Biochimica et Biophysica Acta 1425 (1998) 361^368
Biological characterization of a novel mammalian antimicrobial peptide Renato Gennaro a , Marco Scocchi
b; c
, Laura Merluzzi c , Margherita Zanetti
b; c;
*
a
b
Dipartimento di Biochimica, Bio¢sica e Chimica delle Macromolecole, Universita© di Trieste, I-34127 Trieste, Italy Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, AREA Science Park, Padriciano, 99 I-34012 Trieste, Italy c Dipartimento di Scienze e Tecnologie Biomediche, Universita© di Udine, I-33100 Udine, Italy Received 2 June 1998; revised 13 July 1998; accepted 4 August 1998
Abstract A putative antimicrobial peptide of 34 residues was recently deduced from a bovine cathelicidin gene sequence and named BMAP-34. A peptide based on the deduced sequence was chemically synthesized and used to study the localization, structure and biological activities of BMAP-34. A Western blot analysis using antibodies raised to the synthetic peptide showed that BMAP-34 is stored as proform in the cytoplasmic granules of bovine neutrophils. CD spectroscopy indicates that the peptide assumes an amphipathic K-helical conformation, as also predicted by secondary structure analysis. The peptide exerts a broad spectrum antimicrobial activity against both Gram-negative and Gram-positive organisms, and is not active against eukaryotic cells. When tested on Escherichia coli ML-35, the kinetics of bacterial killing and of inner membrane permeabilization are slower than those observed for other K-helical peptides derived from cathelicidins. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Cathelicidin ; Antimicrobial peptide; K-Helical peptide; Membrane permeabilization; (Cow)
1. Introduction Antimicrobial peptides are small and amphipathic molecules produced by animals and plants as part of the immune defense system [1]. In general, these peptides are cationic and exert their antibiotic activity by permeabilizing bacterial membranes [1,2]. A variety of these peptides have been identi¢ed in leukocytes as derived from prepropeptides of the cathelicidin family [3]. Members of this family show a conserved Nterminal `cathelin' domain (propiece) of about 100 residues and a C-terminal cationic antimicrobial domain of varied length (12^100 residues). Most often,
* Corresponding author. Fax: +39 (40) 398990; E-mail:
[email protected]
a proteolytic cleavage site for elastase is placed between the propiece and the C-terminal peptide [3], allowing for release of the active peptide [4^6]. Cathelicidin-derived peptides show a remarkable structural diversity and exhibit a wide spectrum antimicrobial activity in vitro at Wmolar concentrations. According to common structural features, these peptides have been grouped into K-helical, Cys-rich, Proand Arg-rich, and Trp-rich peptides [3]. We have recently reported the sequence of a novel bovine cathelicidin, as deduced from the gene sequence [7]. The predicted polypeptide includes a prepropiece of 131 residues and a C-terminal putative antimicrobial domain of 34 residues. The peptide was named BMAP-34 (bovine myeloid antimicrobial peptide of 34 residues) after the putative antimicrobial domain. In this report, a synthetic peptide based on the
0304-4165 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 0 8 7 - 7
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sequence of the putative BMAP-34, and antibodies raised in rabbit to the synthetic peptide have been used to determine the structure, biological activity and cellular localization of BMAP-34. The results obtained indicate that BMAP-34 is stored as proform in the cytoplasmic granules of bovine neutrophils. The peptide assumes an amphipathic K-helical conformation common to other antimicrobial peptides, and exerts a broad spectrum antibacterial activity against both Gram-negative and Gram-positive organisms. Conversely, eukaryotic cells are not affected by this peptide. 2. Materials and methods 2.1. Chemicals Fmoc-protected amino acids, PAL-PEG resin and coupling reagents were from PerSeptive Biosystems (Framingham, MA, USA). Acetonitrile, dimethylformamide, dichloromethane, N-methyl-2-pyrrolidone and dimethylsulfoxide were from Lab-Scan (Dublin, Ireland). Tri£uoroacetic acid, tri£uoroethanol and N-methylmorpholine were purchased from Janssen Chimica (Beerse, Belgium). Mueller^Hinton broth, bacto-agar, dextrose, mycological peptone and yeast extract powder were purchased from Difco Laboratories (Detroit, MI, USA). All other chemicals were of analytical grade. 2.2. Sequence analysis Secondary structure prediction of proBMAP-34 was carried out using the PHD neural network [8]. Database searches were performed by using the BLAST network service of the National Center for Biotechnology Information. 2.3. Peptide synthesis and puri¢cation A BMAP-34 peptide including the C-terminal residues of the polypeptide predicted from the gene sequence, was synthesized by the solid phase method on a Milligen 9050 synthesizer using the Fmoc chemistry. Polyethylene glycol-polystyrene (PEG-PS) resin (0.17 mmol g31 ) with a PAL linker, was used as support to obtain a C-terminally amidated peptide.
