synthetic peptides corresponding to the β-hairpin loop of rabbit defensin np-2 show antimicrobial...

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Synthetic Peptides Corresponding to the b-Hairpin Loop of Rabbit Defensin NP-2 Show Antimicrobial Activity S. Thennarasu and R. Nagaraj 1 Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad - 500 007 (A.P.), India Received November 30, 1998 Mammalian defensins, a class of antibacterial pep- tides, are composed of 29 –35 amino acids with six cys- teines which form three disulfide bonds. Structural studies indicate a triple stranded b-sheet structure with a well defined b-hairpin loop at the C-terminal region. It is demonstrated in this report that 18 and 26 residue synthetic peptides corresponding to the b-hairpin region, constrained by a single disulfide bond, have potent antimicrobial activity without he- molytic activity. Circular dichroism spectroscopy in- dicates that the single S–S bridge appears to constrain the peptides to a b-structure. Peptides corresponding to the b-hairpin region of defensins could thus be at- tractive candidates as therapeutic agents as well as good model compounds for investigation of the vari- ous physiological actions of defensins. © 1999 Academic Press Defensins are a family of antimicrobial, cationic pep- tides composed of 29-34 amino acids (1). They are a part of the molecular armaments of polymorphonucle- ated neutrophils in humans, rabbit, guinea pig and rat that kill microorganisms by a mechanism independent of oxygen (1,2). They have six cysteine residues result- ing in three disulfide bridges which are essential for antimicrobial activity. Mammalian defensins exhibit broad spectrum in vitro antimicrobial activity (1,2). The potency among various defensins vary consider- ably with rabbit neutrophil peptide (NP-2) being the most potent. The crystal structure of human defensin HNP-3 has been determined (3). The structure indi- cates that the molecule adopts a triple stranded b-sheet conformation. Detailed NMR studies indicate that all mammalian defensins including the recently isolated b-defensins have very similar b-sheet struc- tures (4 – 6). Recent studies indicate that defensins and defensin-like molecules are also present in epithelial cells (7,8) and human reproductive tissues (9). The observations that human b-defensin-1 is a salt sensi- tive antibiotic in human lung that is inactivated by cystic fibrosis (10) and b-defensin 2 is induced on con- tact with human skin (11) suggest that this class of peptides could have therapeutic potential. However, as defensins are composed of 35 amino acids or more and have three disulphide bridges, their effective therapeu- tic use would not be tenable. Although the structures of naturally occuring defensins have been investigated extensively (6,12–17), there have been no structure– function studies involving their variants or truncated analogs. In this report, we describe the biological ac- tivities of synthetic peptides corresponding to the b-hairpin region 11-33 of the rabbit defensin NP-2. The primary structures of the peptides are summarized in Table 1. EXPERIMENTAL PROCEDURES Peptides P18L and P26L were synthesized by solid-phase methods on a semi-automated LKB Biolynx 4175 Peptide Synthesizer using fluorenylmethoxycarbonyl (Fmoc) chemistry (18). The SH of cysteine residues were protected with acetamidomethyl(Acm) group. The pep- tides were cleaved from the resin by treatment with a mixture containing trifluoroacetic acid (TFA), thioanisole, metacresol and ethanedithiol (10:1:1:1v/v). The peptides were checked for purity on a fast performance liquid chromatography (FPLC) system using reversed-phase pep RPC HR 5/5 columns from Pharmacia. The sol- vents were A, 0.1% TFA in water and B, 0.1% TFA in acetonitrile. The thiol protecting group at cysteine was removed by treatment with mercuric acetate (19). Disulphide bonds were formed by dimeth- ylsulfoxide oxidation of peptides (1 mg/mL) using a 20% v/v solution of dimethylsulfoxide in water (20). The peptides were characterized by amino acid analysis on a LKB 4151 Alpha Plus amino acid analyzer and sequence analysis on a Applied Biosystems 473 A Protein Sequencer. Antibacterial assays were performed as described earlier (21). Different bacterial strains from mid-logarithmic phase cultures were inoculated into LB medium to a concentration of Ca 10 5 CFU/mL. After 6-7h incubation at 37°C, the A 600 values of the cultures were determined along with the control culture where no peptide was added. Aliquots which showed maximum inhibition of growth were appropriately diluted and plated on to nutrient agar plates and incubated at 37°C for 15h. Peptide concentrations of aliquots which showed no colony-forming units were taken as the minimal inhibi- tory concentration (MIC). The experiments were performed in dupli- cates and three times independently. 1 To whom correspondence should be addressed. Fax: 91-40- 717195. E-mail: [email protected]. Biochemical and Biophysical Research Communications 254, 281–283 (1999) Article ID bbrc.1998.9933, available online at http://www.idealibrary.com on 281 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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Biochemical and Biophysical Research Communications 254, 281–283 (1999)

Article ID bbrc.1998.9933, available online at http://www.idealibrary.com on

ynthetic Peptides Corresponding to the b-Hairpin Loopf Rabbit Defensin NP-2 Show Antimicrobial Activity

