Identification and functional characterization of novel scorpion venom peptides with no disulfide bridge from Buthus martensii Karsch

Peptides 25 (2004) 143–150

Identification and functional characterization of novel scorpion venom peptides with no disulfide bridge from Buthus martensii Karsch夽 Xian-Chun Zeng∗,1 , San-Xia Wang, Yan Zhu2 , Shun-Yi Zhu, Wen-Xin Li∗,3 Department of Biotechnology, Institute of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China Received 8 October 2003; received in revised form 10 December 2003; accepted 10 December 2003

Abstract The scorpion venom peptides with no disulfide bridge are rarely identified and poorly characterized so far. Here, we report the identification and characterization of four novel disulfide-bridge-free venom peptides (BmKa1, BmKa2, BmKb1 and BmKn2) from Buthus martensii Kasch. BmKa1 and BmKa2 are very acidic and hydrophilic, showing no any similarity to other proteins, whereas BmKb1 and BmKn2 both are basic, ␣-helical peptide with an amidated C-terminus, showing a little homology with other peptides. Functional tests with synthetic peptide showed that BmKn2 has strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, whereas BmKb1 has weak activity in inhibiting the growth of these bacteria. © 2004 Elsevier Inc. All rights reserved. Keywords: Amphipathic; Antibacterial; Acidic peptide; Venom peptide; Molecular evolution

1. Introduction Scorpion venoms are rich sources of bioactive peptides with 13–76 amino acid residues, which can be classified into two main types: toxins with three to five disulfide bridges, and venom peptides without disulfide bridge [23]. Most scorpion toxins described are ligand peptides that can recognize and specifically interact with ion channels of excitable or non-excitable cells including Na+ , K+ , Cl− and Ca2+ -channels [6,13,17,20,23]. Na+ -channel specific toxins are composed of 60–76 amino acid residues cross-linked by four disulfide bridges; whereas the K+ and Cl− -channel toxins are about 28–41 residues stabilized by three or four disulfide bridges. The toxins specific for Ca2+ -channels have variable amino acid lengths. Very recently, an anticoagulant-peptide-like scorpion toxin compacted by five disulfide bridges has been described, which 夽 GenBank accession numbers: BmKa1, AF146743 (BmK2 renamed as BmKa1); BmKa2, AF150012; BmKb1, AF543048; BmKb1∗ , AF159979 (toxin peptide 6 renamed as BmKb1∗ ); BmKn2, AY323830. ∗ Corresponding authors. E-mail addresses: [email protected] (X.-C. Zeng), [email protected] (W.-X. Li). 1 Present address: Lab of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA. Tel.: +1-301-496-1079; fax: +1-301-402-1519. 2 Present address: Department of Orthodontics, Stomatological College, Fourth Military Medical University, Xi’an 710032, PR China. 3 Tel.: +86-27-87682831; fax: +86-27-87649146.

0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.12.003

is composed of 64 residues [21]. Until now, nearly several hundreds disulfide-bridged scorpion toxins have been characterized from over 20 species of scorpions. The scorpion venom peptides without disulfide bridges, which have been poorly identified and characterized so far, form another interesting family of venom compounds. About nine such peptides have been identified from scorpions, including a bradykinin-potentiating peptide (K12) from Buthus occitanus [10], six antimicrobial peptides: Pandinin from Pandinus imperator [3], Hadrurin from Hadrurus aztecus [16], IsCT1 and IsCT2 both from Opisthacanthus madagascariensis [4,5], Opistoporin 1 and 2 both from South-African scorpion Opistophtalmus carinatus [11], and two deduced peptide (BmKbpp and BmKn1) with unknown function identified by gene cloning from B. martensii Karsch [22,26]. B. martensii Karsch is a representative scorpion species in China. The scorpion venom or the whole tail has been used as a main Chinese traditional medicine to treat some neurological diseases such as stroke, rheumatism and tetanus for more than 2000 years. Therefore, it is very interesting to identify the pharmacologically effective components, and gain further insight into their functions and the mechanism of their actions. Up to now, nearly 70 disulfide-bridged toxins have been identified and characterized from B. martensii Karsch. However, there are a lot of compounds still to be discovered in the venom. In this study, we describe identification and characterization of four novel linear venom peptides from the venom gland of B. martensii

