Enhancement of the activity of scorpion short-chain antimicrobial peptides by addition of basic residues at the C-terminus

Abstract

Scorpion venom contains antimicrobial peptides (AMPs). These AMPs are classified into three families based on peptide length: long-chain (>35 residues), intermediate-chain (20–35 residues), and short-chain (13–19 residues) AMPs. Previously, we identified both short- and intermediate-chain AMPs from the venom of the scorpion Isometrus maculatus. A comparative analysis of their antibacterial activities revealed that short-chain AMPs exhibit relatively weaker activity than intermediate-chain AMPs. A structural comparison indicated that intermediate-chain AMPs possess a longer C-terminal region enriched in basic residues, a feature absent in short-chain AMPs. Removal of this C-terminal basic region from intermediate-chain AMPs resulted in a marked loss of antibacterial activity. Conversely, the addition of basic residues at the C-termini of short-chain AMPs significantly enhanced their activity. These results demonstrate that basic residues in the C-terminal region of scorpion intermediate-chain AMPs are crucial for antibacterial activity.

Introduction

Scorpions use venom to capture prey and defend themselves against predators. Scorpion venoms comprise a rich repertoire of bioactive peptides, among which neurotoxic and antimicrobial peptides represent two major functional classes.¹⁾ The neurotoxic peptides primarily target ion channels, such as Na⁺, K⁺ and Ca²⁺ channels, thereby altering neuronal excitability and leading to paralysis or other neurophysiological effects in prey.²⁾ Scorpion venom also contains antimicrobial peptides (AMPs).³⁾ Some of these AMPs not only exhibit insecticidal activity but also enhance the activity of other neurotoxic peptides synergistically in the venom.⁴⁾ Scorpion AMPs are divided into several families based on their chain length.⁵⁾ The long-chain family comprises peptides with more than 35 residues, the intermediate-chain family includes peptides containing 20–35 residues, and the short-chain family consists of peptides with 13–19 residues. These peptides typically adopt an amphipathic α-helical structure, in which one side of the peptide is hydrophilic and the other is hydrophobic; this feature is necessary for interaction with cell membranes and subsequent pore formation.⁶⁾ Due to their diverse structures and biological functions, scorpion AMPs serve as useful molecular leads for the development of pesticides and therapeutic agents.^7–9) However, some AMPs exhibit toxicity toward mammalian cells, with hemolytic activity being a particular concern.¹⁰⁾ Therefore, the rational design of AMPs that enhance antimicrobial potency while minimizing hemolytic activity is essential for their practical application.

In our previous study, we identified three AMPs (Im-4, Im-5, and Im-6) from the venom of the scorpion Isometrus maculatus (Fig. 1).¹¹⁾ Im-4 and Im-6 are short-chain AMPs consisting of 17 and 16 residues, respectively, whereas Im-5 consists of 25 residues and belongs to the intermediate-chain family. Compared with Im-5, Im-4 and Im-6 exhibited relatively weak antibacterial activity, particularly against the Gram-negative bacterium Escherichia coli (Table 1). In contrast, Im-5 showed potent antibacterial activity against both Gram-negative (E. coli) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) with minimum inhibitory concentrations (MICs) in the low micromolar range (1–10 µM). Structural comparison of these peptides reveals that Im-5 possesses a longer C-terminal region enriched in basic residues, a feature not observed in Im-4 or Im-6. Accordingly, the higher antibacterial activity of Im-5 compared with Im-4 and Im-6 was attributed to its C-terminal basic residues.

Fig. 1. Amino acid sequences of scorpion short-chain and intermediate-chain AMPs. Basic residues are highlighted in red.

Table 1. Biological activities and physicochemical parameters of the synthesized peptides.

