Identification and structural analysis of the antimicrobial domain in hipposin, a 51-mer antimicrobial peptide isolated from Atlantic halibut

Biochimica et Biophysica Acta 1699 (2004) 221 – 227 www.bba-direct.com

Identification and structural analysis of the antimicrobial domain in hipposin, a 51-mer antimicrobial peptide isolated from Atlantic halibut Gunn Alice Birkemo a,*, Dimitris Mantzilas a, Torben Lu¨ders b, Ingolf F. Nes b, Jon Nissen-Meyer a a

Program for Biochemistry and Molecular Biology, Department of Molecular Biosciences, University of Oslo, Oslo, Norway b Department of Chemistry, Biotechnology and Food Science, Agricultural University of Norway, A˚s, Norway Received 4 November 2003; received in revised form 27 February 2004; accepted 2 March 2004 Available online 20 March 2004

Abstract Hipposin is a potent 51-mer antimicrobial peptide (AMP) from Atlantic halibut with sequence similarity to parasin (19-mer catfish AMP), buforin I (39-mer toad AMP), and buforin II (an active 21-mer fragment of buforin I), suggesting that the antimicrobial activity of these peptides might all be due to a common antimicrobial sequence motif. In order to identify the putative sequence motif, the antimicrobial activity of hipposin fragments against 20 different bacteria was compared to the activity of hipposin, parasin and buforin II. Neither parasin nor the 19-mer parasin-like fragment HIP(1 – 19) (differs from parasin in only three residues) that is derived from the N-terminal part (residues 1 – 19) of hipposin had marked antimicrobial activity. In contrast, the fragment HIP(16 – 36) (identical to buforin II) that is derived from the middle part of hipposin (residues 16 – 36) had such activity, indicating that this part of hipposin contained an antimicrobial sequence motif. The activity was enhanced when the parasin-like N-terminal sequence was also present, as the fragment HIP(1 – 36) which consists of residues 1 – 36 in hipposin was more potent than HIP(16 – 36). Extending HIP(1 – 36) with three C-terminal residues—thereby constructing the buforin I-like peptide HIP(1 – 39) (differs from buforin I in only three residues)—increased the activity further. Also, the presence of the C-terminal part of hipposin (residues 40 – 51) increased the activity, as hipposin was clearly the most potent of all the peptides that were tested. Circular dichroism structural analysis of the peptides revealed that they were all non-structured in aqueous solution. However, trifluoroethanol and the membrane-mimicking entities dodecylphosphocholine micelles and negatively charged liposomes induced (amphiphilic) a-helical structuring in hipposin. Judging from the structuring of the individual fragments, the tendency for a-helical structuring appeared to be greater in the C-terminal and the buforin II-like middle region of hipposin than in the parasin-like N-terminal region. D 2004 Elsevier B.V. All rights reserved. Keywords: Antimicrobial peptide; Histone; Halibut; Hipposin

1. Introduction Gene-encoded, ribosomally synthesized antimicrobial peptides (AMPs) are widely distributed in nature, being produced by prokaryotes, plants, and a wide variety of animals, both vertebrates and invertebrates [1– 3]. These peptides apparently represent an important defense against microorganisms. AMPs usually contain between 20 and 60 amino acid residues and they are often cationic and amphiphilic or hydrophobic, which reflects the fact that many of these peptides kill their target cells by permeabilizing the * Corresponding author. Tel.: +47-22-85-66-09x73-02; fax: +47-22-8544-43. E-mail address: [email protected] (G.A. Birkemo). 1570-9639/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2004.03.001

cell membrane. Their positive charge presumably facilitates interactions with the negatively charged bacterial phospholipid-containing membranes and/or acidic bacterial cell walls, whereas their amphiphilic or hydrophobic character enables membrane permeabilization. Examples of AMPs are the cecropins found in insects [4], the magainins found in frogs [5], and the defensins found in mammals [6,7]. The skin and skin mucus of several fish species have been shown to contain AMPs. These include the 33-mer pardaxin of Red Sea Moses sole [8 – 11], the 25-mer pleurocidin of winter flounder [12,13], the 19-mer parasin of catfish [14], the C-terminally amidated 22-mer moronecidin of hybrid striped bass [15], and the recently identified 51-mer hipposin of Atlantic halibut [16]. Other characterized AMPs from fish are misgurin, a 21-residue

