A novel ranacyclin-like peptide with anti-platelet activity identified from skin secretions of the frog Amolops loloensis

Gene 576 (2016) 171–175

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Research paper

A novel ranacyclin-like peptide with anti-platelet activity identified from skin secretions of the frog Amolops loloensis Xue Hao a,b,1, Xiaopeng Tang a,b,1, Lei Luo a,b, Yuming Wang c, Ren Lai a,d, Qiumin Lu a,d,⁎ a

Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming 650223, Yunnan, China Graduate University of Chinese Academy of Sciences, Beijing 100009, China Clinical Laboratory, The Second Affiliated Hospital of Kunming Medical University, China d Joint Laboratory of Natural Peptide of University of Science and Technology of China & Kunming Institute of Zoology, Chinese Academy of Sciences, China b c

a r t i c l e

i n f o

Article history: Received 12 August 2015 Received in revised form 24 September 2015 Accepted 1 October 2015 Available online 9 October 2015 Keywords: Amolops loloensis Amphibian Skin secretion Anti-platelet

a b s t r a c t Albeit many bioactive peptides have been reported from amphibian skins, no anti-platelet peptide has been identified till to date. Here, an anti-platelet peptide, namely Zongdian platelet inhibitor (ZDPI), with the molecular weight of 1798.6 Da, was purified and characterized from skin secretions of the frog, Amolops loloensis. The amino acid sequence of ZDPI was determined as FRGCWLKNYSPRGCL-NH2 by combination methods of Edman degradation, mass spectrometry analysis and carboxypeptidase Y treatment revealing that it is composed of 15 amino acid residues with two cysteines formed an intra-molecular disulfide bridge and C-terminal amidation. cDNA encoding ZDPI precursor was cloned from skin cDNA library of A. loloensis. The precursor is composed of 63 amino acid (aa) residues including the predicted signal peptide (22 aa), an acidic spacer peptide (19 aa), and mature ZDPI. BLAST search indicates that ZDPI belongs to antimicrobial peptide family of ranacyclin, peptide leucine arginine or odorranain. It was found to inhibit ADP-induced platelet aggregation in a dose-dependent manner. At the concentration of 32 μg/ml, ZDPI completely inhibited platelet aggregation induced by ADP. To the best of our knowledge, this is the first report about an anti-platelet peptide from amphibian skin secretions. Considering its strong inhibitory ability on platelets and simple structure, ZDPI might be an excellent candidate or template to develop anti-thrombosis agent. In addition, the discovery of anti-platelet peptide in the frog skin increases biological function spectrum of amphibian skin peptides. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Amphibians are well-known vertebrates which have adapted to live in both water and on land. Notably, amphibian skins are naked as well as directly exposed to environments. Their skins appear to be fragile and easy to be injured by noxious factors, such as predators, microorganisms, parasites, and some physical injuries. Most bioactive compounds in amphibian skins mainly exert defensive functions and many of them are bioactive peptides (Clarke, 1997; Xu and Lai 2015). More than ten types of bioactive peptides containing N2000 members have been identified, such as antimicrobial peptides, bradykinins, tachykinins, serine protease inhibitors, neurotoxins, anti-oxidant peptides, bombesins, and woundhealing peptides. Hundreds of antimicrobial peptides were identified Abbreviations: aa, amino acid; TTX-S, tetrodotoxin-sensitive; VGSC, voltage-gated sodium channel; RP-HPLC, reverse phase high performance liquid chromatography; MALDITOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; PPP, platelet-poor plasma; PRP, platelet-rich plasma. ⁎ Corresponding author at: Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming 650223, Yunnan, China. E-mail address: [email protected] (Q. Lu). 1 These authors contributed equally to this paper.

http://dx.doi.org/10.1016/j.gene.2015.10.003 0378-1119/© 2015 Elsevier B.V. All rights reserved.

