. A similar motif was observed in PdT-1, whereas acidic residues also preceded mature peptides in bradykinin and phyllokinin precursors.21,27 All these peptides were susceptible to hydroxyprolination, suggesting that this motif may further enhance the modification of this region. Unusually, Pha-T1 was the only tryptophyllin-like peptide identified by cDNA sequencing, despite several rounds of cloning using a variety of primers that targeted different regions of the signal peptide. This would appear to suggest that the T-2 and T-3 peptides have a heterogeneous signal peptide, though it may simply be attributed to poor annealing at the 3′-end of the primer, owing to the change of a few nucleotides. Another reason for their absence may be the relatively low expression of the peptides. Highly expressed peptides featuring homologous signal peptides would be preferentially primed, leading to the masking of any lower expressed products. Attempts to design primers based on the mature peptide sequences proved unfruitful, owing to their short size and high degeneracy. With at least 13 of these peptides identified to date in this species, it is obvious that their production serves a role of some importance. The activities of many of the peptides have yet to be properly established. Previous experiments have demonstrated that several of the T-2 peptides reported here (PhaT2-3 and Pha-T2-4) effect gastric emptying and liver protein synthesis,4,23 whereas PdT-1 has been demonstrated to be a potent relaxant of arterial smooth muscle.14 Due to its homology with PdT-1, it seems highly likely that Pha-T1 would share a similar effect. Interestingly, many of the peptides described here were found to exist as homologous isoforms. A similar pattern was observed in the production of amphibian bradykinins,34 whereby multiple isoforms were specifically tailored to target endogenous receptors in a variety of predators, including reptiles, birds and mammals.21,35-37 It may well be that the production of tryptophyllins are mediated in a similar fashion, making them attractive candidates for further research. Hyposins. The five novel hyposins described in the current study were all primarily identified by Q-TOF MS/MS followed by de novo sequencing. The peptides are listed and aligned in Table 3. Like the tryptophyllins, the peptides were easily sequenced owing to their short size and excellent fragmentation profiles, to which b- and y-ion assignments could be easily attributed (Figure 2). Four residues were conserved in all five peptides, though hyposin-HA1 and hyposin-HA2 appeared to feature a deletion of residue seven. Residue substitutions among the peptides were quite common, though they did not appear to alter the chemistry of the peptides. All five peptides

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Figure 4. Alignment of nucleotide sequences of Tryptophyllin-like peptides. Sequences are aligned alongside PdT-1 from Pachymedusa dacnicolor. Bases conserved in all three prepro-sequences are highlighted in black whereas those in gray highlight consensus bases.

Figure 5. Alignment of amino acid sequences of Tryptophyllin-like peptides. Sequences are aligned alongside PdT-1 from Pachymedusa dacnicolor. Residues conserved in all three prepro-sequences are highlighted in black whereas those in gray highlight consensus amino acids.

contained a high proportion of basic residues near the Cterminus. Extensive trawling of on-line databases failed to produce any peptides with significant homology. The biological function of these peptides is yet to be established. Attempts to clone the hyposin precursors using primers which targeted the conserved precursor domains of bradykinins and dermaseptins failed, suggesting that the peptides featured a different signal peptide. Instead, a primer was designed based on the mature hyposin-HA1 peptide. This strategy proved more successful and generated a 3′-RACE product containing a single copy of the mature peptide, along with a short 3′-untranslated

region and poly-A tail (data not shown). The cDNA-derived sequence confirmed the positions of the isobaric leucine and isoleucine residues, as well as verified the presence of an amidation modification at the C-terminus, with the penultimate glycyl residue appropriately positioned to act as an amide donor. Comparison of the cloned hyposin-HA1 sequence with the other four hyposins elucidated their isobaric residue positions and post-translational modifications. Four of the peptides were assumed to feature C-terminal amidations, while the reshuffling of C-terminal amino acids in hyposin-HA5 appeared to affect its modification. Like the tryptophyllins, the Journal of Proteome Research • Vol. 6, No. 9, 2007 3611

research articles slight heterogeneity displayed within these peptides appears to imply that they too are constructed to target the specificity of a variety of receptors. Further work, including 5′-RACE sequencing and bioactivity assays would yield further information on these peptides, giving a greater understanding of both their processing and functional applications. Dermorphin. One of the peptides identified during the current study was [Hyp]6-dermorphin. This peptide is widely expressed in numerous frogs from the Phyllomedusa genus, including P. sauvagei, P. rohdei, and P. burmeisteri.24,25,38 Again, this peptide was easily identified by QTOF MS/MS de novo sequencing experiments, as shown in Figure 3a. Post-translational modifications, including hydroxyprolination of the proline residue and C-terminal amidation were assumed based on comparison with known sequences, whereas the second residue was also assumed to have been modified to a D-Ala, in keeping with previously identified amphibian opiate peptides. The peptide appears to form part of the frog’s defensive strategy against natural predators, with effects including catalepsy, rigidity, and sedation observed in rats. Extensive in vitro and in vivo tests have demonstrated that this peptide is a highly potent and selective µ-opiate receptor agonist, inducing an analgesic effect up to 100 times more powerful than morphine in humans.39,40 The modifications on this peptide are understood to play an important role in its enhanced potency and increased stability.41 Indeed, analogs of the peptide that lacked the C-terminal amide were shown to have reduced affinity for the µ-receptor (30-100 times lower), whereas synthetic peptides that lacked the D-amino acid were completely devoid of any opiate activity.38,40 In an attempt to clone the cDNA sequence encoding this peptide, a degenerate primer was constructed based on dermorphin precursors from P. sauvagei,27 A. annae, and P. dacnicolor.42 Cloning efforts ultimately proved unsuccessful, owing to the low expression of the dermorphin peptide and the preferential binding of the primer to cDNAs encoding higher expressed peptides with similar coding regions. Miscellaneous Peptides. Although efforts to characterize the dermorphin precursor proved unsuccessful, they did succeed in cloning a cDNA encoding a novel phenylalanine-rich peptide, PRP-HA1. This peptide had been partially identified by QTOF MS/MS sequencing, though the derived precursor sequence confirmed the presence of a C-terminal amide and differentiated the leucine residues from isoleucine residues. High levels of homology were observed in the signal peptidecoding region, the acidic spacer coding region and the 3′untranslated region with cDNAs encoding dermorphins, dermaseptins, and phylloseptins, though no significant homology was observed in the region encoding the mature peptide itself. The absence of a structural homolog made it very difficult to speculate the nature and function of this peptide. Two other novel peptides, a tyrosine-rich peptide and a glycine-rich peptide were also identified in the current study and named TRP-HA1 and GRP-HA1 respectively. Both peptides were identified solely by QTOF MS/MS de novo sequencing (data not shown). The well-populated fragmentation profiles generated from the peptides greatly eased the allocation of both b- and y-ions for structural determination. TRP-HA1 was found to be homologous to a peptide previously isolated from P. sauvagei,4 though the function of this peptide is unknown. Database searches failed to reveal any peptides with significant homology to GRP-HA1. Further work needs to be done on these peptides to ascertain their functional application, though the 3612