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peptide isolated from homogenized mudfish [17], and bass hepcidin, a cysteine-rich 21-residue member of the hepcidin family isolated from bass gill extracts [18]. These peptides kill a wide range of Gram-positive and Gramnegative bacteria, apparently by permeabilizing the targetcell membrane. Some of the peptides are also active against fungi [14,17]. Only pardaxin has significant hemolytic activity, which might limit its applications as an antimicrobial agent [11]. Some of the AMPs isolated from fish show sequence similarities to AMPs isolated from other organisms. Pleurocidin is similar to ceratotoxin and dermaseptin isolated from medflies and frogs, respectively [19,20]. The fish peptides parasin and hipposin show sequence similarities to each other and to buforin I [16], a 39-residue AMP isolated from Asian toad [21,22], as all three peptides are derived from the N-terminal region of histone H2A. The presence of common sequence motifs in hipposin and the smaller AMPs, parasin and buforin I, suggests that the antimicrobial activity of hipposin is due to only a part of the sequence of hipposin. In this study, we have examined the activity and secondary structure of peptide fragments derived from different parts of hipposin and located an antimicrobial region in the middle of hipposin.

2. Materials and methods 2.1. Microorganisms The following bacteria were used for testing the antimicrobial activity of hipposin, hipposin fragments, and parasin I. Halibut pathogens Vibrio anguillarum HI-644, V. anguillarum HI-610 and Vibrio salmonicida HI-651 were isolated from salmon, cod and halibut, respectively, identified and kindly provided by Øyvind Bergh, Institute of Marine Research, Bergen, Norway. Aeromonas salmonicida subsp. salmonicida 1851/97 V 3819, V. anguillarum 1190/ 97(3767) and Yersina ruckeri V3775 were obtained from the Section for Fish Health at the National Veterinary Institute in Oslo, Norway. Bacillus thuringiensis (ATCC 10792), Bacillus subtilis (ATCC 6633), Pseudomonas flourescences F283-V5, Pseudomonas sp. 92, Staphylococcus

aureus (ATCC 6538), Staphylococcus epidermis (ATCC 14990) and Listeria ivanovii Li4 were obtained from the ˚ s, Norway. EscherNorwegian Food Research Institute, A ichia coli DH5a, E. coli pHK22 Col.V, E. coli (ATCC 14763), Enterococcus faecalis LMGT2708, Enterococcus avicum LMGT2810, Shigella soneii (ATCC 11060), and Lactobacillus corenyformis subsp. torquens LMGT2308 were obtained from the Laboratory of Microbial Gene ˚ s. Except Technology, Agricultural University of Norway, A for L. corenyformis, V. anguillarum HI-644, V. anguillarum HI-610 and V. salmonicida HI-651, these bacteria were all cultured in 3% (w/v) trypticase soy broth (TSB) at 37 jC. V. anguillarum HI-644, V. anguillarum HI-610 and V. salmonicida HI-651 were cultured in TSB containing 1% (w/v) NaCl at 15– 18 jC, while L. corenyformis was cultured in de Man, Rogase, Sharpe broth (MRS) at 30 jC. All strains were stored at
73 jC until use. 2.2. Peptide preparation and determination of peptide concentration Hipposin and fragments of hipposin (Fig. 1), parasin and pleurocidin were synthesized and partly purified at the University of Newcastle, Molecular Biology Unit, UK. The peptides were purified to apparent homogeneity (as judged by analytical reversed phase chromatography and mass spectrometry analyses) by chromatography on a C18 reversed phase column (Vydac, 218TP54; 4.6  250 mm) ¨ KTA-chromatography system (Amersham Biousing the A sciences). The column was equilibrated with 0.1% TFA and the peptides were eluted from the column with a gradient of 0 – 45% (v/v) isopropanol containing 0.1% (v/v) TFA. The peptides were detected by measuring the ultraviolet absorbance at 214, 254 and 280 nm. The purity and/or identity of the peptides were verified by analytical reversed phase chromatography on a Sephasil C18 column (Amersham Biosciences) using the SMART system (Amersham Biosciences), by mass spectrometry using a matrix-assisted laser desorption ionization Voyager-DERP mass spectrometer (Perseptive Biosystems) with a-cyano-4-hydroxycinamic acid as matrix, and by amino acid composition analysis. The amino acid composition analysis was performed at the Biotechnology Center, University of Oslo

