Peptides 53 (2014) 30–41

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Genomic and functional characterization of three new venom peptides from the scorpion Heterometrus spinifer夽 Shifen Wu 1 , Yao Nie 1 , Xian-Chun Zeng ∗ , Hanjun Cao, Lei Zhang, Lingli Zhou, Ye Yang, Xuesong Luo, Yichen Liu State Key Laboratory of Biogeology and Environmental Geology & Department of Biological Science and Technology, School of Environmental Studies, China University of Geosciences (Wuhan), Wuhan 430074, People’s Republic of China

a r t i c l e

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Article history: Received 9 October 2013 Received in revised form 20 December 2013 Accepted 20 December 2013 Available online 31 December 2013 Keywords: Antimicrobial peptide Cysteine-free peptide Genomic organization Heterometrus spinifer Scorpion Structure/function relationship

a b s t r a c t Three new cysteine-free venom peptides, which are referred to as Heterin-1, Heterin-2 and Spiniferin, respectively, were identified from the scorpion Heterometrus spinifer. Heterin-1, Heterin-2 and Spiniferin contain 43, 24 and 13 amino acid residues, respectively. Genomic analysis showed that the genomic organizations of the three peptides are consistent with those of the known Na+ , K+ or Cl− -channel specific toxins from scorpions; this suggests that the genes of the cysteine-free and cysteine-rich peptides from scorpions were derived from a common ancestor. Antimicrobial assay demonstrated that Heterin-1 possesses potent activities against both Gram-positive and Gram-negative bacteria. Among the tested bacterial species, Heterin-1 is the most active against Bacillus megaterium and Micrococcus luteus with MICs of 4.0 ␮M and 4.0 ␮M, respectively. Heterin-2 is able to potently inhibit the growth of Gram-positive bacteria with MICs from 5.6 ␮M to 30.0 ␮M; however, it has weaker activities against the tested Gramnegative bacteria. It is interesting to see that deletion of the C-terminal random coiled tail (KKD) in Heterin-2 markedly changed the antimicrobial specificity and activity of the peptide. Spiniferin has very weak antimicrobial activities against both Gram-positive and Gram-negative bacteria. We found that introducing three net charges into the polar face of Spiniferin significantly increased its antimicrobial activity against the majority of the tested bacteria; however, in some instances, net charge on the polar face is not important for the antimicrobial activity of the peptide. These studies have expanded our understanding of the diversity, evolution and structure/function relationships of the cysteine-free peptides from scorpions. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Scorpion venom peptides are powerful weapons for scorpions to capture prey and defend themselves against biological enemies. These peptides can be grouped into two classes: disulfide-bridged and non-disulfide-bridged peptides [36]. Disulfide-bridged peptides (DBPs) from scorpions are always 23–76 amino acid residues long and stabilized by 3–5 disulfide bridges [2,31,39,44]. The majority of DBPs from scorpions is able to modulate the physiological functions of Na+ , K+ , Ca2+ or Cl− channels, and is toxic to insects, mammals or crustaceans [2,9,11,31].

夽 The cDNA sequences of Heterin-1, Heterin-2 and Spiniferin have been deposited in the GenBank database under the accession numbers of KC538867, KC538869 and KC538871, respectively. Their genomic sequences are also deposited in the database under the accession numbers of KC538868, KC538870 and KC538872, respectively. ∗ Corresponding author. Tel.: +86 27 67883481. E-mail address: [email protected] (X.-C. Zeng). 1 These authors contributed equally to this work. 0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2013.12.012

Because of their diverse specificities and potencies, these peptides provide invaluable tools for investigations of the structures and functions of different ion channels [11]. In addition to the ion channel modulators, scorpion venom also contains a variety of other DBPs, including proteinase inhibitors, defensins, antimicrobial peptides and orphan peptides [3,31,39]. Some of the DBPs demonstrate great potentials to be effective therapeutic agents for the treatment of autoimmune diseases, glioma, pain and epilepsy [6,14,18,33]. Scorpion venom also contains a vast diversity of nondisulfide-bridged peptides (NDBPs) [31,36]. Until now, at least 30 different NDBPs have been identified from scorpions [4,5,7,8,12,15,17,22,24,25,29,30,32,34–38,40,41,43]. The majority of these peptides possess antimicrobial or/and immune-regulatory activities. Some of them were shown to have bradykininpotentiating activity [40]. It is noteworthy that some NDBPs are able to potently inhibit the growth of some antibiotic-resistant pathogens, and thus have potentials to be clinically applied [12,43]. Most NDBPs are cationic, ␣-helical and amphipathic molecules. Here, we described the cloning, genomic analysis, functional characterization and structure/function relationship analysis of

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Table 1 Primers employed for genomic amplification. Primer

Sequence (5 –3 )