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Karsch; moreover, we further synthesized and functionally characterized two new amphipathic peptides of them. 2. Materials and methods 2.1. Construction and analysis of a scorpion venom gland cDNA library Scorpions of B. martensii Karsch were obtained from Henan Province, China, and fed in our lab for 3 weeks. Venom glands were cut-off 2 days after extraction of their venom to allow the toxin-producing cells of the glands to enter a secretory phase, and frozen immediately in liquid nitrogen. Total RNA was extracted from homogenized gland tissues using a modified single-step method. Poly(A)+ mRNA was purified by a PolyATract mRNA Isolation Kit (Promega). A scorpion venom gland cDNA library was constructed using SuperScript Plasmid System (GIBCO/BRL). Briefly, first-strand cDNA was synthesized from 6 ␮g mRNA by standard reverse transcription with Not I primer-adapter. Double-stranded cDNA was prepared with Escherichia coli DNA polymerase I, E. coli DNA ligase and T4 DNA polymerase. After addition of Sal I-adapter, the double-stranded cDNA was cut by Not I. The Nucleon QC system (Amersham) was used to remove the residual adapters and other low molecular weight DNAs. The cDNA was then ligated into the Not I–Sal I-cut plasmid pSPORT1. Cloned cDNA was introduced into E. coli DH5␣ by transformation. The 32 P-labeled first strand cDNA was analyzed by alkaline agarose electrophoresis to estimate the size range of products synthesized. 2.2. Screening of the cDNA library A subtraction strategy combined with “size-selective” random sequencing was used to screen the cDNA library, which included three steps as followed. a. Analysis of insert size of clones from the cDNA library PCR amplification was used to determine the insert size of 5000 clones from the cDNA library. The forward primer was T7 promoter universal primer; and the reverse primer was 5 -AGCGGCCGCCCT(15) -3 , corresponding to a partial sequence of the pSPORT1 vector and 3 -terminal poly(A) sequence. The PCR templates were prepared by a boiling method. The PCR products were analyzed by electrophoresis on 2.0% agarose gel. The cDNA clones with the insert size between 350 and 450 bp were chosen for further screening. b. Removal of the cDNAs code for disulfide-bridged peptide toxins A mixture of cDNA probes including the cDNAs of BmKT, BmKM4, BmKCT, BmKK1, MkTxII, BmKBT, BmKAPi, BmK␤-CT, BmTX2 and BmP01and BmP02 [8,15,19,23,24,25,27] (excluding their 5 UTR, 3 UTR and signal peptide region) were prepared by PCR

and radiolablled with [␣-32 P]dCTP by nick-translation (Boehringer Mannheim, Germany) as described by user’s instruction. About 3000 clones from the first step were transferred to Hybond-N nylon filters and hybridized with the probes. The negative clones were chosen for random sequencing. c. DNA sequencing About 100 independent clones were picked randomly from the second step. Plasmid DNA was prepared using QIAGEN Plasmid Mini kit (QIAGEN GmbH, Germany). Nucleotide sequences were determined with the universal M13 forward and reverse primers from both directions using ABI PRISM® BigDyeTM Primer Cycle Sequencing Kit and ABI PRISM 377 DNA sequencer. 2.3. Bioinformatics analysis Open reading frame was determined by ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf). Sequence similarity search was performed using BLAST programs (http://www.ncbi.nlm.nih.gov/BLAST). Multiple alignment of sequences was done by Launcher (http://dot.Imgen.bcm. tmc.edu:9331/multi-align). Signal peptides were predicted using SignalP (http://www.cbs.dtu.dk/services). Physicochemical property was analyzed by ProtParam (http://www. expasy.ch/tools/protparam). Amphipathic ␣-helix was predicted by GCG-Lite program. 2.4. 5 -race If a sequenced cDNA is only a partial fragment, it is necessary to get its full-length sequence by 5 -race. A gene-specific primer was designed and synthesized based on the determined sequence. Total RNA of scorpion venom gland was prepared by TRIZOL LS Reagent (GIBCO/BRL). The SMART RACE kit (Clontech, USA) was used to synthesize the first-strand cDNA and prepare PCR amplification according to the product’s instruction manual. The PCR products were purified by QIAquick Spin kit (Qiagen GmbH, Germany) and cloned for sequencing. 2.5. Peptide synthesis Peptide was synthesized by solid phase methods using Fmoc N-terminal protected amino acids on an Applied Biosystems model 433-A peptide synthesizer, and was purified by reverse-phase high performance liquid chromatography (RP-HPLC). The purified peptide was confirmed by mass spectrometry analysis and amino acid composition analysis. 2.6. Antimicrobial assay The antimicrobial activity of synthetic peptides was tested against a series of Gram-positive strain and Gram-negative strain (shown in Table 1). The bacteria were grown at