from amphibian skins (Duda et al., 2002; Grevelink et al., 1993; Lai et al., 2002; Li et al., 2007a, 2007b; Zasloff, 1987); more than 100 bradykinins and tachykinins, which are capable to induce algesia and smooth muscle contraction, were reported from amphibian skins (Conlon, 1999; Li et al., 2006; Severini et al., 2002; Liu et al., 2007; Yasuhara et al., 1979); many serine protease inhibitor peptides with anti-parasite activity were found in amphibian skins (Yan et al., 2012). Recently, more than 20 anti-oxidant peptides have been identified from skin secretions of the frog Rana pleuraden (Yang et al., 2009), which exhibit strong as well as rapid activity to scavenge reactive oxygen species (ROS), revealing a novel skin anti-oxidant system. A neurotoxin (anntoxin) capable of inhibiting tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel (VGSC) has been purified and characterized from the skin secretions of the tree frog, Hyla annectans (You et al., 2009). Anntoxin contains analgesic property, which eases sufferings of frogs, when they are subjected to injuries. In addition, several woundhealing peptides have been reported from frog and salamander skins (Liu et al., 2014; Mu et al., 2014; Tang et al., 2014). Therefore, amphibian skins could be a pool of bio-chemical compounds. Up to now, many peptides in amphibian skins are thought to remain unidentified as well as uncharacterized. In this study, a novel anti-platelet peptide (ZDPI) was purified and characterized from skin secretions of rufous-spotted torrent

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frog, A. loloensis. Importantly, the current work introduces a new group of bioactive peptides in amphibian skin. 2. Materials and methods 2.1. Collection of frog skin secretions Adult A. loloensis frogs (both sexes, n = 30; weight range 30–40 g) were collected from outdoors in Yunnan Province of China. Skin secretions were collected according to the methods described in our previous report (Hao et al., 2012). Skin secretions were mainly stimulated by anhydrous ether to exude copious secretions in the dorsal region of each frog. The skin secretions were washed by 0.1 M phosphate buffered saline (PBS), pH 6.0, containing 1% (v/v) protease inhibitor cocktail (Sigma) and collected. Pooled solution was immediately centrifuged to remove debris. The supernatants were lyophilized and kept at −20 °C till use. All the animal experiments were approved by Kunming Institute of Zoology, Chinese Academy of Sciences. 2.2. Peptide purification Lyophilized frog skin secretion sample (3 g, total absorbance at 280 nm is 460) was dissolved in 10 ml PBS. The sample was then applied to a Sephadex G-50 gel filtration column (Superfine, GE healthcare, 2.6 cm diameter, 100 cm length), which was equilibrated with 0.1 M PBS. Sample fraction was performed by eluting the column using 0.1 M PBS buffer. Each eluted fraction with 3.0 ml was collected and the absorbance of the eluate was measured at 280 nm. The antiplatelet activity of fractions was determined as described below. The protein peak containing anti-platelet activity was pooled, lyophilized, and resuspended in 2 ml of 0.1 M PBS, and purified further by C18 reverse phase high performance liquid chromatography (RP-HPLC, Waters C18, 30 cm × 0.46 cm) column. 2.3. Structural analysis Pulsed liquid-phase sequencer (Applied Biosystems, model 491) was used to analyze amino sequence of ZDPI by Edman degradation. Purity and molecular weight of ZDPI was determined by mass spectrometry analysis, which was conducted on a MALDI-TOF-MS mass spectrometer (matrix-assisted laser desorption ionization timeof-flight, AXIMA CFR, Kratos Analytical) by using a positive ion and linear mode, with specific operating parameters including a 20 kV ion acceleration voltage, 50-time accumulation for single scanning, and ±0.1% accuracy of mass determinations. Polypeptide mass standard (Kratos Analytical) was used as external standard. 2.4. Construction and screening of the skin cDNA library As described in our previous report (Hao et al., 2012), total RNA was extracted from the frog skin by using RNeasy Mini Kit (QIAGEN, Germany) following instructions of the manufacturer. The cDNA was synthesized by using a SMART™ PCR cDNA Construction kit (Clontech, Palo Alto, CA, USA) containing cDNA 3′SMART CDS primer IIIA, SMART IV oligonucleotide, and advantage polymerase. The synthesized cDNA was used as template for PCR to screen the cDNA encoding the precursor of ZDPI. Two pairs of oligonucleotide primers, (S1: 5′-TT(T/C)(A/ C)G(A/G/C/T)GG(A/G/C/T)TG(T/C)TGG(T/C)T(A/G/C/T)AA(A/G)AA-3′ and primer II A: 5′-AAGCAGTGGTATCAACGCAGAGT-3′; S2: 5′-(A/G/C/ T)AG(A/G)CA(A/T/C/G)CC(A/T/C/G)C(G/T)GG (A/G/C/T)GA(G/A)TA(G/ A)TT-3′ and primer II A) were used in PCR reactions. Primers S1 and S2 are designed from the amino acid sequence of ZDPI determined by Edman degradation. The DNA polymerase used was advantage polymerase from Clontech. The PCR conditions were 2 min at 94 °C, followed by 30 cycles of 10 s at 92 °C, 30 s at 50 °C, and 40 s at 72 °C. Finally, the PCR products were cloned into pGEM®-T Easy vector (Promega,