Usage

1F 1R 2F 2R 3F 3R

CGAGGCTTCAAGCAAAAATGA GGACAAACAGATGCTTTAAGG AGCCATGCAGTACAAGACATT TTACTGAAGCTGCGACAGTAA TGTATCAGATCATCCAGCATG ATTACACATCGAGCTTATCAC

Genomic amplification for Heterin-1 Genomic amplification for Heterin-1 Genomic amplification for Heterin-2 Genomic amplification for Heterin-2 Genomic amplification for Spiniferin Genomic amplification for Spiniferin

three novel cysteine-free peptides from the scorpion Heterometrus spinifer, which were referred to as Heterin-1, Heterin-2 and Spiniferin, respectively. Genomic analysis demonstrated that the genes of all three peptides contain a phase 1 intron inserted in the nucleotide sequence coding for the N-terminal region of the signal peptide. This genomic organization of the cysteine-free peptides is similar to those of the known cysteine-rich toxins from scorpions described so far; this strongly suggests that the genes of the cysteine-free peptides could share a common evolutionary origin with those of the cysteine-rich toxins from scorpions. Functional assay showed that Heterin-1 possesses potent antimicrobial activities against both Gram-positive and Gram-negative bacteria. It has relatively low hemolytic activity against human red blood cells. Heterin-2 showed stronger antimicrobial activities against Gram-positive bacteria than Gram-negative bacteria. It has high hemolytic activity against human red blood cells. Secondary structure prediction indicated that Heterin-2 consists of an ␣-helical domain and a random coiled hydrophilic tail (KKD). We found that deletion of KKD from the molecule markedly changed its antimicrobial specificity and activity, and significantly reduced its hemolytic activity against human red blood cells. Spiniferin exhibited very weak antimicrobial activities against both Gram-positive and Gram-negative bacteria. It has extremely weak hemolytic activity against human red blood cells. We found that introduction of two residue mutations into the polar face of Spiniferin, which resulted in the change of net charge from 0 into +3, markedly enhanced its antimicrobial activities against the majority of the tested bacteria; however, for some of the tested bacterial species, net charge on the polar face is not important for the antimicrobial activity of the peptide. Our investigations gained new insights into the evolution and the structure/function relationship of the cysteine-free antimicrobial peptides from scorpions, and provided alternative templates for the development of new antibiotics. 2. Materials and methods 2.1. Animal Specimens of H. spinifer were purchased from the Guangdong province, southern China. The specimens were kept in an escapeproof tank provided with foods and water. 2.2. Construction of a cDNA library from the venom glands of the scorpion

2.3. Screening of the cDNA library A size-selective sequencing strategy was utilized to screen the cDNA library as described previously [41]. DNA fingerprint and PCR strategies were used to exclude duplicate sequences. The obtained cDNAs were sequenced by Nanjing GenScript Biotechnology Corporation (Nanjing, China). 2.4. Genomic cloning of Heterin-1, Heterin-2 and Spiniferin Genomic DNA of the scorpion H. spinifer was prepared as described previously [42]. The genomic sequences of the peptides were obtained by PCR amplifications. Primers were designed based on the sequences of 5 and 3 UTRs of the cDNAs. The primers specific for Heterin-1, Heterin-2 and Spiniferin are listed in Table 1. PCR product was separated by gel electrophoresis, and further purified using Gel Extraction Kit (Omega, USA). The purified DNAs were ligated into pMD19-T (TaKaRa, Japan) according to the instruction manual. The ligated vectors were transformed into the E. coli DH5␣ competent cells. For each gene, five positive clones were picked up for sequencing. 2.5. Bioinformatics analysis Open reading frame (ORF) was determined using ORF Finder. Signal peptide was predicted using SignalP 4.0. Protein sequence homology search was performed using the BLAST sever. Multiple sequences alignments were performed using ClustalW2. Secondary structure was predicted using the NPSA server. Helical wheel projections were determined using HeliQuest. Biochemical properties of peptides were predicted using ProtParam. 2.6. Peptide synthesis Peptides were synthesized using the solid-phase method on the Applied Biosystems peptide synthesizer (model 433-A). Synthesized peptides were purified by reverse-phase high performance liquid chromatograph (RP-HPLC), and verified by mass spectrometry and amino acid composition analysis. Quantitative amino acid analysis is the only method, which enables the net peptide content to be determined. Here, the amount of each amino acid is measured after total acid hydrolysis, the sum total of which gives the amount of peptide in the product. 2.7. Antimicrobial assay

Ten specimens of the scorpion H. spinifer were stimulated by electrical stimulation, which is capable of activating transcription of the genes encoding venom peptides. Two days later, telsons of the scorpions were cut off and thoroughly ground into fine powders in liquid nitrogen. Total RNA was extracted from the homogenized tissues with TRIZOL. Polyadenylated mRNA was purified using a TaKaRa mRNA purification kit. A cDNA library was constructed using the SuperScriptTM cDNA library construction kit according to the instruction for users. A cDNA library was generated by transforming the ligated cDNA into the Escherichia coli DH5␣ competent cells.