Fig. 1. The amino acid sequence of hipposin [16], fragments of hipposin, parasin [14] and buforin I [22] and II [32]. The gray shading indicates basic residues and there are no acidic residues. The black shading indicates residues that differ from the corresponding residues in hipposin, but note that residue 1 in parasin is also basic. Only the first and last residues are shown for the hipposin fragments, since their sequences are identical to the corresponding sequence in hipposin.

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(Oslo, Norway), as described by Martı´nez-Cuesta et al. [23]. Briefly, the lyophilized peptides were hydrolyzed in 6 M HCl in a vacuum at 110 jC for 24 h, and the analysis was performed on an Applied Biosystems Amino Acid Analyzer. Peptide concentrations were determined by amino acid composition analysis. For peptides with UV absorption at 280 nm, the peptide concentrations were confirmed by measuring the UV absorption at 280 nm and converting it to the peptide concentration using molecular extinction coefficients, calculated from the contribution of individual amino acid residues. 2.3. Radial diffusion antimicrobial activity assay The antimicrobial activity of the fragments was determined by the radial diffusion assay as described by Lehrer et al. [24]. A 20 ml culture of target cells in mid-logarithmic phase was washed with 10 mM sodium phosphate, pH 7.4, and resuspended in 10 ml of the same buffer. A cell suspension containing 1  106 bacterial colony-forming units (CFUs) was added to 6 ml of under-layer agar (10 mM sodium phosphate, 0.06% (w/v) TSB (MRS for L. corenyformis), 1% (w/v) agarose, pH 6.5) and the mixture was poured into a Petri dish. Samples containing 4, 2, 1 and 0.5 nmol of each peptide were lyophilized and resuspended in 4 Al 0.01% acetic acid, resulting in samples with peptide concentrations of 1.0, 0.5, 0.25 and 0.13 mM, respectively. Each sample was then added to a 3-mm well that was made on the solidified under-layer agar. After incubation for 3 h at 18, 30 or 37 jC, the under-layer agar was covered with a nutrient-rich top-agar overlay (6% (w/v) TSB (MRS for L. corenyformis), 1% (w/v) agarose) and incubated overnight at 25 jC (18 jC for V. salmonicida HI-651 and V. anguillarum HI-644 and HI-610). Antimicrobial activity was determined by observing the zone of suppression of bacterial growth around the 3-mm diameter wells. Pleurocidin (0.05 mg/ml, 4 Al) was used as a positive control, and acetic acid (0.01%, 4 Al) was used as a negative control. 2.4. Liposome preparation Single-bilayer phospholipid vesicles were prepared essentially according to the procedure of Batztri and Korn [25]. Twenty-four micromoles of 1-palmitoyl-2-oleoyl-snglycero-3-[phospho-L-serine] (POPS; Avanti Polar Lipids, Inc., Alabama USA) or 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC; Avanti Polar Lipids) dissolved in chloroform was carefully dried under a stream of ultra-pure nitrogen. The dried lipids were redissolved in 1 volume of absolute ethanol and dried again. Subsequently, the lipids were redissolved in 200 Al of absolute ethanol and slowly (about 100 Al/min), and at constant speed, injected into 4 ml of 10 mM potassium phosphate (pH 7.4) at room temperature. The ethanol was removed by dialysis against 10 mM potassium phosphate (pH 7.4).