The couplings were performed with N-hydroxybenzotriazole and 2-(1H-benzotriazole-1-yl)-1,1,3,3tetramethyluroniumtetra£uoroborate (TBTU) or with O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetra£uoroborate (HATU) (residues 12^18 and 27^29), in the presence of 0.6 M N-methylmorpholine. To increase the yield of the synthesis, a 4:1 (v/v) mixture of N-methyl-2-pyrrolidone and dimethylsulfoxide was used as solvent to improve peptide solvation, the column temperature was increased to 45³C and the resin was washed with the so called `magic mixture' (N-methyl-2-pyrrolidone/N,N-dimethylformamide/dichloromethane, 1:1:1 (v/v), with 1% Triton X-100 and 2 M ethylencarbonate) before each coupling [9]. Amino acid side chains were protected as follows: 2,2,5,7,8-pentamethylchroman-6sulfonyl (Arg), t-butyl (Asp, Glu, Ser), trityl (Gln), t-butyloxycarbonyl (Lys). Deprotection and cleavage from the resin were carried out using a mixture of tri£uoroacetic acid, ethanedithiol, water and triisopropylsilane (92.5:2.5:2.5:2.5, v/v) for 3 h at room temperature. The peptide was then repeatedly extracted with ethyl ether and puri¢ed by reverse phase HPLC on a C18 Delta-Pak column (19U300 mm, Waters, Bedford, USA), using an appropriate 0^ 60% water/acetonitrile gradient in 0.1% tri£uoroacetic acid. 2.4. CD spectroscopy CD spectra were recorded on a Jasco J-600 spectropolarimeter using a 2 mm pathlength cell. Peptide samples (5^25 WM) were dissolved in 5 mM sodium phosphate bu¡er, pH 7.0, in the absence or presence of tri£uoroethanol up to 45% (v/v). The K-helical content was estimated using the equation ([a]3[a]rc )/([a]K 3[a]rc ), where [a] is the mean molar ellipticity per residue at 222 nm, in deg cm2 dmol31 , and [a]rc and [a]K are the estimated molar ellipticities, respectively, for a random coil (31000 deg cm2 dmol31 ) and for a 100% helical peptide (336 500 deg cm2 dmol31 ) [10]. 2.5. Analytical assays Peptide concentration was determined by measuring the absorbance of Phe at 257 nm using an extinction coe¤cient of 195.1 M31 cm31 [11]. The mo-
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lecular mass of BMAP-34 was determined with an API I ionspray mass spectrometer. Antibodies to BMAP-34 were raised in rabbit by repeated i.m. injections of the synthetic BMAP-34 peptide, and Western analysis was performed as previously described [4]. 2.6. Antimicrobial activity and membrane permeabilization Antibacterial and antifungal activities of the puri¢ed peptide were determined as minimum inhibitory concentration (MIC) by the microdilution susceptibility test in 96-well microdilution plates. The following bacterial strains were tested, under the assay conditions previously described [12]: Escherichia coli ATCC 25922, ML-35, D21 and D22, Salmonella typhimurium ATCC 14028, a clinical isolate of Salmonella enteritidis, Pseudomonas aeruginosa ATCC 27853, Serratia marcescens ATCC 8100, Staphylococcus aureus ATCC 25923, and two methicillin-resistant clinical isolates carrying the mecA gene (provided by L. Dolzani, Department of Biomedical Sciences, University of Trieste, Italy), Staphylococcus epidermidis ATCC 12228, and Bacillus megaterium Bm11. The antifungal activity was determined using a clinical isolate of Candida albicans. The assay conditions were similar to those used for bacteria [12], except that C. albicans was grown and tested in Sabouraud liquid medium and the MIC was determined after incubation at 30³C for 36^48 h. Outer and inner membrane permeabilization of E.