Antimicrobial activity of the synthetic peptides was assessed against a set of Gram-positive and Gram-negative bacteria. Bacteria were grown in the 1× BactoTM Trytic Soy Broth (TSB) medium at 37 ◦ C until the OD600 value of the culture reached approximate 0.4. The bacterial suspension was diluted with the same medium to a final OD600 of 0.002. 20 ␮l of each peptide solution prepared by serial dilution was added to a 96-well plate containing 180 ␮l of the diluted bacterial culture. The plate was incubated at 37 ◦ C for 12 h. Inhibition of bacterial growth was determined by measuring optical density (OD) at  = 600 nm with an ELISA plate reader (Infinite

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Table 2 The sequences of the native peptides and their analogs. Peptide

Amino acid sequence

Molecular mass (Da)

Net charge

Heterin-1 Heterin-2 Heterin-2M Spiniferin Spiniferin-M

GVWDWLKKTAKNVWNSDIVKQLKGKAINAAKNYVAEKIGATPS-NH2 FWGALAKGALKLIPSLVSSFTKKD-NH2 FWGALAKGALKLIPSLVSSFT-NH2 ILGEIWKGIKDIL-NH2 ILGKIWKGIKNIL-NH2

4742.54 2576.47 2205.26 1496.90 1494.95

+5 +3 +2 0 +3

200 PRO, Tecan Austria GmbH). Minimum inhibitory concentration (MIC) was thus calculated.

2.8. Hemolysis assay Hemolytic activity of the synthetic peptides was determined by measuring the release of hemoglobin from the human red blood cells. Cells were rinsed 2–3 times using 0.9% NaCl. Washed cells were suspended in the same solution at a density of 1 × 107 –108 cells/mL. Cells were mixed with different concentrations of peptides. Controls for zero and 100% hemolysis were the cells without addition of the tested peptides, and the cells with 10% Triton X-100, respectively. The mixture was incubated at 37 ◦ C for 30 min. Samples were then centrifuged at 10,000 × g for 5 min. Supernatant was measured at  = 570 nm using a spectrophotometer (UNICO, UV-4802). Percent hemolysis was thus calculated.

3. Results 3.1. Identification of three novel venom peptides from the scorpion Sequencing of approximate 500 selected clones from the cDNA library revealed that we obtained the full-length cDNAs of three new cysteine-free peptides from the scorpion H. spinifer, which were referred to as Heterin-1, Heterin-2 and Spiniferin (Figs. 1–3). The sequences and biochemical properties of the three peptides are listed in Table 2.

3.3. Characterization of the precursor of Heterin-2 The cDNA of Heterin-2 is 302 nucleotides long containing a 5 UTR of 4 nucleotides, an open reading frame of 222 nucleotides and 3 UTR of 76 nucleotides (Fig. 2A). A predicted polyadenylation signal (AATAAA) is located 59 nucleotides downstream of the stop codon. The putative precursor of Heterin-2 contains 73 amino acid residues, including a typical signal peptide of 22 residues, a mature peptide of 24 residues and a propeptide of 27 residues. Protein sequence homology search revealed that Heterin-2 shows 92%, 71% and 58% sequence identities to Pandinin-2 from the scorpion P. imperator, Ponericin-W5 from the ant Pachycondyla goeldii and OcyC3 from the scorpion Opisthacanthus cayaporum, respectively [5,28,32] (Fig. 2B). Thus, Heterin-2 is a new member of the 4th family of NDBPs from scorpions. We classified it as NDBP4.2 (Fig. 2B). It was predicted that the secondary structure of Heterin-2 consists of a typical ␣-helix domain (residues 2–21) flanked by the N- and C-terminal random coiled regions (Fig. 2C). Helical-wheel diagram for the helix region of Heterin-2 showed that the peptide has distinct hydrophobic and hydrophilic faces, and thus is an amphipathic molecule (Fig. 2D). It was shown that Pandinin-2 has potent antimicrobial activities against a wide range of Gram-positive bacteria. Thus, it is likely that Heterin-2 is also an antimicrobial peptide. It is noteworthy that both Heterin-2 and Pandinin-2 have a random coiled, hydrophilic tail KKD. In this study, we tried to figure out how the antimicrobial and hemolytic activities of Heterin-2 changed if this tail was deleted from the molecule.