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2.5. Circular dichroism Circular dichroism (CD) spectra were recorded by using a Jasco J-810 spectropolarimeter (Jasco International Co., Ltd., Tokyo, Japan) calibrated with ammonium D-camphor10-sulfonate (Icatayama Chemicals, Tokyo Japan). Measurements were performed with 1.4 mM liposomes or various concentrations of TFE (0– 50% (v/v)) or DPC (0– 16 mM) at 23 jC by using a quartz cuvette (Starna, Essex England) with a path length of 0.1 cm. All the measurements were performed with a peptide concentration of 0.10 mg/ml in 10 mM potassium phosphate buffer (pH 7.4). Samples were scanned five times at 20 nm/min with a band width of 1 nm and a response time of 1 s, over the wavelength range 190– 260 nm. The data were averaged and the spectrum of a sample-free control sample was subtracted. The a-helical content of the various peptides was calculated after smoothing (means-movement, convolution width 5) from mean residual ellipticity at 222 nm ([h]222) using the formula fH ¼ ½h222 =½
40000ð1
2:5=nÞ where fH and n represent the a-helical content and the number of peptide bonds, respectively [26]. All measurements were conducted at least twice.

3. Results 3.1. The peptides that were studied In order to identify the region(s) of hipposin that exerts antimicrobial activity, the activity of various synthetic fragments of hipposin was determined and compared to the activity of hipposin and parasin. Unacetylated hipposin (which has the same activity as the natural acetylated form [16]), parasin, and four fragments of hipposin were all synthesized and tested for activity. The peptide samples were all judged by analytical reversed phase chromatography to be more than 90% pure, and the purity and identity of the peptides were verified by mass spectrometry and amino acid composition analysis (results not shown). The four fragments derived from hipposin were: HIP(1– 19), a 19-mer fragment with a sequence identical to that of the N-terminal region of hipposin; HIP(16 –36), a 21-mer fragment with a sequence identical to that of the middle part of hipposin; HIP(1 –36), a 36-mer fragment with a sequence identical to that of the first (from the N-terminus) 36 residues in hipposin; and HIP(1 – 39), a 39-mer fragment with a sequence identical to that of the first (from the N-terminus) 39 residues in hipposin (Fig. 1). These fragments were chosen on the basis of their sequence similarities to other AMPs. HIP (1 –19) is the hipposin analogue of parasin (the two peptides differ only in three residues) and similarly, HIP(1– 39) and HIP(16– 36) are the hipposin-analogues of buforin I and II,

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peptide concentrations using 20 different Gram-negative and Gram-positive bacteria (Table 1). Consistent with earlier results [16], hipposin was active at all concentrations tested against 19 of the 20 target cells, the insensitive bacteria being Y. ruckeri (V3775) (Table 1). Parasin and its hipposinanalogue, HIP(1– 19), had similar potencies and activity spectra (Table 1), indicating that the sequence differences between these two peptides at positions 1, 6 and 10 (Fig. 1)

respectively (Fig. 1). Buforin II is an active fragment derived by cleavage of buforin I with endoproteinase Lys-C [22]. 3.2. Antimicrobial activity of the hipposin fragments, hipposin and parasin The antimicrobial activity of hipposin, parasin and the four hipposin fragments was determined at four different

Table 1 Antimicrobial activity of hipposin analogues and parasin Microorganism

Zone-widtha (mm) Hipposin (mM)

Parasin (mM)

HIP(1 – 19) (mM)