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coli ML-35 was evaluated by following the unmasking of L-lactamase and L-galactosidase activities using respectively 7-(thienyl-2-acetamido)-3-[2-(4-N,Ndimethyl-aminophenylazo)-pyridinium methyl]-3-cephem-4-carboxylic acid (PADAC) and o-nitrophenyl-L-D-galactopyranoside (ONPG) as substrates, as previously described [13]. The kinetics of bacterial killing was evaluated using E. coli ML-35. Log-phase bacteria (2^4U105 cfu ml31 ) were incubated with 5 WM peptide in Mueller^ Hinton broth. Aliquots were removed at ¢xed time intervals, appropriately diluted, plated on Mueller^ Hinton agar and the colony forming units counted after 16^18 h incubation at 37³C. The hemolytic activity was evaluated by determining hemoglobin release of 10% (v/v) suspensions of fresh human or bovine erythrocytes at 415 nm, as previously described [13]. Melittin was used as positive control. 3. Results 3.1. Western analysis of BMAP-34 Analysis of the bovine cathelicidin gene family revealed a putative novel cathelicidin (pre-proBMAP34) of 165 amino acid residues [7]. The polypeptide showed a conserved cathelin-like prepropiece, followed by a C-terminal putative antimicrobial domain (peptide sequence) of 34 residues predicted to assume an K-helical conformation. This novel cathelicidin
Fig. 1. Alignment of the sequence of the precursor of BMAP-34 with those of the other bovine cathelicidins, i.e. BMAP-27, BMAP28, indolicidin, dodecapeptide, Bac7 and Bac5 [3,7,18]. Dashes indicate residues that are identical between BMAP-34 precursor and at least one of the other polypeptides. Gaps are indicated by dots. Mature antimicrobial peptides derived from each cathelicidin are shown in bold.
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Fig. 2. Western blot analysis of proBMAP-34 and BMAP-34 peptide. Lysates of total bovine neutrophils (lanes 1 and 2) and of neutrophil granules (lanes 3 and 4) were acid-precipitated with 10% TCA (lanes 1 and 3) or solubilized with 1% Triton X-100 (lanes 2 and 4). Protein was separated by SDS-PAGE, electroblotted on nitrocellulose paper and immunostained using rabbit antibodies to synthetic BMAP-34 peptide. Molecular weight markers are indicated on the right.
adds to the previously identi¢ed bovine members (Fig. 1), with 76^82% sequence identity in the preproregion and no signi¢cant homology in the C-terminal antimicrobial domain. The gene is expressed in myeloid bone marrow cells, as demonstrated by Northern analysis [7]. The transcript was also detected by RT-PCR in spleen and testis, but not in mature neutrophils [7], where the known bovine cathelicidins are stored as propeptides [3]. We looked further at the expression of this gene in the transcript-positive tissues, trying to correlate the presence of the transcript with that of the protein. To this aim, a Western analysis of spleen and testis was performed using antibodies raised in rabbit to synthetic BMAP-34 peptide. The proform was not detected in total lysates of these organs (not shown), despite the presence of a BMAP-34 transcript. This result may be explained assuming that the proportion of BMAP-34-producing cells in these organs is small, relative to the total cell population, and detection of the proform may thus require enrichment of these cells. Conversely, analysis of total neutrophils and of neutrophil granule lysates revealed a single band with an apparent size of approximately 15 kDa, comparable with that calculated for proBMAP-34 (Fig. 2, lanes 1 and 3). These data, together with the absence of BMAP-34 transcript in mature neutrophils, are in keeping with previous observations indicating that the bovine cathelicidins are mostly synthesized in the proliferative stage of neutrophil maturation and accumulate as proforms in
the large granules [4]. Only when neutrophils, and neutrophil granules, were solubilized in non-denaturing conditions, a band with the size of the mature peptide was detected (Fig. 2, lanes 2 and 4). This result, and the presence of a putative cleavage site for elastase as the C-terminal residue of the propiece (Fig. 1) suggest that the activation of BMAP-34 is brought about by a mechanism similar to that observed for other cathelicidin-derived peptides that are cleaved o¡ from the precursor by elastase upon release of this enzyme from the azurophil granules [4^ 6]. 3.2. Structural analysis and chemical synthesis The sequence of BMAP-34 shows four negatively, in addition to 12 positively charged residues. The presence of four acidic amino acids is quite uncommon for antimicrobial peptides. All over, BMAP-34 is highly cationic, with a net positive charge of eight (or nine, assuming the K-amino group to be protonated), and highly hydrophilic, as also indicated by a mean residue hydrophobicity of 30.503 calculated using the hydrophobicity consensus scale of Eisen-
Fig. 3. Circular dichroism spectra of BMAP-34 peptide (25 WM) recorded in 5 mM sodium phosphate bu¡er, pH 7.0 in the absence (3W3W) or the presence of 15% (^ ^ ^) and 45% (99999) TFE. The inset shows the variation of the mean molar ellipticity per residue at 222 nm (3[a]222 ) with increasing TFE concentrations.