3.4. Characterization of the precursor of Spiniferin 3.2. Characterization of the precursor of Heterin-1 The cDNA of Heterin-1 is 363 nucleotides long containing a 5 UTR of 17 nucleotides, an open reading frame of 252 nucleotides and a 3 UTR of 94 nucleotides, with a polyadenylation signal (AATAAA) located 75 nucleotides downstream of the stop codon (Fig. 1A). Analysis of the predicted precursor of Heterin-1 indicated that it consists of 83 amino acid residues, including a signal peptide of 22 residues, a mature peptide of 43 residues with no disulfide bridges, and a propeptide of 18 residues. Protein homology search showed that the amino acid sequence of Heterin-1 exhibits 73%, 73%, 68%, 45% and 37% identities to those of Opistoporin-1 and Opistoporin-2 from Opistophtalmus carinatus [24], Pandinin-1 from Pandinus imperator [5], Vejovine from Vaejovis mexicanus [17], and Hadrurin from Hadrurus aztecus [34], respectively (Fig. 1B). It has been shown that all these peptides belong to the 3rd family of NDBPs from scorpions. Therefore, Heterin-1 is a new member of NDBP3 , and is accordingly named as NDBP3.14 (Fig. 1B). Secondary structure prediction suggested that Heterin-1 consists of an ␣-helical domain (residues 3–38) flanked by two short random coiled segments (residues 1–2 and 39–43) (Fig. 1C). Helical-wheel diagrams showed that the helix (residues 3–38) has distinct hydrophilic and hydrophobic faces, and thus is amphipathic (Fig. 1D).

The cDNA of Spiniferin is 342 nucleotides long, which encodes a precursor of 67 amino acid residues (Fig. 3A). The precursor of Spiniferin contains a signal peptide of 23 residues, a mature peptide of 13 residues and a propeptide of 31 residues with a typical posttranslational processing signal (GKR). According to the described propeptide processing rule in scorpions, the GKR containing sequence GKRGLNDLSDLDELFDGEISKADLDFLREIM would be removed during the posttranslational processing; this would lead to the C-terminal amidation of the mature peptide [25,43]. Therefore, the mature peptide of Spiniferin consists of 13 amino acid residues (ILGEIWKGIKDIL) with an amidated C-terminus. As shown in Fig. 3B, Spiniferin shows 62%, 62%, 54%, 54%, 54% and 54% sequence identities to IsCT, Pantinin-2, IsCT2, OcyC1, Pantinin-3 and UyCT3 [7,8,22,32,43], respectively. Spiniferin also shares 54%, 50%, 50%, 46%, 38% and 31% identities to UyCT5, OcyC2, Pantinin-1, Hp1090, Meucin-13 and Hp1035, respectively [15,22,32,35,43]. Among these peptides, OcyC1 and OcyC2 have not yet been functionally characterized. This suggests that Spiniferin is a new member of the 5th family of NDBPs from scorpions. We thus classified it as NDBP5.24 (Fig. 3B). Secondary structure prediction showed that Spiniferin is an ␣helical molecule. Helical-wheel projection of Spiniferin revealed that the molecule has distinct hydrophilic and hydrophobic faces, and thus is an amphipathic molecule (Fig. 3C and D).

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Fig. 1. Analysis of the cDNA and amino acid sequence of Heterin-1. (A) The cDNA sequence encoding the precursor of Heterin-1. The deduced amino acid residues are shown below the corresponding nucleotide codons. The amino acid sequence is numbered starting from the N-terminal residue of the peptide; the first residue of the mature peptide is numbered as +1, whereas the signal peptide is numbered as minus. The signal peptide is underlined; a putative polyadenylation signal is marked by italics. The propeptide was highlighted with blue color. (B) Multiple sequence alignment of Heterin-1 and the other members of subfamily NDBP3 from scorpions, such as Hadrurin (GenBank accession number P82656), Parabutoporin (P83312), BmKbpp (Q9Y0X4), Pandinin-1 (P83239), Opistoporin-1 (AY427948), Opistoporin-2 (P83314), Hge029 (EL698903), Opistoporin-4 (AY427949), Con13 (FM998759), C22 (AGA82757), Im-1 (P0CF38), Vejovine (ADZ24463) and Mauriporin (CCN80315). Alignment was performed using ClustalW. Conserved residues are highlighted in blue color. (C) Predicted secondary structure of Heterin-1. C and H stand for coiled coil and ␣-helix structures, respectively. (D) Helical-wheel diagram for the helix region of Heterin-1. Nonpolar and hydrophobic residues are highlighted in yellow color; basic residues are in blue color; small neutral residues are in gray color; acidic residues are in red color; polar residues are in purple color; hydrophilic residues are in green color. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Characterization of the precursor of Heterin-2. (A) The cDNA sequence encoding the precursor of Heterin-2. (B) Multiple sequence alignment of Heterin-2 and the other members of subfamily NDBP4 from scorpions, such as Pandinin-2 (P83240) and OcyC3 (C5J888). (C) Secondary structure prediction of Heterin-2. (D) Helical-wheel diagram for the helix region of Heterin-2.