1.0 0.5 0.25 0.13 1.0 0.5 0.25 0.13 1.0 Gram-positive Bacillus subtilis Bacillus thuringiensis Enterococcus faecalis Enterococcus avicum Lactobacillus corenyformis Listeria ivanovii Staphylococcus aureus Staphylococcus epidermis Gram-negative Aeromonas salmonicida Escherichia coli ATCC 14763 Escherichia coli DH5a Escherichia coli pHKK22 Pseudomonas fluorescences Pseudomonas sp. Shigelli soneii Vibrio anguillarum 1190/97 Vibrio anguillarum (HI-644) Vibrio anguillarum (HI-610) Vibrio salmonicida (HI-651) Yersina ruckeri a

0.5

HIP(16 – 36) (mM)

0.25 0.13 1.0

0.5

HIP(1 – 36) (mM)

0.25 0.13 1.0

0.5

HIP(1 – 39) (mM)

0.25 0.13 1.0

0.5 0.25 0.13

7.5 6.3 5.3 2.5 2.0 1.8

4.8 1.3

3.8b 2.8b 2.5b 2.3b 4.0b 3.0b 2.3b 1.5b 3.5 3.0 2.5 NZc NZ NZ NZ NZ NZ NZ NZ NZ NZ NZ

2.0 NZ

5.8 5.3 4.8 4.3 0.3d 0.0d 0.0d 0.0d

5.8 5.5 5.0 2.3 2.0 1.8

4.5 0.8

4.0 3.3 2.8

2.0

3.0b NZ NZ

NZ

3.3b NZ NZ

NZ

NZ NZ NZ

NZ

0.8d 0.5d 0.5d 0.3d

2.3 2.0 1.5

1.3

5.8 5.5 5.0

4.5

NZ NZ NZ

NZ

NZ NZ NZ

NZ

4.0

3.5

2.8

2.3

3.3

3.0

2.8

2.3

3.0 2.5 2.3

1.8

9.5 8.8 8.0

6.5

1.5b 1.0b 0.5b 0.3b 1.0b 0.5b 0.3b 0.3b 8.3

6.8

5.8

3.8

9.5

9.0

8.0

7.3

10.0 9.3 8.5

7.3

5.0 4.5 4.0 5.3 4.5 4.0

3.5 3.8

NZ NZ NZ NZ NZ NZ

NZ NZ

NZ NZ NZ NZ NZ NZ

NZ NZ

1.8 1.0 NZ NZ NZ NZ

NZ NZ

4.5 4.8

4.0 4.3

3.5 4.0

3.3 3.8

4.8 4.3 3.8 5.0 4.8 4.3

3.5 3.8

4.5 4.0 3.5

3.0

NZ NZ NZ

NZ

NZ NZ NZ

NZ

1.0

0.8

NZ

NZ

3.3

2.8

2.3

1.8

3.3 2.8 2.5

2.0

3.0 2.8 2.5

2.3

NZ NZ NZ

NZ

NZ NZ NZ

NZ

1.5

0.8

NZ

NZ

0.5d 0.5d 0.3d 0.0d

3.0 2.8 2.5

1.8

7.5 6.8 5.8

5.0

6.3b 5.5b 4.4b 3.8b 6.0b 5.5b 4.0b 3.8b 4.3

3.3

3.0

2.5

6.0

5.3

4.5

4.0

6.0 4.8 3.8

3.5

6.3 5.5 4.8

4.5

3.3b 3.0b 2.5b 2.0b 3.0b 2.8b 2.0b 2.0b 3.8

3.3

2.8

2.0

5.0

4.5

4.3

4.0

5.3 5.0 4.5

4.0

7.3 6.8 6.0

5.0

4.5b 3.5b 3.3b 2.8b 4.8b 3.5b 3.0b 2.5b 4.3

3.8

3.3

2.8

5.8

5.0

4.3

3.8

6.0 4.8 4.0

3.5

5.5 4.8 4.5

3.8

2.3b 2.0b 1.5b 1.3b 2.0b 2.0b 1.5b 0.8b 4.5

4.3

3.0

2.5

4.5

4.0

3.5

3.3

5.8 5.0 4.3

3.5

5.5 5.0 4.5 4.3 3.8 3.3 5.0 4.8 4.3

3.8 2.5 4.0

2.0b 1.5b 1.0b 0.8b 1.3b 1.0b 0.8b 0.5b 4.5 NZ NZ NZ NZ NZ NZ NZ NZ 1.5 NZ NZ NZ NZ NZ NZ NZ NZ 1.0