Peptide	Sequencea)	Antibacterial activity, MIC (µM)			Hemolysis EC50 (µM)	Net charge	Hydrophobicity	Hydrophobic moment
		E. coli	S. aureus	B. subtilis
Im-5	FLGSLFSIGSKLLPGVIKLFQRKKQ	10 b)	5 b)	1 b)	28b)	+5	0.555	0.432
Im-5(−)	FLGSLFSIGSKLLPGVIK-NH2	>100	>100	100	NTd)	+3	0.768	0.493
Im-4	FIGMIPGLIGGLISAIK-NH2	>200b)	10b)	5b)	>30 (0.84%)b)	+2	0.907	0.597
Im-4(+)	FIGMIPGLIGGLISAIKGRRRR	13	6	1.5	43	+5	0.517	0.455
Im-6	FFFLPSLIGGLVSAIK-NH2	>100b)	50b)	2.5b)	>25 (2.7%)b)	+2	0.953	0.431
Im-6(+)	FFFLPSLIGGLVSAIKGRRRR	13	6	1.5	40	+5	0.534	0.324
AamAP2	FPFSLIPHAIGGLISAIK-NH2	120c)	48c)	NTd)	100–120c)	+2	0.850	0.395
AamAP2(+)	FPFSLIPHAIGGLISAIKGRRKR	20	5	5	>100 (12%)	+5	0.490	0.303

a) Additional sequences are underlined. b) Data from Ref. 11. c) Data from Ref. 13. d) NT: not tested.

In the present study, we investigated the effects of adding basic residues at the C-termini of short-chain AMPs, such as Im-4 and Im-6, on their antibacterial activity. In addition, the effects of this modification on their hemolytic activity were also examined.

Materials and methods

Peptide synthesis

Peptides were synthesized by the Fmoc-based solid-phase method using automated microwave-assisted synthesizer (Liberty Lite, CEM, Matthews, NC, USA), in which N,N′-diisopropylcarbodiimide and Oxyma Pure were used for coupling reactions. Rink Amide ProTide Resin (LL) and Fmoc-Arg(Pbf)-ProTide Resin (LL) (CEM) were used for the synthesis of C-terminal amide and acid peptides, respectively. The synthesized peptides were purified on a flash chromatography system Isolera Spektra (Biotage, Uppsala, Sweden). An RP column (Sfär, C18 D, 30 g, Biotage) was used for separation. The column was eluted with 0.1% trifluoroacetic acid (TFA) in H₂O and 0.1% TFA in CH₃CN at a flow rate of 25 mL/min using a linear gradient of 10–50% of CH₃CN over 25 min. Fractions containing a desired peptide were collected and lyophilized. The purity of the synthesized peptides was examined using LC/MS. The analysis was carried out on an LCMS-2020 mass spectrometer (Shimadzu, Kyoto, Japan).

Antibacterial activity

Antibacterial activity was evaluated against one Gram-negative bacterium, Escherichia coli NBRC 3972, and two Gram-positive bacteria, Staphylococcus aureus NBRC 13276 and Bacillus subtilis NBRC 3009 (NITE Biological Resource Center, Chiba, Japan) by a liquid growth inhibition assay. Each bacteria strain was cultivated overnight at 37°C in petri dish in aerobic conditions. The bacteria were then grown in liquid LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl). Minimal inhibitory concentrations (MICs) were determined using a 2-fold microtiter broth dilution assay. Aliquots of each sample (10 µL) were incubated with 90 µL of a suspension of a mid-logarithmic phase culture of bacteria in a 96-well plate at a starting absorbance of A₅₉₅=0.001 in LB medium for 20 hr at 37°C with continuous shaking. Inhibition of growth was monitored by measuring the absorbance at 595 nm using a Benchmark microplate reader (Bio-Rad, Hercules, CA, USA). Experiments were performed in triplicate and repeated at least three times. MICs are expressed as the lowest concentration causing 100% growth inhibition.

Hemolytic activity

Fresh sheep red blood cells (sRBCs) were washed three times with PBS (35 mM phosphate buffer and 150 mM NaCl, pH 7.2) by centrifugation at 2000×g for 5 min and resuspended in PBS. Aliquots of each sample in PBS (50 µL) were added to 50 µL of sRBC suspension [4% (v/v) in the final] in a microtube and incubated for 1 hr at 37°C. PBS or 0.1% Triton X-100 were used as the negative and positive controls, respectively. The samples were centrifuged at 2000×g for 5 min. The supernatant was transferred to a 96-well plate to monitor hemoglobin release by measuring the absorbance of the supernatant at 450 nm using a Benchmark microplate reader (Bio-Rad). The experiments were performed in triplicate and repeated three times. The hemolysis rate was calculated using the following equation, where A_P, A_T, and A₀ represent the absorbance of the peptide sample, the positive control, and the negative control, respectively.