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Thompson et al.

fact that they are expressed quite highly in the secretion would imply that they play a role of some importance. In conclusion, the present study successfully identified 22 peptides, including 20 novel structures from at least 6 different structural classes. Peptides were largely identified by QTOF MS/ MS combined with cDNA sequencing, giving a detailed description of both their amino acid sequences and posttranslational modifications. Furthermore, corroboration with known sequences on on-line databases aided the allocation of modifications to peptides which had not been identified via cDNA sequencing. All the peptides identified in the current study were obtained from a single secretion, causing the minimum amount of discomfort to the test organism. This application could be further extended to collecting secretions from animals in the field,21 reducing the need to house and feed animals of interest and giving a more definitive account of the peptides which are produced in the animal’s natural environment. The combined cDNA cloning and proteomic approach described here and in our related papers34,43 has yielded almost 50 novel peptides and structural variants from at least seven different structural classes from the defensive skin secretome of this little-studied South American leaf frog. Recent studies by our group on the skin secretions of this frog have yielded data on bradykinin-like peptides34 and antimicrobial peptides.43 Whereas the latter were investigated for their activity, pharmacological properties of the other newly discovered molecules will be the focus of future study. Their short sequences make them ideal candidates for solid-phase peptide synthesis to conduct rigorous bioactivity assays. It has been speculated that many naturally occurring substances mimic endogenous substances found in mammals and are not only selective of specific receptors, but in many cases display greater specificity to endogenous mammalian receptors,44 making them interesting candidates for novel drug design and increasing our understanding of the tertiary structure of receptors.

Acknowledgment. We thank the Department of Education and Learning (DEL) for Northern Ireland for funding the PhD studentship of Alan Hunter Thompson. We also gratefully acknowledge the advice of Prof. Stephanie McKeown in completion of aspects of this project. References (1) Erspamer, V.; Erspamer, G. F.; Severini, C.; Potenza, R. L.; Barra, D.; Mignogna, G.; Bianchi, A. Toxicon 1993, 31, 1099. (2) Simmaco, M.; Mignogna, G.; Barra, D. Biopolymers 1998, 47, 435. (3) Clarke, B. T. Biol. Rev. 1997, 72, 365. (4) Erspamer, V.; Melchiorri, P.; Falconieri Erspamer, G.; Montecucchi, P. C.; de Castiglione, R. Peptides 1985, 6, 7. (5) Macfoy, C.; Danosus, D.; Sandit, R.; Jones, T. H.; Garraffo, H. M.; Spande, T. F.; Daly, J. W. Z. Naturforschung C. 2005, 60, 932. (6) Chen, T.; Orr, D. F.; Bjourson, A. J.; McClean, S.; O’Rourke, M.; Hirst, D. G.; Rao, P.; Shaw, C. FEBS J. 2002, 269, 4693. (7) Chen, T.; Li, L.; Zhou, M.; Rao, P.; Walker, B.; Shaw, C. Peptides 2006, 27, 42. (8) Escoubas, P.; Sollod, B.; King, G. F. Toxicon 2006, 47, 650. (9) Soares, M. R.; Oliveira-Carvalho, A. L.; Wermelinger, L. S.; Zingali, R. B.; Ho, P. L.; Junqueira-de-Azevedo Ide, L.; Diniz, M. R. Toxicon 2005, 46, 31. (10) Borges, A.; Alfonzo, M. J.; Garcia, C. C.; Winand, N. J.; Leipold, E.; Heinemann, S. H. Toxicon 2004, 43, 671. (11) Dai, L.; Corzo, G.; Naoki, H.; Andriantsiferana, M.; Nakajima, T. Biochem. Bioph. Res. Co. 2002, 293, 1514. (12) Chen, T.; Farragher, S.; Bjourson, A. J.; Orr, D. F.; Rao, P.; Shaw, C. Biochem. J. 2003, 371, 125. (13) Erspamer, V.; Falconieri Erspamer, G.; Cei, J. M. Comp. Biochem. Phys. C. 1986, 85, 125.

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