3.8 2.9 NZ NZ NZ NZ

2.0 NZ NZ

4.5 4.0 3.8 3.3 0.5d 0.3d 0.0d 0.0d 4.8 4.3 3.8 3.5

5.0 4.8 4.0 4.5 3.8 3.3 5.8 4.8 4.3

3.5 2.3 3.5

5.5 5.0 4.3

3.8

1.3b NZ NZ

NZ

NZ NZ NZ

NZ

1.0

NZ NZ

NZ

3.5

3.3

3.0

2.3

4.0 3.5 3.0

2.8

6.8 5.8 5.0

4.5

NZ NZ NZ

NZ

NZ NZ NZ

NZ

4.5

3.5

3.0

2.8

5.0

4.3

4.0

3.8

6.3 5.5 4.5

4.3

4.0 3.8 3.3

2.8

NZ NZ NZ

NZ

NZ NZ NZ

NZ

1.8

1.3

NZ

NZ

4.3

3.8

3.0

2.3

4.0 3.0 2.3

1.8

NZ NZ NZ

NZ

NZ NZ NZ

NZ

NZ NZ NZ

NZ

NZ NZ NZ

NZ

NZ NZ NZ

NZ

NZ

NZ NZ

NZ

The zone-width is defined as the distance from the center of the well to the outer boarder of the zone minus the radius of the well (1.5 mm). If nothing else is indicated by superscript letters, the zones were clear and of the indicated diameter both after overnight and 48 h incubation. b Zone was hazy, but visible after overnight incubation. No visible zone after 48 h incubation. c NZ: No zone detected. d Clear zone surrounded by a hazy zone after overnight incubation. The number indicates the diameter of the clear zone. The diameter of the hazy zone was 2 – 3 mm. The clear zone was still clear after 48 h, whereas the surrounding hazy zone was not.

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HIP(1– 39) that differs from buforin I in only three residues) increased the activity further. HIP(1 –39) had more than 10 times higher activity against 4 (B. thuringiensis, E. faecalis, A. salmonicid, and S. soneii) of the target cells than HIP(1– 36), and the potency towards the other cells was similar (within about 2-fold) for the two fragments (Table 1). Also the presence of the C-terminal part of hipposin (residues 40– 51) increased the activity, as hipposin was clearly the most potent of all the peptides that were tested. Hipposin was 5– 10 times more potent than HIP(1– 39) towards 6 (E. faecalis, E. avicum, S. epidermis, E. coli ATCC 14763, E. coli pHK22, V. anguillarum HI-644) of the 20 target cells and had similar potency (within about 2-fold) toward the other cells (Table 1). Y. ruckeri was insensitive to all the peptides at all peptide concentrations that were tested (Table 1). Fig. 2. CD spectra of hipposin (0.10 mg/ml) in the presence of 0 – 50% (v/v) TFE. Each sample was scanned five times at 20 nm/min with a band width of 1 nm and a response time of 1 s over the wavelength range 190 – 260 nm. The CD measurements were performed at 23 jC.