Helical wheel projection

The helical wheel projection was generated using the online software HeliQuest (https://heliquest.ipmc.cnrs.fr) along with calculations of hydrophobicity and hydrophobic moment.

Results and discussion

Design of peptides

We first synthesized an Im-5 analog to investigate the role of the C-terminal basic region. In this analog, the LFQRKKQ sequence at the C-terminal region of Im-5 was removed, and the C-terminus was amidated, as observed in Im-4 and Im-6. This analog was designated Im-5(−) (Table 1). Next, we synthesized analogs of Im-4 and Im-6 to examine the effect of adding basic residues at their C-termini. Analysis of the precursor sequences of scorpion short-chain AMPs, including Im-4 and Im-6, revealed that their C-terminal propeptide regions contain sequences with four consecutive basic residues [RXXR (X=R or K)] (Table S1). This sequence is known as a cleavage motif for post-translational processing by furin.¹²⁾ Although mature scorpion short-chain AMPs in the venom lack this motif after processing, we hypothesized that adding this motif would confer an effect similar to that of the C-terminal region of Im-5. Accordingly, the GRRRR sequence was introduced at the C-termini of Im-4 and Im-6, and the resulting analogs were designated Im-4(+) and Im-6(+), respectively (Table 1). Furthermore, a similar modification was performed on another short-chain AMP, AamAP2, which was identified from the venom of Androctonus amoreuxi (Table 1).¹³⁾ AamAP2 has been reported to exhibit relatively weak antimicrobial activity against both E. coli and S. aureus. In this case, the GRRKR sequence was introduced at the C-terminus based on the precursor sequence, and the resulting analog was designated AamAP2(+).

Antibacterial activity

Im-5(−), which lacks C-terminal basic residues, showed no or only marginal antibacterial activity against the three bacterial species tested. In contrast, Im-4(+) and Im-6(+), which contain additional basic residues at the C-terminus, exhibited significant antibacterial activity comparable to that of Im-5 (Table 1). Specifically, Im-4(+) showed more than a 20-fold increase in activity against the Gram-negative bacterium E. coli, as well as 2- and 4-fold increases in activity against the Gram-positive bacteria S. aureus and B. subtilis, respectively. Similarly, Im-6(+) exhibited more than a 10-fold increase in activity against E. coli, and 10- and 2-fold increases in activity against S. aureus and B. subtilis, respectively. The addition of the basic residues at the C-terminus of AamAP2 also enhanced its antibacterial activity, consistent with the results observed for Im-4 and Im-6. AamAP2(+) exhibited a 6-fold increase in activity against E. coli, and 10-fold higher against S. aureus. Although antibacterial activity against B. subtilis has not previously been reported for AamAP2, AamAP2(+) also showed significant activity against B. subtilis.

Antibacterial activity of peptides is influenced by factors such as chain length, net charge, hydrophobicity, and amphiphilicity.^6,14,15) Changes in peptide length can affect the effectiveness of AMPs, as a sufficient length is required for a peptide to span the lipid bilayer and stabilize pore formation. Likewise, variations in net positive charge influence the electrostatic interaction of AMPs with anionic bacterial membranes. In the present study, the addition of basic residues at the C-termini of Im-4, Im-6, and AamAP2 significantly enhanced their antibacterial activity against both Gram-negative and Gram-positive bacteria. This modification increased the chain length and net charge but did not affect the hydrophobic moment (i.e., amphiphilicity); rather, it slightly decreased it (Table 1). This observation is supported by helical wheel projections of the modified peptides (Fig. 2), which show that the basic residues at the C-terminal region are not confined to the hydrophilic side but distributed across both faces of the helix. These findings suggest that the basic residues in the C-terminal region contribute to antibacterial activity through a mechanism other than enhancement of amphiphilicity.

Fig. 2. Comparison of helical wheel projections of scorpion AMPs with and without C-terminal basic residues. Arrows indicate the hydrophobic moment.