3.5. Genomic organization of the genes encoding the three peptides The gene of Heterin-1 consists of two exons interrupted by an intron of 540 bp (Fig. 4A). The intron was inserted in the nucleotide sequence coding for the C-terminal region of the signal peptide; the split site is between the first (G) and the second base (T) of a codon encoding the residue Val located at position −7 of the precursor. Similarly, the Heterin-2 gene contains a single intron of 904 bp, which is located in the signal peptide-encoding region, inserted between the first (G) and second base (T) of a codon coding for the residue Val at position −7 (Fig. 4B). The Spiniferin gene consists of two exons disrupted by an intron of 455 bp located in the nucleotide sequence encoding the C-terminal region of the

signal peptide, inserted between first (A) and second base (T) of a codon coding for the residue Met at position −7 of the signal peptide (Fig. 4C). The intron of each gene begins with GT and ends with AG. It is noteworthy that the genes of Na+ , K+ and Cl− -channel specific toxins from scorpions described so far also consist of two exons interrupted by an intron located in the signal peptideencoding region of the precursor [1,10,16,21,23,26]. This suggests that the genomic organizations of the cysteine-free peptides are highly consistent with those of the cysteine-rich toxins from scorpions (Fig. 5). Therefore, it is likely that the genes of these two classes of scorpion venom peptides could be derived from a common evolutionary ancestor by gene duplication and subsequent mutations.

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Fig. 3. Analysis of the precursor of Spiniferin. (A) The cDNA sequence encoding the precursor of Spiniferin. (B) Multiple sequence alignment of Spiniferin and the other members of subfamily NDBP5 from scorpions, such as BmKn1 (Q9GQW4), IsCT (Q8MMJ7), IsCT2 (Q8MTX2), BmKn2 (Q6JQN2), Hge027 (P0C8W1), Hge028 (P0C8W2), OcyC1 (C5J886), OcyC2 (C5J887), Hp1090 (P0DJ02), Hp1035 (P0DJ03), VsCT1 (I0DEB5), VsCT2 (I0DEB6), VmCT1 (I0DEB3), VmCT2 (I0DEB4), UyCT3 (AGA82755), UyCT5 (AGA82756), UyCT1 (AGA82754), Meucin-13 (E4VP07), StCT1 (P0DJO3), StCT2 (P0DJO4), Pantinin-1 (AGK88380), Pantinin-2 (AGK88381) and Pantinin-3 (AGK88382). (C) Predicted secondary structure of Spiniferin. (D) Helical-wheel diagram for the helix region of Spiniferin.

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Fig. 4. The genomic organizations of Heterin-1 (A), Heterin-2 (B) and Spiniferin (C). Nucleotide sequences of exons are shown in capital letters, whereas that of intron is in lower-case letters.

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Fig. 4. (Continued ).

Fig. 5. A comparison of the genomic organizations of Heterin-1, Heterin-2 and Spiniferin genes with those of Na+ , K+ and Cl− -channel toxin genes.

3.6. Antimicrobial and hemolytic activities of Heterin-1 Because Heterin-1 is homologous to some known antimicrobial peptides, it is likely that it also possesses antimicrobial activities. The antimicrobial activity of Heterin-1 was tested against a set of Gram-positive and Gram-negative bacteria (Table 3). We found that Table 3 Antimicrobial activity of Heterin-1.a Microorganism Gram-positive bacteria Staphylococcus aureus AB 94004 Bacillus megaterium AB 90008 Micrococcus luteus AB2010179 Gram-negative bacteria Escherichia coli DH5␣ Pseudomonas fluorences PF Pseudomonas putida PP Enterobacter cloacae AB 2010162 Salmonella enterica AB 2010185 a

The data were obtained using 1× TSB.

MIC (␮M) Heterin-1 42.0 4.0 4.0 25.0 26.0 27.0 40.0 15.0

Heterin-1 possesses broad-spectrum activities against both Grampositive and Gram-negative bacteria. For Gram-positive bacteria, Heterin-1 is able to potently inhibit the growth of Bacillus megaterium and Micrococcus luteus with the MICs of 4.0 ␮M and 4.0 ␮M, respectively; it possesses relatively weaker activity against Staphylococcus aureus with a MIC of 42.0 ␮M. For Gram-negative bacteria, Heterin-1 displayed intermediate antimicrobial activities against the tested bacteria. The MIC values of Heterin-1 are 15.0 ␮M for Salmonella enterica, 25.0 ␮M for E. coli DH5␣, 26.0 ␮M for Pseudomonas fluorences, 27.0 ␮M for Pseudomonas putida and 40.0 ␮M for Enterobacter cloacae. We further evaluated the cytotoxicity of the peptide in human red blood cells. As shown in Fig. 6A, Heterin-1 is not able to induce detectable hemolysis at a concentration of less than 2.5 ␮M. At concentrations of 5.0 ␮M, 10.0 ␮M, 20.0 ␮M and 40.0 ␮M, Heterin-1 induces 20.1%, 54.2%, 74.9% and 88.7% hemolysis of human red blood cells, respectively. The amino acid sequence of Heterin-1 shows 73% identities to that of Opistoporin-1. However, the hemolytic activity of Heterin1 is much stronger than that of Opistoporin-1. We observed that Opistoporin-1 has a proline at position 19, probably bending the