did not affect the activity. Somewhat surprisingly, however, neither parasin nor HIP(1– 19) showed marked antimicrobial activity (Table 1), suggesting that the N-terminal region is not the major antimicrobial domain of hipposin. Although inhibitory zones were detected at all peptide concentrations tested against 7 of the 20 target cells after overnight incubation (Table 1), all of these zones were hazy—in contrast to the clear inhibitory zones obtained with the other active peptides—and no visible zones were detected after incubation for a total of 48 h. Parasin and HIP(1 – 19) thus only slightly delayed the growth of the sensitive cells. In contrast to the N-terminally derived parasin-like HIP(1 – 19) fragment, the HIP(16 – 36) fragment derived from the middle of hipposin had marked antimicrobial activity. HIP(16 – 36) produced clear zones (no visible bacterial growth) at all concentrations tested against 9 of 20 target cells even after 48 h incubation and only against 4 of 20 target cells was no activity detected (Table 1). The middle part of hipposin (residues 16 – 36) thus contained an antimicrobial sequence motif. The activity was, however, enhanced when the parasin-like N-terminal sequence was also present, as the fragment HIP(1– 36) that consists of residues 1 –36 in hipposin was much more potent than HIP(16 –36). HIP(1 – 36) had detectable activity against three target cells (B. thuringiensis, E. faecalis and S. aureus) that were insensitive to HIP(16– 36) at all peptide concentrations tested (Table 1). Moreover, HIP(1 –36) was at least 10 times more active than HIP(16 – 36) against 7 (B. subtilis, L. ivanovii, S. epidermis, E. coli DH5a, V. salmonicida HI-651, and V. anguillarum 1190/97(3767) and HI-644), and about 5– 10 times more active against 4 (L. corenyformis, E. coli ATCC 14763, E. coli pHK22, and V. anguillarum HI-610) other target cells (Table 1). Only against 1 of the 20 target cells (Y. ruckeri) was HIP(1– 36) completely inactive at all concentrations tested (Table 1). Extending HIP(1 –36) with three C-terminal residues (thereby constructing the buforin I-like peptide

3.3. Circular dichroism The structures of hipposin and its fragments were analyzed by CD spectrometry under various conditions. The CD spectra of all the peptides in pure water and in 10 mM potassium phosphate (pH 7.4) were characteristic of a nonstructured conformation (Fig. 2), with a-helical contents of not more than 1– 4% (Table 2). TFE induces and stabilizes ahelical structure in peptides that have an intrinsic tendency to adopt this type of secondary structure [27 – 29]. In the presence of TFE, hipposin yielded CD spectra typical for an a-helical peptide (Fig. 2), with a calculated helical content of about 48% at 50% TFE (Table 2). DPC micelles and anionic liposomes (POPS), but not cationic liposomes (POPC), also induced helical structuring in hipposin (Table 1). The helical content appears to be in the middle and C-

Table 2 Helical content of hipposin analogues under various solvent conditions Solvent and concentration a-Helical content of Hipposin HIP HIP HIP HIP (1 – 19) (16 – 36) (1 – 36) (1 – 39) (%) (%) (%) (%) (%) Trifluoroethanol (% v/v) 0.0 25 50.0

4 32 48

1 3 7

2 10 13

3 18 31

2 21 32

Dodecylphosphocholine (mM) 0.0 8.0 12.0 16.0

4 21 21 21

1 1 1 2

2 11 10.5 12

3 8 10 10

2 11 13 13

4

1

2

1

3

20 4

7 1

11 2

9 3

12 2

Potassium phosphate (10 mM) POPSa (1.4 mM) POPCb (1.4 mM) a b

POPS = 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine]. POPC = 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

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terminal parts of hipposin, since HIP(1– 19) had compared to the other peptides a relative low helical content (Table 2).