The importance of basic residues in the C-terminal region has also been investigated in other AMPs. ToAP2 is a peptide identified from the venom of the scorpion Tityus obscurus and shows high sequence similarity to Im-5 (Fig. 1).¹⁶⁾ It has been demonstrated that the substitution of two Lys residues in the C-terminal region of ToAP2 with Leu results in a loss of antifungal activity.¹⁷⁾ However, because two other amino acid residues in the central region were simultaneously substituted in this analog, it remains unclear whether the observed loss of activity is attributable specifically to replacement of the basic residues in the C-terminal region with neutral residues. Furthermore, the effect of this substitution on antibacterial activity has not yet been elucidated. Melittin, a peptide identified from bee venom, also contains a C-terminal basic region similar to that of Im-5 (Fig. 1).¹⁸⁾ This peptide is known to exhibit not only antibacterial activity but also hemolytic activity. It has been reported that removal of the basic residues in the C-terminal region of melittin reduces its cytolytic activity; however, the effect of this modification on antibacterial activity has not been clearly described.¹⁹⁾ Thus, the contribution of the C-terminal basic residues to antibacterial activity has not been fully elucidated. In the present study, we clearly demonstrated the importance of basic residues for antibacterial activity by removing the C-terminal basic region from Im-5 and by adding basic residues to the C-termini of Im-4 and Im-6. Regarding the functional role of C-terminal basic residues, previous studies have shown that they contribute to the initial membrane association in melittin.²⁰⁾ Similarly, the C-terminal basic residues of Im-5, Im-4(+), Im-6(+), and AamAP2(+) are likely to exert a comparable effect during the early stages of membrane interaction.

The basic sequence introduced at the C-termini of the short-chain AMPs was based on that of the propeptide region of their precursors, as described above. This region is absent from the precursor of intermediate-chain AMPs, including Im-5 (Table S1). The molecular diversity of venom peptides arises from single-base substitutions, deletions, insertions, and frameshifts in gene sequences.^21,22) Therefore, it is conceivable that, during evolution, intermediate-chain AMPs underwent mutations in the C-terminal propeptide region, resulting in the incorporation of basic residues at the C-terminus without removal of cleavage motifs such as RXXR.

Hemolytic activity

It is well known that an increase in the number of basic residues in AMPs can enhance both antibacterial and hemolytic activities.²³⁾ In this context, Im-5 exhibits relatively higher hemolytic activity than Im-4 and Im-6, suggesting that the basicity of the C-terminal region of Im-5 may contribute to its hemolytic activity. Therefore, the effects of adding basic residues at the C-termini of Im-4, Im-6, and AmAP2 on hemolytic activity were investigated (Table 1). Im-4(+) and Im-6(+) exhibited higher hemolytic activity than their respective parent peptides, with activity levels comparable to that of Im-5. In contrast, AmAP2(+) exhibited only marginal hemolysis (12%) even at 100 µM. Although the hemolytic activity of AmAP2 shown in Table 1 was taken from the literature,¹³⁾ the addition of basic residues at its C-terminal region did not appear to affect its hemolytic activity. Previous studies have reported that the position of proline residues in AMPs significantly influences hemolytic activity.^24,25) Unlike Im-4 and Im-6, AmAP2 contains an additional Pro residue in its N-terminal region, which might explain why AmAP2(+) did not exhibit high hemolytic activity despite the introduction of basic residues at its C-terminus. Further investigation is required to clarify the effects of the Pro residues on hemolytic activity in AMPs.

Conclusion

In this study, we aimed to elucidate the factors responsible for the differences in antibacterial activity between the short-chain and intermediate-chain AMPs in scorpion venom. Removal of the basic residues present in the C-terminal region of Im-5, an intermediate-chain AMP, resulted in an almost complete loss of antibacterial activity. In contrast, the addition of basic residues at the C-termini of the short-chain AMPs, such as Im-4, Im-6, and AmAP2, significantly enhanced their antibacterial activity. These findings indicate that the presence of basic residues in the C-terminal region of scorpion intermediate-chain AMPs is crucial for antibacterial activity, particularly against the Gram-negative bacterium E. coli. The addition of basic residues at the C-terminus also led to increased hemolytic activity in Im-4 and Im-6. On the other hand, no significant effect on hemolytic activity was observed following the addition of basic residues at the C-terminus of AmAP2. Further insight into the structural differences among these peptides may provide a rational strategy for designing AMPs with potent antimicrobial activity while minimizing toxicity to mammalian cells.

Electronic supplementary materials

The online version of this article contains supplementary material, which is available at https://www.jstage.jst.go.jp/browse/jpestics/