X.-C. Zeng et al. / Peptides 25 (2004) 143–150 Table 1 Minimal inhibitory concentration (MIC) of synthetic BmKb1 and BmKn2 (␮g/ml) Bacterial strain Gram-positive Staphylococcus aureus Micrococcus luteus Bacillus subtillis Gram-negative Escherichia coli Pseudomonas aeroginosa

BmKn2

BmKb1

0.6 8.0 5.0

16 81.5 48.8

1.5 21.3

18.1 90.8

37 ◦ C in LB medium to an approximate D600 of 0.8 and then diluted in the same medium to a D600 of 0.002 (105 –106 colony-forming units/ml). 10 ␮l of different concentrations of peptides was added to 90 ␮l of the diluted bacteria culture. The mixtures were incubated in 96-well plates at 37 ◦ C with continuous shaking. Inhibition of growth was determined by measuring the absorbance at 620 nm, and the minimal inhibitory concentration (MIC), expressed as the lowest concentration that causes 100% inhibition of growth, was achieved.

3. Results 3.1. Construction and analysis of a scorpion venom gland cDNA library To get ideal sequencing data, the quality of cDNA library is critical. The alkaline electrophoresis analysis of 32 P-labeled first strand cDNA showed a major class of cDNAs ranging in size from 300 to 550 nucleotides was observed (data not shown). Almost all the precursors of scorpion venom peptides characterized so far are composed of 50–96 amino acid residues [13]. Including their 3 UTR and 5 UTR, the intact mRNAs encoding these precursors should be approximately 300–600 nucleotides. Therefore, our cDNA library is composed of cDNA inserts that are nearly full-length copies of the mRNAs from which they were derived. Moreover, we amplified 10 rarely-expressed toxin peptide genes: BmKK2, BmKK4, BmKBT, BmP01, BmP02, MkTxII, BmKAPi, BmKM4, BmTx2, and BmKT [8,15,19,23,24] from the cDNA library revealing the presence of full-length cDNAs for these peptides (data not shown). These data suggested that the cDNA library we constructed is of high quality. 3.2. Screening the cDNA library and sequencing After removing the majority of known disulfide-bridged neurotoxin clones by hybridization, we sequenced 100 clones with insert from 300 to 450 bp and got four novel cDNAs from the cDNA library, three of them are full-length