Fig. 1. Purification of ZDPI from skin secretions of A. loloensis. A: Gel filtration chromatography. Sephadex G-50 column (Superfine, Amersham Biosciences, 2.6 cm × 100 cm), equilibrated and developed with 0.1 M PBS. B: Fraction VI eluted from Sephadex G-50 column was loaded onto C18 reverse phase high performance liquid chromatography (RP-HPLC, Hypersil BDS C18, 30 cm × 0.46 cm) column, equilibrated with 0.1% (v/v) TFA/water, elution was performed with an acetonitrile liner gradient. The peptide peak containing activity to inhibit platelet was marked by an arrow. C: The eluted peak containing activity to inhibit platelet from ‘A’ was finally purified by RP-HPLC (Hypersil BDS C18) with the same elution condition as ‘A’. The purified peptide with antiplatelet activity is indicated by an arrow. D: MALDI–TOF–MS analysis of native ZDPI.

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Madison, WI). DNA sequencing was performed on an Applied Biosystems DNA sequencer. 2.5. Platelet aggregation assays Platelet aggregation inhibiting activities were determined according to the methods described previously (Grevelink et al., 1993). Human plasma and platelets were obtained from Kunming Blood Center of Yunnan province. Platelet-poor plasma (PPP) was prepared by centrifuging the plasma at 4000 rpm for 10 min at 22 °C and used within 4 h. Platelet-rich plasma (PRP) was prepared by adding platelet to the PPP and placed at room temperature. Platelet aggregation was monitored by light transmission in an aggregometer (Plisen, Beijing, China) with continuous stirring at 37 °C. All experiments were performed in triplicate. 2.6. Peptide synthesis ZDPI (FRGCWLKNYSPRGCL-NH2) was synthesized in GL Biochem (Shanghai) Ltd. (Shanghai, China). Its purity was greater than 98% determined by RP-HPLC and MALDI-TOF mass spectrometry. The synthetic peptide contained an intra-molecular disulfide bond. 3. Results 3.1. Purification of anti-platelet peptide Similar to our previous report (Hao et al., 2012), according to absorbance at 280 nm, the supernatant of A. loloensis skin secretions was fractionated into six fractions by Sephadex G-50 (Fig. 1A), and in platelet aggregation assay, fraction-VI (marked by an arrow) was found to show anti-platelet activity. For further separation, fraction-VI was then applied to a C18 RP-HPLC column. More than 15 peaks were eluted