. Thennarasu and R. Nagaraj1

entre for Cellular and Molecular Biology, Uppal Road, Hyderabad - 500 007 (A.P.), India

eceived November 30, 1998

observations that human b-defensin-1 is a salt sensi-tctpdhtnefatbpT

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Mammalian defensins, a class of antibacterial pep-ides, are composed of 29–35 amino acids with six cys-eines which form three disulfide bonds. Structuraltudies indicate a triple stranded b-sheet structureith a well defined b-hairpin loop at the C-terminal

egion. It is demonstrated in this report that 18 and 26esidue synthetic peptides corresponding to the-hairpin region, constrained by a single disulfideond, have potent antimicrobial activity without he-olytic activity. Circular dichroism spectroscopy in-

icates that the single S–S bridge appears to constrainhe peptides to a b-structure. Peptides correspondingo the b-hairpin region of defensins could thus be at-ractive candidates as therapeutic agents as well asood model compounds for investigation of the vari-us physiological actions of defensins. © 1999 Academic

ress

Defensins are a family of antimicrobial, cationic pep-ides composed of 29-34 amino acids (1). They are aart of the molecular armaments of polymorphonucle-ted neutrophils in humans, rabbit, guinea pig and rathat kill microorganisms by a mechanism independentf oxygen (1,2). They have six cysteine residues result-ng in three disulfide bridges which are essential forntimicrobial activity. Mammalian defensins exhibitroad spectrum in vitro antimicrobial activity (1,2).he potency among various defensins vary consider-bly with rabbit neutrophil peptide (NP-2) being theost potent. The crystal structure of human defensinNP-3 has been determined (3). The structure indi-

ates that the molecule adopts a triple stranded-sheet conformation. Detailed NMR studies indicatehat all mammalian defensins including the recentlysolated b-defensins have very similar b-sheet struc-ures (4–6). Recent studies indicate that defensins andefensin-like molecules are also present in epithelialells (7,8) and human reproductive tissues (9). The

1 To whom correspondence should be addressed. Fax: 91-40-17195. E-mail: [email protected].

281

ive antibiotic in human lung that is inactivated byystic fibrosis (10) and b-defensin 2 is induced on con-act with human skin (11) suggest that this class ofeptides could have therapeutic potential. However, asefensins are composed of 35 amino acids or more andave three disulphide bridges, their effective therapeu-ic use would not be tenable. Although the structures ofaturally occuring defensins have been investigatedxtensively (6,12–17), there have been no structure–unction studies involving their variants or truncatednalogs. In this report, we describe the biological ac-ivities of synthetic peptides corresponding to the-hairpin region 11-33 of the rabbit defensin NP-2. Therimary structures of the peptides are summarized inable 1.

XPERIMENTAL PROCEDURES

Peptides P18L and P26L were synthesized by solid-phase methodsn a semi-automated LKB Biolynx 4175 Peptide Synthesizer usinguorenylmethoxycarbonyl (Fmoc) chemistry (18). The SH of cysteineesidues were protected with acetamidomethyl(Acm) group. The pep-ides were cleaved from the resin by treatment with a mixtureontaining trifluoroacetic acid (TFA), thioanisole, metacresol andthanedithiol (10:1:1:1v/v). The peptides were checked for purity on

fast performance liquid chromatography (FPLC) system usingeversed-phase pep RPC HR 5/5 columns from Pharmacia. The sol-ents were A, 0.1% TFA in water and B, 0.1% TFA in acetonitrile.he thiol protecting group at cysteine was removed by treatmentith mercuric acetate (19). Disulphide bonds were formed by dimeth-lsulfoxide oxidation of peptides (1 mg/mL) using a 20% v/v solutionf dimethylsulfoxide in water (20). The peptides were characterizedy amino acid analysis on a LKB 4151 Alpha Plus amino acidnalyzer and sequence analysis on a Applied Biosystems 473 Arotein Sequencer.Antibacterial assays were performed as described earlier (21).ifferent bacterial strains from mid-logarithmic phase cultures were

noculated into LB medium to a concentration of Ca 105 CFU/mL.fter 6-7h incubation at 37°C, the A600 values of the cultures wereetermined along with the control culture where no peptide wasdded. Aliquots which showed maximum inhibition of growth wereppropriately diluted and plated on to nutrient agar plates andncubated at 37°C for 15h. Peptide concentrations of aliquots whichhowed no colony-forming units were taken as the minimal inhibi-ory concentration (MIC). The experiments were performed in dupli-ates and three times independently.

0006-291X/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 254, No. 2, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CD measurements were carried out on a JASCO J-20 spectropo-arimeter which was calibrated with 102(1) camphorsulfonic acid.he spectra were normalized for concentration (determined by aminocid analysis) and path length to obtain mean residue ellipticity afterase line correction. All the measurements were done at 25°C inotassium phosphate buffer (PPB) (10mM; pH 7.4) and trifluoroetha-ol (TFE). Peptide concentrations were 20 mM.