4. Discussion Published data on the activities of parasin, hipposin and buforin I indicate that these three peptides have similar antimicrobial potencies, despite their different lengths [14,16]. This could possibly be attributed to a common antimicrobial domain, since the three peptides show sequence similarities due to the fact that they are all derived from the N-terminal region of histone H2A, a protein that was recently shown to also possess antimicrobial activity [30]. Of the 19 residues in parasin, 18 and 16 are identical to those found in the N-terminal region of, respectively, buforin I and hipposin (Fig. 1). Of the 39 residues in buforin I, 36 are identical to those found in the N-terminal region of hipposin (Fig. 1). One might, consequently, expect that the antimicrobial activity of hipposin and buforin I is due to the parasin-like sequence present in their N-terminal region. However, neither parasin nor HIP(1– 19), which is derived from the parasin-like N-terminal part of hipposin, had marked antimicrobial activity. This was somewhat surprising, since parasin has been reported to be very potent against a wide spectrum of microorganisms; about 10 – 100 times more potent than magainin II and 2– 8 times more potent than buforin I [14]. This discrepancy does not seem to be due to the use of different antimicrobial activity assays, since similar radial diffusion assays were used in this and previous studies [14]. Moreover, no marked salt concentration-dependent variations (from about 40 to 240 mM NaCl) in the activity of these peptides were observed (data not shown). Buforin II is a 21-mer fragment generated by cleaving buforin I with endoproteinase Lys-C and it corresponds to residues 16– 36 in buforin I. Buforin II was found to be very active, in fact about twice as potent as buforin I [22], indicating that the antimicrobial activity of buforin I is localized to its C-terminal half rather than its N-terminal parasin-like sequence. Consistent with these results, we found that antimicrobial activity was associated with HIP(16– 36), whose sequence is identical to that of buforin II (Fig. 1), thus localizing an antimicrobial domain to the middle part of hipposin (residues 16 –36). However, somewhat contrary to what was expected from earlier results showing that buforin II is more potent than buforin I [22], we found that HIP(16 –36) was markedly less potent than the buforin I-like HIP(1– 39) fragment (differs from buforin I in only three residues; Fig. 1) and hipposin. Thus, additional sequences, both in the parasin-like N-terminal part of hipposin (since HIP(1– 36) was more potent than HIP(16– 36)) and in the C-terminal part of hipposin (since HIP(1– 39) and especially hipposin were more potent than HIP(1 – 36)), are also important for the activity as they increase the potency and broaden the antimicrobial spectra.

There was no synergy between the 19-mer and 21-mer fragments when used together as loose peptides (data not shown). Hipposin is similar to other AMPs in being very cationic (15 of 51 residues are basic and there are no acidic residues) and in that it may become amphiphilic. An axial projection diagram with 170j rotation per residue reveals that the Nterminal parasin-like region from residue 1 to 19 will become amphiphilic if it adapts a h-strand secondary structure [14], whereas an Edmundson a-helical wheel diagram reveals that the region from residue 27 to residue 48 will become amphiphilic if it adapts an a-helical structure (Fig. 3). The middle and C-terminal parts of hipposin indeed had a greater intrinsic tendency to adopt a helical structure than the N-terminal parasin-like sequence when exposed to membrane-mimicking environments. Helical structuring of the middle part of hipposin is consistent with data on the structure of buforin II [31,32]. Buforin II, whose sequence is identical to that of the middle part of hipposin, forms an amphiphilic structure consisting of an extended helical region in residues 5 to 10, a hinge in residue 11, and a C-terminal regular a-helical region from residue 12 to 21 [31,32]. It is likely that the middle part of hipposin adopts a similar or identical structure in view of their common sequence. The cationic and apparent amphiphilic character of hipposin suggests that hipposin may—as is the case for most AMPs—readily associate with and possibly permeabilize target cell membranes. The cationic character would enable the peptide to bind to the anionic surface of target cells and the amphiphilic character would then enable the peptide to interact with and permeabilize the cell membrane. However, mode of action studies suggest that buforin II kills target cells not by membrane permeabilization, but rather by penetrating into cells and then binding to DNA and/or RNA [31,33]. The cationic character of most AMPs should enable them to bind to DNA and RNA if the peptides penetrate the

Fig. 3. Edmundson a-helical wheel representation of the amphiphilic region in hipposin. The amphiphilic region starts with residue 27 and ends with residue 48. The black areas indicate the hydrophobic residues. Glycine is considered a neutral residue and is shown with both black and white circles.

G.A. Birkemo et al. / Biochimica et Biophysica Acta 1699 (2004) 221–227

cell membrane and it will be of great interest to determine if this novel mechanism is the manner by which other AMPs, including hipposin and its active peptide fragments, kill their target cells.

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Acknowledgements [18]

This work was supported by a grant from the Research Council of Norway. [19]

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