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from this separation as indicated in Fig. 1B. The eluted peak (marked by an arrow) was found to exert antiplatelet activity, which was collected and purified further on the same C18 RP-HPLC column to obtain pure peptide (Fig. 1C). The purified peptide (marked by an arrow) was named Zongdian platelet inhibitor (ZDPI). 3.2. Amino acid sequence of anti-platelet peptide The complete amino acid sequence of ZDPI is FRGCWLKNYSPRGCL determined by automated Edman degradation. The theoretical molecular weight (MW) of linear ZDPI is 1800.13 Da, which did not match well with the observed [M + H]+ of 1798.587 Da from MALDI–TOF–MS (Fig. 1D). Considering that ZDPI contains two cysteine residues, which may form an intra-molecular disulfide, its MW should be 1798.13 Da. In addition, carboxypeptidase Y treatment did not lead to the release of free amino acids, indicating that the C-terminal end of the peptide was amidated. The observed molecular masses (1797.587 Da) of ZDPI with an amidated C-terminus and an intra-molecular disulfide from MALDI–TOF mass spectrometry are matched well with the calculated molecular mass (1797.13 Da). 3.3. cDNA cloning Upon screening of the skin cDNA library, several clones containing inserts of around 280 base pairs were identified and isolated. Both strands of these clones were sequenced. The cDNA encoding ZDPI was found to contain an open reading frame of 281 nucleotides. It encodes a pro-peptide of 63 amino acids (Fig. 2A). It comprises a predicted signal peptide, an acidic spacer peptide, and a mature ZDPI composed of 15 amino acid residues. The deduced amino acid sequence from the cDNA is identical to ZDPI sequence determined by automated Edman degradation. By BLAST search, ZDPI was found to show similarity to antimicrobial peptide family of ranacyclin (He et al., 2013), peptide leucine

Fig. 2. The nucleotide sequence encoding ZDPI precursor and the deduced amino acid sequence of the precursor polypeptide, and their sequence comparison with other amphibian skin peptides. A: The nucleotide sequence encoding ZDPI precursor. The bar (-) indicates stop condon. The mature sequence is boxed. B: The sequence comparison of ZDPI with other amphibian skin peptides. The identical amino acid residues indicated by a star (*). Ranacyclin-AJ (He et al., 2013), peptide leucine arginine (Salmon et al., 2001) or odorranain-B1 (Li et al., 2007a, 2007b) were used for sequence comparison.

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Fig. 3. Inhibition of ZDPI on platelet aggregation induced by ADP. 0.3 ml PRP with 10 μl different samples were incubated at 37 °C with continuous stirring for 5 min before tests. Detection began immediately at the time ADP was added into the system and stopped at 300 s. Salt water (0.9%) was used as the control.

arginine (Salmon et al., 2001) or odorranain (Li et al., 2007a, 2007b) (Fig. 2B). 3.4. Inhibition on platelet aggregation Aggregation of platelet is the pathophysiologic basis of thrombosis and ADP is the key factor that causes platelet aggregation. The effect of ZDPI was tested on platelet aggregation induced by ADP. As illustrated in Fig. 3, ZDPI inhibited ADP-induced platelet aggregation in a dosedependent manner. At the concentration of 8, 16, and 32 μg/ml, ZDPI inhibited platelet aggregation for approximately 36, 57, and 89%, respectively, during 300 s. The IC50 was calculated to be 12.27 μg/ml. 4. Discussion In order to protect themselves from the harmful environment, amphibian skins are equipped with an excellent chemical defense system containing many pharmacological compounds, especially geneencoding bioactive peptides with diversity. Based on reports during past two decades, thousands of these peptides including antimicrobial peptides, bradykinins, antioxidant peptides, wound-healing peptides, immunomodulatory peptides and so on (Xu and Lai, 2015). However, novel peptides from amphibian skins with anti-platelet aggregation activity remain unknown. In this report, a novel peptide named ZDPI containing merely 15 amino acids was purified and characterized from skin secretions of frog, A. loloensis. Functionally, it was found to inhibit platelet aggregation induced by ADP (Fig. 3). Structurally, ZDPI belongs to the peptide family of ranacyclin (He et al., 2013), peptide leucine arginine (Salmon et al., 2001) or odorranain (Li et al., 2007a, 2007b). This peptide family contains a trypsin-inhibitory loop composed of 11 amino acids including an intra-molecular disulfide bridge between two cysteine residues. Trypsin-inhibitory loop endows those peptides with resistance against trypsin (Li et al., 2007a, 2007b). Besides, previous work revealed that ranacyclins contained strong antimicrobial activity. However, kinetic assay indicate that ZDPI may not effectively resist trypsin since its Ki on trypsin was 2.6 mg/ml. In addition, ZDPI was not considered to contain antimicrobial activity, as it couldn't inhibit growth of four kinds of bacteria including Escherichia coli, Bacillus subtillis, Candida albicans and Staphylococcus aureus even at a high concentration of 1 mg/ml (data not shown). Some may raise doubts here, why they share the same structural characterization but meanwhile exerting different function? Compared with three other peptides (Fig. 2B), two