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cDNAs encoding three venom peptides without disulfide bridge (referred as to BmKa1, BmKa2 and BmKb1, respectively), the last one code for the 3 -partial sequence of a peptide (referred to as BmKn2). The full-length cDNA of BmKn2 was completed by overlapping this sequence and the 5 -partial sequence obtained by 5 race. 3.3. Sequence analysis 3.3.1. Structural features of BmKa1, BmKa2, BmKb1 and BmKn2 precursors As shown in Fig. 1, each of the four cDNAs is composed of three parts: 5 UTR, an open reading frame and 3 UTR. Their non-coding regions have the following similar structural features: (a) the flanking nucleotides of initiation codon ATG are AAA/G (adenines); (b) their putative polyadenylation signals (AATAAA) are found at similar positions, 14, 11, 11 and 11 nucleotides upstream from the poly(A) tail, respectively; (c) their 3 UTRs are rich in AAA or TTT motifs which might involve in the control of mRNA stability [2]. The cDNAs code for four precursors each including a putative signal peptide and a premature or mature peptide. The signal peptides of the four precursors have four common properties that satisfy the requirements for a functional signal sequence. (a) The signal peptide cleavage site is at a small neutral residue (Gly, Ala, Ser and Ala, respectively); (b) there is one or two positively charged residue(s) (Lys or Arg) at its N-terminus which is known to improve the export efficiency of most eukaryotic signal peptides [12]; (c) it has one negatively charged residue (Glu or Asp) at its C-terminal part except for BmKb1; (d) it has a hydrophobic core consisting of Leu, Phe or Val. All these structural features are similar to the described disulfide-bridged toxin precursors as well as to the known venom peptide precursors without disulfide bridge from scorpions, suggesting the two classes of scorpion venom peptides might have a similar gene expression regulation mechanism [4,8,13,23–25]. 3.3.2. BmKa1 and BmKa2, two highly acidic peptides The BmKa1/BmKa2 precursor contains a signal sequence of 19/24 residues and a mature peptide of 38/50 residues. A search for amino acid sequence homology indicated that both peptides showed no any similarity to other proteins. The two peptides have an irregular amino acid composition, both being extremely rich in acidic residues: Asp and Glu (Asp+Glu: 39 and 36%, respectively). It suggested that they are highly acidic and hydrophilic. Their calculated isoelectric points (IP) are 3.39 and 2.69, respectively, which are the lowest among the scorpion venom peptides identified so far. Secondary structure prediction showed that Bmka1 is composed of 100% coil structures, whereas BmKa2 contains three random coiled regions (residues 1–8, 17–31 and 43–50, respectively) separated by two ␣-helical domains (residues 8–16 and 32–42). The helical wheel diagram (figures not shown) for the two ␣-helical domains showed their helix

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surfaces have distinct hydrophobic and hydrophilic regions, demonstrating an amphipathic nature of BmKa2. Based on the molecule’ s nature of BmKa1, we proposed one hypothesis for its function: balancing the pH value of venom liquid in scorpions and acting as synergists of other peptides. Because the vast majority of scorpion venom peptides are basic, acidic peptides might be neces-

sary to keep the liquid’s pH in a right range. Although the sequences are quite different, the amino acid composition and IP value of BmKa2 is similar to those of the acidic antimicrobial lipopeptides isolated from Bacillus subtilis [1]. Further investigation will be interesting to unravel whether BmKa2 represent a new class of antimicrobial peptides from scorpions.

(A)

TAGAA

-19

-15

-10

-5

ATG AAA CCT GGA GTA TTC TTT CTG TTG TTT CTC TTG GTT GCT GCC ATG M

K

P

R

-1

+1

V

F

F

L

L

F

L

L

+5

V

A

A

M

+10

ATA GAG ACT GGC GAA AGT GAA GAA AAT GAA GAA GGA TCT AAT GAA AGT I

E

T

G

E

S

+15

E

E

N

E

E

+20

G

S

N

E

S

+25

GGT AAA TCT ACA GAA GCA AAA AAC ACA GAT GCA TCA GTC GAC AAC GAA G

K

S

T

E

+30

A

K

N

+35

T

D

A

S

V

D

N

E

+38

GAT TCA GAT ATT GAT GGT GAT TCT GAT TGA TGT TTC ATT GCT AAT TTT D

S

D

I

D

G

D

S

D

end

ATCATTTCTGAAATCGGTTTTAGCTACTGAAATGTGTATTGAAAAAATCTTTTAC TGAAATGAAGTGAATAAAATAATTATTTGCAT poly(A)

(B)