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Fig. 6. The hemolytic activity of the peptides. (A) The hemolytic activity of Heterin-1. (B) The hemolytic activity of Heterin-2M and Heterin-2. (C) The hemolytic activity of Spiniferin and Spiniferin-M.

␣-helix in two segments, which could cause less hemolytic activity when compared with Heterin-1.

Table 4 Antimicrobial activity of Heterin-2 and Heterin-2M.a Microorganism

3.7. Deletion of KKD markedly changed the antimicrobial specificity and activity of Heterin-2 We found that Heterin-2 is capable of inhibiting the growth of the tested Gram-positive bacteria (Table 4). Heterin-2 possesses the strongest antimicrobial activity against S. aureus with a MIC of 5.6 ␮M; it has relatively weaker activity against B. megaterium and M. luteus with MIC values of 30.0 ␮M and 15.8 ␮M, respectively. Heterin-2 also possesses antimicrobial activities against some of the tested Gram-negative bacteria (Table 4). The MIC values are 15.8 ␮M for E. coli DH5␣, 20.0 ␮M for P. putida, 30.0 ␮M for S. enterica and 40.0 ␮M for P. fluorences. However, Heterin-2 does not have any detectable activity against Klebsiella oxytoca and E. cloacae at the concentration of 45.0 ␮M. Therefore, Heterin-2 generally possesses much lower antimicrobial activities against Gram-negative bacteria than against Gram-positive bacteria. Secondary structure prediction showed that Heterin-2 has a highly hydrophilic random coiled tail (KKD) at the C-terminus of

MIC (␮M) Heterin-2

Gram-positive bacteria Staphylococcus aureus AB 94004 Bacillus megaterium AB 90008 Micrococcus luteus AB2010179 Gram-negative bacteria Escherichia coli DH5␣ Pseudomonas fluorences PF Pseudomonas putida PP Klebsiella oxytoca AB 2010143 Enterobacter cloacae AB 2010162 Salmonella enterica AB 2010185 a

Heterin-2M

5.6 30.0 15.8

6.9 1.5 1.5

15.8 40.0 20.0 >45.0 >45.0 30.0

40.0 >50.5 35.0 >45.0 >45.0 40.0

The data were obtained using 1× TSB.

the peptide. This tail may significantly increase the hydrophilicity of the molecule. In order to investigate how the hydrophilic tail affects the antimicrobial specificity and activities of the peptide, we synthesized a KKD tail-deleted peptide, which was named as Heterin-2M (Table 2). Compared with Heterin-2, Heterin-2M

S. Wu et al. / Peptides 53 (2014) 30–41 Table 5 Antimicrobial activity of Spiniferin-M and Spiniferin.a Microorganism

MIC (␮M) Spiniferin-M

Gram-positive bacteria Staphylococcus aureus AB 94004 Bacillus megaterium AB 90008 Micrococcus luteus AB2010179 Gram-negative bacteria Pseudomonas putida PP Pseudomonas fluorences PF Salmonella enterica AB 2010185 Escherichia coli DH5␣ Enterobacter cloacae AB 2010162 a

Spiniferin

12.0 50.0 2.0

>82.0 41.0 82.0

16.0 32.0 35.0 82.0 >82.0

>82.0 >82.0 >82.0 82.0 >82.0

39

The hemolytic activities of both Spiniferin and Spiniferin-M in human erythrocytes were determined (Fig. 6C). We found that Spiniferin is not able to induce detectable hemolytic reaction at concentrations up to 48.0 ␮M. However, Spiniferin-M possesses much higher hemolytic activity in human red blood cells. At a concentration of less than 3.0 ␮M, Spiniferin-M is not able to induce significant hemolysis. At the concentrations of 6.0 ␮M, 12.0 ␮M, 24.0 ␮M and 48.0 ␮M, Spiniferin-M induced 1.7%, 19.1%, 31.9% and 56.6% hemolysis of the cells, respectively. These data suggests that net charge on the polar face of a peptide is indispensable to the hemolytic activity. Increasing net charge on the polar face of the amphipathic ␣-helix significantly increased the hemolytic activity.

The data were obtained using 1× TSB.