amino acids (aa) at the N-terminal of ZDPI (FRGCWLKNYSPRGCLNH2) were lacked and several other sites were substituted in the rest part of the sequence. Antimicrobial peptides often take an amphiphilic helix structure. The substituted leucine and glycine (both are nonpolar amino acids) may insert into the hydrophilic side, which interferes the formation of the amphiphilic structure, results in no detectable antimicrobial activity. Furthermore, loss of trypsin-inhibitory activity may be due to the low similarity with odorranain-B1, though the trypsin-inhibitory loop seems to still exist in ZDPI. Anti-platelet activity of ZDPI reported here increases functional scopes of amphibian skin peptides. As a short peptide composed of only 15 amino acid residues, ZDPI has several advantages over larger biologics, including being less expensive to produce, ship and store. Thus, the efficacy of ZDPI combined with ease of production makes it an excellent lead or template for development of novel anti-thrombosis agents. Conflicts of interest The authors claim no conflicts of interest. Acknowledgement This work was supported by MOST (2013CB911300), NSFC (U1132601, 31200590), CAS (SAJC201308) and Yunnan Province (2011CI139, 2012BC009). References Clarke, B., 1997. The natural history of amphibian skin secretions, their normal functioning and potential medical applications. Biol. Rev. Camb. Philos. Soc. 72, 365–379. Conlon, J., 1999. Bradykinin and its receptors in non-mammalian vertebrates. Regul. Pept. 79, 71–81. Duda Jr., T.F., Vanhoye, D., Nicolas, P., 2002. Roles of diversifying selection and coordinated evolution in the evolution of amphibian antimicrobial peptides. Mol. Biol. Evol. 19, 858–864. Grevelink, S., Youssef, D., Loscalzo, J., Lerner, E., 1993. Salivary gland extracts from the deerfly contain a potent inhibitor of platelet aggregation. Proc. Natl. Acad. Sci. 90, 9155–9158. Hao, X., Yang, H., Wei, L., Yang, S., Zhu, W., Ma, D., Yang, H., Lai, R., 2012. Amphibian cathelicidin fills the evolutionary gap of cathelicidin in vertebrate. Amino Acids 43, 677–685. He, X., Yang, S., Wei, L., Liu, R., Lai, R., Rong, M., 2013. Antimicrobial peptide diversity in the skin of the torrent frog, Amolops jingdongensis. Amino Acids 44, 481–487. Lai, R., Zheng, Y., Shen, J., Liu, G., Liu, H., Lee, W., Tang, S., Zhang, Y., 2002. Antimicrobial peptides from skin secretions of Chinese red belly toad Bombina maxima. Peptides 23, 427–435. Li, J., Liu, T., Xu, X., Wang, X., Wu, M., Yang, H., Lai, R., 2006. Amphibian tachykinin precursor. Biochem. Biophys. Res. Commun. 350, 983–986.

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