AACTTACTTCTGTTTTTAATTTCAAGTAGTCAAA

-24

-20

-15

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ATG TCT TCT AAA ACA CTA TTA GTA CTG CTA CTG GTC GGT GTA TTA GTT M

S

S

K

T

L

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L

-1

+1

L

L

V

G

V

L

V

+5

TCT ACT TTC TTC ACG GCC GAT GCC TAT CCT GCA TCA ATG GAT AAT TAT S

T

F

F

T

A

+10

D

A

Y

P

A

+15

S

M

D

N

S

+20

GAT GAT GCT TTA GAA GAA TTG GAC AAT CTT GAT TTA GAT GAT TAT TTC D

D

A

L

E

+25

E

L

D

N

L

+30

D

L

D

D

Y

+35

F +40

GAC TTA GAA CCT GCC GAT TTC GTT TTA TTA GAC ATG TGG GCA AAT ATG D

L

E

P

A

D

F

V

L

+45

L

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M

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M

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TTG GAA AGT TCG GAC TTT GAT GAT ATG GAA TAA TTT TCA TTA AAA AAT L

E

S

S

D

F

D

D

M

E

end

TAAATTTGTATTTAAAAATCTGGCTTGTAATATTTTTCCTTTTTTGAAAATAAAGT

TAGATTATTPoly(A)

Fig. 1. cDNA and protein sequences of BmKa1 (A), BmKa2 (B), BmKb1 (C) and BmKn2 (D). Each deduced protein is shown below its corresponding nucleotide sequence, and is numbered on top from the N-terminal amino acid residue of the peptide; the signal peptide is underlined; the potential polyadenylation signal AATAAA is in italics. For (C) and (D), the post-translational processing signal: Gly-Arg-Arg or Gly-Lys-Arg is indicated in bold italics; amino acid residues of mature BmKb1/BmKn2 are printed in boldface.

X.-C. Zeng et al. / Peptides 25 (2004) 143–150

(C)

147

GAATATTCGAAACTCGGCCAAG

-22

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-15

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ATG GAA ATA AAG TAT CTT CTT ACC GTC TTC TTA GTC CTG CTA ATA GTG M

E

I

K

Y

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L

L

-1

+1

T

V

F

L

V

L

L

I

V +10

+5

TCC GAT CAT TGC CAA GCA TTT CTG TTT TCT CTA ATA CCA TCA GCC ATC S

D

H

C

Q

F

A

L

F

+15

S

L

I

P

S

A

+20

I

+25

AGC GGG CTC ATC AGC GCT TTT AAA GGA AGA AGG AAA AGA GAT TTG AAT S

G

L

I

S

A

F

K

G

+30

R

R

K

R

D

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L

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GGC TAT ATA GAC CAC TTC AAG AAT TTT AGA AAA CGT GAT GCC GAA TTG G

Y

I

D

H

N

F K

+45

F

R

K

R

D

A

E

L

+50

GAA GAA TTA CTT TCT AAA CTA CCA ATT TAT TAA CTT CTT TAC GTA CTA E

E

L

L

S

K

L

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end

CAGTCGTTAGTCTCTCTTCCGGTCGTTTCCTAATATAAATTAAAAATTATAAACTGTTGG

TGAAATTAATAAATATTTTTTCTTPoly(A)

(D)

ATAAA

-23

-20

-15

-10

ATG AAA TCT CAG ACC TTT TTC CTT CTT TTT

CTA GTT GTT TTA TTA TTA

M

L

K

S

Q

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F

-5

F

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-1

+1

L

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S

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D

F

A

V

V

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L

L

+5

GCA ATT TCA CAA TCA GAC GCT TTC ATT A

F

I

GGT GCT ATT GCT CGT CTT CTC G

+15

A

I

A

R

L

+20

L +25

TCT AAA ATT TTT GGA AAA AGA AGT ATG GGA GAT ATG GAT ACT ATG AAG S

K

I

F

G

K

R

S

M

+30

G

D

M

D

T

+35

M

K

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TAC TTA TAT GAT CCA AGT TTG AGT GCA GCT GAT TTG CAA ACC TTA CAA Y

L

Y

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S L

S

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L

Q

+47

AAA CTA ATG GAA AAT TAC TGA TTA TTT GAT TTT AAA ATT GTT ATC TGT K

L

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end

TAGTTTAATTTAAATTGATTTTAAATTTTAGATAATAAATATTTCTTTTGpoly(A)

Fig. 1. (Continued ).