4. Discussion possesses much stronger antimicrobial activities against B. megaterium (MIC 1.5 ␮M) and M. luteus (MIC 1.5 ␮M) than Heterin-2; the MIC values of Heterin-2M are approximate 19.0- and 9.5-fold lower than those of Heterin-2 for the two strains of bacteria, respectively (Table 4). However, compared with Heterin-2, Heterin-2M exhibits weaker activity againt all other tested Gram-positive and Gramnegative bacteria. Therefore, deletion of the KKD tail from Heterin-2 markedly changes the antimicrobial specificity and activities of the molecule. Moreover, we found that Heterin-2M has much lower hemolytic activity than Heterin-2 toward human red blood cells (Fig. 6B). At the concentrations of 0.8 ␮M, 1.6 ␮M, 3.2 ␮M and 6.4 ␮M, Heterin2 induces 2.0%, 10.6%, 49.3% and 94.0% hemolysis of the cells, respectively; however, at the same concentrations, Heterin-2M only induces 0%, 1.5%, 22.1% and 50.1% hemolysis of the cells, respectively. These data indicated that deletion of the C-terminal KKD tail from Heterin-2 markedly reduces the cytotoxicity of the molecule in human red blood cells. 3.8. Antimicrobial and hemolytic activity of Spiniferin and its analog Spiniferin is a typical ␣-helical and amphipathic molecule. However, this peptide contains zero net charge. Thus, it is likely that Spiniferin has very weak antimicrobial activities. We found that Spiniferin possesses weak activities against Gram-positive bacteria (Table 5). Spiniferin inhibits the growth of B. megaterium and M. luteus at MICs of 41.0 ␮M and 82.0 ␮M, respectively. It does not possess any detectable activity against S. aureus at 82.0 ␮M. For the tested Gram-negative bacteria, Spiniferin has no inhibitory effect on P. fluorences, P. putida, E. cloacae and S. enterica at 82.0 ␮M; it has very weak activity against E. coli DH5␣ (MIC 82.0 ␮M). This suggests that Spiniferin has very weak antimicrobial activities against both Gram-positive and Gram-negative bacteria. In order to explore the effects of net charges on the antimicrobial and hemolytic activities of Spiniferin, we designed an analog of Spiniferin (referred to as Spiniferin-M) by substituting the residues E4 and D11 on the polar face with K4 and N11, respectively, while keeping the nonpolar face unchanged (Table 2). The substitutions resulted in a net charge increases from 0 to +3. As shown in Table 5, Spiniferin-M possesses much higher activities than Spiniferin against the majority of the tested bacteria, including S. aureus, M. luteus, P. putida, P. fluorences and S. enterica. However, Spiniferin-M has antimicrobial activities similar to those of Spiniferin against E. coli DH5␣ and B. megaterium. Therefore, in most instances, increasing the net charge on the polar face made the analog possess much high antimicrobial activity; however, it is less frequent that the net charge on the polar face is not important for the antimicrobial activity of Spiniferin.

4.1. The genes of cysteine-rich and cysteine-free peptides from scorpions could share a common ancestor Scorpions are considered to be living fossils as they have existed on the earth since the Silurian period, around 430 million years ago. During their long-term evolution, scorpions successfully adapted to different harsh environments, and kept their morphology almost unchanged. One key to their survival in many tough conditions is a group of toxic peptides in their venom glands, which are the powerful weapons for scorpions to capture prey and defend themselves. These toxins can be deadly to many organisms, including insects, mammals, crustaceans and microorganisms [31]. It was found that many types of venom peptides are effectively tailored to their users’ lifestyles and unique microbial environments. This raised a question: what is the molecular mechanism that drove the functional evolution of the scorpion venom peptides? It was shown that the DBPs diverged by gene duplication and subsequent accumulation of mutations in the sequences [13]. However, it is unclear how the genes of NDBPs diversified. Moreover, the evolutionary relationship between DBPs and NDBPs from scorpions remains to be elucidated. Here, we found that the genes of Heterin-1, Heterin2 and Spiniferin, which represent three different subfamilies of NDBPs from scorpions, share a common genomic organization. This suggests that the evolution of scorpion NDBPs seems to have followed the “standard model” of gene evolution: duplication of the ancestral gene, followed by subsequent mutations and natural selection based on the needs of the scorpions. Moreover, the genomic organizations of the three NDBPs are highly consistent with those of the known DBPs from scorpions described so far. Therefore, the genes of cysteine-rich and cysteine-free peptides from scorpions could share a common ancestor.