ESULTS AND DISCUSSION

Analysis of NMR data suggests that the b-hairpinegion in NP-2 (as well as in other defensin) is struc-urally better defined than other regions (15). The-hairpin region in NP-2 is composed of residues R15 to31 (Table 1). The segment L11-E14 is presumed toccur in a b-turn conformation. We have examined theiological activities and structures of synthetic linearnd cyclic peptides corresponding to the b-hairpin re-ion of NP-2. Peptide P18L corresponds to the segment17-R33. Peptide P26L includes the b-turn region inP-2. P18L and P26L were cyclized by a single S-Sridge with the cysteines positioned at the N and-terminal regions of the peptide as shown in Table 1.The antimicrobial activities of linear and cyclic pep-

ides region are summarized in Table 2. P18C and26C show antibacterial activity against gram nega-ive and gram positive microorganisms. The linear pep-

Primary Sequences of Rabbit Defensin NP-2 and SyntheticPeptides Corresponding to Its C-Terminal Segment

Peptide Sequencea

NP-2 VVCACRRALCLPLERRAGFCRIRGRIHPLCCRR1 5 10 15 20 25 30

P18C CAGFMRIRGRIHPLCMRRAcm Acm

P18L CAGFMRIRGRIHPLCMRRP26C ALCLPLERRAGFMRIRGRIHPLCMRR

Acm AcmP26L ALCLPLERRAGFMRIRGRIHPLCMRR

a Acm, acetamidomethyl.

TAB

Antimicrobial A

Peptide

Minimal inhibit

E. coli W160.37 B. subtilis

P18La NA NAP18C 4 2P26La NA NAP26C 11 4

Note. NA, not active.a P18L and P26L showed no activity at concentrations of 30 and 6

282

ides without the disulfide bridge are inactive as in thease of defensins (1,2,6). Peptide P18C which has the-turn segment in NP-2 corresponding to residues2-25 is more active than P26C which has both the-turns corresponding to residues 11-14 and 22-25 inP-2. All the four peptides did not lyse rat erythrocytespto 60 mg/mL.The structures of the peptides were investigated byD spectroscopy. The spectra of P18L in buffer andFE are shown in Fig. 1A. The spectrum in buffer isharacteristic of peptides in random conformation. Thepectrum in TFE shows a minimum ;208 nm, a shoul-er ;220 nm and a cross over ;200 nm which isharacteristic of helical conformation. Estimation ofelical content from the ellipticity at 222 nm (22) yieldsvalue of 30%. The spectra of P18C in buffer and TFE

re shown in Fig. 1B. The spectrum in buffer shows aross-over ;198 nm and a prominent band ;205 nm.n TFE, the spectrum is characterized by a minimum

2

ity of Peptides

concentration (MIC)(mg/ml) against

S. aureus ATCC 8530 P. aeruginosa

NA NA3 9

NA NA11 33

g/ml, respectively.

FIG. 1. Circular dichroism spectra of 18- and 26-residue peptidesn different media. (A) P18L, (B) P18C, (C) P26L, (D) P26C. (- - -),PB; (—), TFE.

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Vol. 254, No. 2, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

light decrease in the intensity of the band ;207 nm asompared to water, there is considerable increase inntensity of the u222 band. The spectra of P26L arehown in Fig. 1C. In buffer, the peptide is unordered.n TFE, the u222 value indicates a helical content of30%. The spectra of P26C in buffer and TFE are

hown in Fig. 1D. The spectra are similar to P18Cualitatively similar but there is a decrease in thentensity of the negative bands as compared to P18C.

hile more definitive data are required to unequivo-ally establish the structures of the peptides, the CData suggests b-structures for P18C and P26C as com-ared to helical structures for P18L and P26L.The broad spectrum antimicrobial activity of the cy-

lic 18 and 26-residue peptide corresponding to the-hairpin region of defensin NP-2 suggests that thisegion could be an important determinant of the anti-acterial activity of defensin NP-2. Since the b-hairpintructural motif is conserved in all mammalian de-ensins (6) as well as in insect (25) and plant defensins26) it is conceivable that this structural motif is anmportant determinant of antimicrobial activity. Since18C and P26C are specifically active only on microor-anisms and do not lyse mammalian cells unlike de-ensins (2), the b-hairpin region could be an attractiveandidate for possible therapeutic applications andlso be of considerable help in understanding how de-ensins exert other physiological responses (2,27) likenhibition of protein kinase C, specific binding to theCTH receptor, blocking steriod genesis, acting as se-

ective chemoattractants for monocytes and neutrophilunctions such as adhesion, superoxide anion genera-ion and phagocytosis.

CKNOWLEDGMENTS

We thank V. M. Dhople for amino acid analysis and M. V. Jagan-adham for sequence analysis. This research was partially sup-orted by a grant from the Department of Science and Technology,ndia.

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