3.3.3. BmKb1 and BmKn2, two basic peptides BmKb1/BmKn2 is composed of a signal peptide of 22/23 residues, and a premature peptide of 52/47 residues that contains a typical processing signal: Gly-Arg-Arg/Gly-Lys-Arg at positions 19–21/14–16. According to the described propeptide cleavage rule, the Gly-Arg-Arg or Gly-Lys-Arg group would be removed during post-translational processing, which would result in the removal of a 31-residue (for

both BmKb1 and BmKn2) propeptide and the C-terminal amidation of a 18- (BmKb1) or 13-residue mature peptide (BmKn2). Similar precursor structure and the same processing mechanism were observed in the precursors of two scorpion linear peptides IsCT1 and IsCT2 from O. madagascariens [4,5]. Comparison of BmKb1 with available protein sequences in GenBank database revealed that BmKb1 showed no homology with any of other proteins but few

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(A) BmKb1 IsCT IsCT2

1

2

3

4

5

F

L

F

S

L

6 I . .

7 P L F

8 S G G

9 A K .

10 I . .

11 S W W

12 G E N

13 L G G

14 I . .

15 S K K

16 A S S

17

18 %

F

K

100

L L

F F

16 22

(B) BmKn2 BmKn1 IsCT IsCT2 Temporin A Temporin B Temporin F Temporin L

1 F . I I . L . .

2 I . L F L L L V

3 G . . . P P P Q

4 A . K . L I L W

5 I V . . . V . F

6 A . W W G G G S

7 R G E N . N K K

8 L . G G V . V F

9 L . I I . . . .

10 S . K K . K . G

11 K . S S G S G R

12 I . L L . L . .

13 F . . . L L L L

% 100 84% 23 30 46 15 33 23

Fig. 2. Protein alignment. (A) Protein sequence alignment of BmKb1 with IsCT and IsCT2. (B) Alignment of BmKn2, BmKn1, IsCT, IsCT2, Temporin A, Temporin B and Temporin F, Temporin L. Amino acids common to BmKb1/BmKn2 are indicated by ‘·’.

identical residues with IsCT1 and IsCT2. The amino acid alignment of the three peptides showed three Ile residues are highly conserved among them suggesting the peptides might be evolutionally or functionally related (Fig. 2A). BmKn2 shows the highest homology with BmKn1 (84%), a deduced peptide with unknown function from B. martensii Karsch [26], Temporin A (46%), F (33%), L (23%), B (15%) from European red frog Rana temporaria [14,18,28], IsCT2 (30%) and IsCT (23%) (Fig. 2B) [10,11]. BmKb1/BmKn2 has a net charge of +1/+2, making them basic peptides. Secondary structure prediction showed that both contain one ␣-helix domain (residues 2–16 and 2–11, respectively), and two flexible random coiled regions at both terminus (BmKb1, residues 1 and 17–18; BmKn2, residues 1 and 12–13). Helical-wheel diagrams for BmKb1and BmKn2 were shown in Fig. 3. Both peptides have a distinct hydrophobic and a hydrophilic region, making both molecules markedly amphipathic. 3.3.4. Antimicrobial activity of BmKb1 and BmKn2 Table 1 shows the MIC of BmKb1 and BmKn2 against a series of Gram-positive and Gram-negative bacteria. Generally speaking, BmKn2 can potently inhibit both Gram-positive and Gram-negative bacteria. Compared to IsCT and IsCT2 [4,5], which are known to be active particularly against Gram-positive bacteria, BmKn2 also displayed good activity against Gram-negative bacteria such as E. coli and P. aeroginosa, showing at least three-fold higher activity against P. aeroginosa or more than two-fold higher activity against E. coli than IsCT or IsCT2 (Fig. 4B). Gram-negative bacteria differ from Gram-positive bacteria in having a smaller cell wall peptidoglycan layer, but possessing an outer membrane in addition to the common cytoplasmic membrane. This structure makes Gram-negative bacteria more difficult to be targeted and more intrinsically resistant to most antibiotics [9]. However, antimicrobial