4.2. Antimicrobial properties of the scorpion antimicrobial peptides Heterometrus is a genus of scorpion belonging to the family Scorpionidae. It is widely distributed across the tropical and subtropical southeastern Asia. It can be easily found in Vietnam and Thailand. Because a lot of environmental bacteria are capable of surviving and proliferating in the tropical and subtropical regions, the scorpions living there should need stronger antimicrobial ability to protect themselves from microbial invasion than those living in cold and dry environments. Therefore, the venom from the scorpion H. spinifer could be a rich source of antimicrobial peptides. Until now, a total of seven different venom peptides have been identified from the venom glands of H. spinifer, including a K+ -channel specific toxin ␬-KTx1.3 and six antimicrobial peptides HsAp, HsAp1, HsAp2, Heterin-1, Heterin-2 and Spiniferin [25,27 and this article]. It is expected that more antimicrobial peptides will be identified

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or isolated from H. spinifer, which could fuel future research in this area. Heterin-1, Heterin-2 and Spiniferin belong to three different subfamilies of cysteine-free peptides from scorpions. All these peptides are predicted to be ␣-helical and amphipathic. Although the exact mode of action of this kind of antimicrobial peptides has not yet been established, it is extensively accepted that the cytoplasmic membrane is the main target of these peptides, whereby accumulation of peptides in the membrane causes increased permeability and loss of barrier function, resulting in the leakage of cytoplasmic components and cell death. Factors that are important for antimicrobial activities include peptide hydrophobicity, positively charged residues, amphipathic nature and secondary structure [19,20]. Heterin-2 contains a random coiled tail (KKD) that significantly reduces the hydrophobicity of the peptide. It is interesting to see that deletion of KKD from the molecule markedly increased the antimicrobial activities against some of the tested Gram-positive bacteria, but slightly reduced the activities of the peptide against the tested Gram-negative bacteria. The hemolytic activity significantly decreased with increasing hydrophobicity. This finding suggests that peptide hydrophobicity is highly associated with the specificity of the peptide; decreasing hydrophilicity could be a good way to reduce toxicity of the antimicrobial peptides to mammalian cells. Spiniferin has no net charge. It possesses very weak antimicrobial activities against both Gram-positive and Gram-negative bacteria, and almost has no toxicity to human red blood cells. Increasing net charge on the polar face of Spiniferin was achieved by mutating E4 and D11 into K4 and N11, respectively. The net charge was thus changed from 0 of Spiniferin into +3 of SpiniferinM. We found that the general antimicrobial and hemolytic activities of Spiniferin-M were much stronger than those of Spiniferin. This finding is consistent with the previous observation that net charge is important for both antimicrobial and hemolytic activities [20]. However, the activities of Spiniferin-M against B. megaterium and E. coli DH5␣ are similar to those of Spiniferin; this suggests that although it is not often seen, in some instances, net charge on the polar face of a peptide is not important for the antimicrobial activity. Heterin-1, Heterin-2 and Spiniferin are 43, 24, and 13 residues long, respectively. BmKbpp is also an antimicrobial peptide with 47 residues from scorpion [40]. Compared the antimicrobial spectrum of these peptides with each other, it can be seen that the peptides with short chain (Spiniferin and Heterin-2) possess stronger activity against Gram-positive bacteria than against Gram-negative bacteria; however, the peptide with long chain (BmKbpp) possesses weaker activity against Gram-positive bacteria than against Gramnegative bacteria. The peptide with intermediate size (Heterin-1) has similar activity against Gram-positive and Gram-negative bacteria. Therefore, chain length of antimicrobial peptide has significant effects on its specificity. 4.3. Update of the classification of the subfamilies NDBP3 , NDBP4 and NDBP5 from scorpions Since we proposed a classification system for the scorpion cysteine-free peptides eight years ago [36], a lot of new NDBPs have been identified or isolated from different species of scorpions. Here, we would like to take this opportunity to update the classification of the peptides belonging to the subfamilies NDBP3 , NDBP4 and NDBP5 . As shown in Fig. 1B, the subfamily of NDBP3 has been expanded to 14 members so far. BmKb1 is an antimicrobial peptide from the scorpion Mesobuthus martensii Karsch [41]. In the previous classification system, it was assigned into the NDBP4 subfamily, and named as NDBP4.2 . However, we found that the cysteine-free

antimicrobial peptides with 17–19 residues from scorpions have high sequence similarity to each other. They have consistent secondary structures that are different from those of other known NDBPs from scorpions (data not shown). Therefore, we propose that these peptides should be classified as an independent subfamily (NDBP7 ). Accordingly, we excluded BmKb1 from NDBP4 . NDBP4 includes 3 members now (Fig. 2B). As shown in Fig. 3B, NDBP5 has been expanded to 24 members and become the largest group among the NDBP subfamilies from scorpions.

Acknowledgments This work was funded by the China National 973 Research Project, Basic Research and Clinical Application of Venom Peptides from Toxic Animals (No. 2010CB529800) (2010–2015) and the Fundamental Research Fund for the Central Universities (No. CUGL100613, No. CUGL110604 and No. CUGL120608) (2010–2013) granted to Prof. X.C. Zeng.

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