cationic peptides have an amphipathic structure that allows them to bind and break across the outer membrane in a self-promoted uptake way [9]. BmKn2 have a higher IP value (11.65) and more positive net charges, hence are more active than IsCT or IsCT2. BmKn2 showed a higher activity against the Gram-positive bacterial strain than against the Gram-negative one. BmKb1 showed weaker activity against both Grampositive and Gram-negative bacteria than BmKn2 (Fig. 4A), IsCT or IsCT2. It seems to be not selective to the stain of bacteria. A number of reports showed that both the amphiphilic ␣-helical conformation and the net positive charges of the peptide are key determinant for the bacterial membrane-perturbing ability [7]. BmKb1’s IP value (9.67) and net charge (+1) are similar (or same) to that of IsCT (IP, 9.51; +1) or IsCT2 (IP, 9.67; +1), and they all have a typical ␣-helical structure, however, their antibacterial activities are quite different. It provides useful information for the designing of effective antimicrobial peptides.

4. Discussion In conclusion, here we first report four novel disulfidebridge-free venom peptides from scorpion. BmKa1 and BmKa2 are highly acidic, which are the first report of this kind. Secondary structure prediction showed they contain 100 and 62% coil structure, respectively. With such structure, the two peptides seem to lack any ability to target any receptor or whole cell. Do they only act as regulators of venom liquid’s pH value in scorpion? It is very fascinating to further investigate their functions. BmKb1 and BmKn2 are two novel cationic, ␣-helical antimicrobial peptides. The antibacterial activity of BmKn2 is much stronger than that of BmKb1 (Fig. 4A). Considering the fact that the

X.-C. Zeng et al. / Peptides 25 (2004) 143–150

149

(A)

100 90 80 70 60 50 40 30 20 10 0

S.

au re us M .l ut eu B. s su bt illi s E P. .c ae ol i ro gi no sa

BmKn2 BmKb1

(B)

80 BmKn2 IsCT IsCT2

70 60 50 40 30 20 10

gi no sa

co li E.

P. ae ro

ub B. s

S.

au re

us

til lis

0

Fig. 4. Comparison of antibacterial activity of BmKn2 with that of BmKb1 (A), or IsCT and IsCT2 (B).

Fig. 3. Helical wheel diagram of BmKb1 (A) and BmKn2 (B). Hydrophobic residues are given in blue letters and boxed; hydrophilic ones are printed in red letters. (For interpretation of the references to color in the figure legend, the reader is referred to the web version of this article.)

classification of scorpion venom peptides is based on the peptide length and primary structure similarity, BmKb1 and BmKn2 could represent two distinct subfamilies of ␣-helical antimicrobial peptides. BmKn2 together with BmKn1, IsCT and IsCT2 form a new class of antimicrobial peptides from scorpion, of which BmKn2 has the strongest activity (Fig. 4B). They have a common post-transcriptional processing and amidation mechanism. BmKb1 represent another novel class of antimicrobial to be discovered. Al-

though the primary structures of BmKa1, BmKa2, BmKb1 and BmKn2 are quite different, their precursor structures share very similar features suggesting they might have a common ancestor in the evolutionary process. It is worthy to mention that we also got an isoform of BmKb1 cDNA (referred as to BmKb1∗ , GenBank accession number: AF159979, sequence not shown here) while we cloned the BmKb1 cDNA. The difference between the two isoforms is that the TAT in positions 217–219 in BmKb1 cDNA mutated into TAA (stop codon) in BmKb1∗ cDNA, which resulted in a premature stop codon, such that a truncated premature peptide: FLFSLIPSAISGLISAFK GRRKRDLNG (the italic residues are processing signal). This genetic polymorphism suggests the disulfide bridge-free venom peptide is undergoing a divergence evolution. More works need to be done to answer the questions: (1) can this premature peptide be normally processed and matured? (2) Is it an evolutionary link between two new kind of peptides or just evolutionary garbage?

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