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berg [14]. Secondary structure prediction analysis based on the PHD pro¢le network [8] indicates a strong potential for helix formation in the segment spanning residues 8^31. Helical wheel projection of the peptide shows the amphipathic nature of the putative helix, with an angle of approximately 200³ subtending the polar face, and the potential for 20 i, i+3/ i+4 ion pairs with a prevalence (12 vs. 8) of complimentary over repulsive pairs. The amphipathicity of the peptide is also suggested by a relatively high mean hydrophobic moment per residue (0.481), as determined according to Eisenberg [14]. When ¢tted to a hydrophobic moment vs. hydrophobicity plot calculated by Tossi et al. for a variety of K-helical antimicrobial peptides [15], BMAP-34 is well into the surface-seeking helices, within a region including peptides with potent antimicrobial, and poor or no hemolytic activity. The putative role of this peptide in host-defense was evaluated by investigating its in vitro biological activity. To this aim, a peptide based on the sequence of BMAP-34 was ¢rst synthesized by the solid phase method using the Fmoc chemistry, and the in vitro Table 1 Minimum inhibitory concentrations (MIC) of BMAP-34 peptide Organism and strain
MICa (WM)
E. coli ATCC 25922 E. coli ML-35 E. coli D21 E. coli D22 S. typhimurium ATCC 14028 S. enteritidis (clinical isolate) S. marcescens ATCC 8100 P. aeruginosa ATCC 27853 S. aureus ATCC 25923 S. aureus Cowan 1 S. aureus (MRSA, clinical isolate) S. aureus (MRSA, clinical isolate) S. epidermidis ATCC 12228 B. megaterium Bm 11 C. albicans (clinical isolate)
1.5 1.5 1.5 1.5 3 3 1.5 v48 3 3 6 6 1.5 3 s 96
a
MIC was de¢ned as the lowest concentration of peptide preventing visible microbial growth after incubation of 16^18 h at 37³C (bacteria) or 36^48 h at 30³C (C. albicans). All the strains were grown in Mueller^Hinton broth, except C. albicans that was grown in Sabouraud medium. Results were determined with 1.0^2.0U105 (bacteria) and 0.25^0.50U105 (C. albicans) colony forming units ml31 and are the mean of at least three independent determinations with a divergence of not more than a MIC value. MRSA, methicillin-resistant S. aureus.
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Fig. 4. Permeabilization of the outer (OM) and inner (IM) membranes of E. coli ML-35. The permeabilization at the indicated peptide concentrations was measured by following spectrophotometrically the unmasking of periplasmic L-lactamase (open bars) and cytoplasmic L-galactosidase (dark bars) activities at 570 and 405 nm, respectively [13]. Mean values of two to three independent experiments are shown. Permeabilization was determined by measuring the variation of absorbance from 7 to 11 min after peptide addition, and expressed as percent of bacteria completely permeabilized by sonication.
antimicrobial and cytotoxic activities of the synthetic BMAP-34 were then determined. By analogy with other cathelicidin-derived peptides, the C-terminal Gly predicted at position 34 was regarded as an amidation signal [3], and the peptide synthesized as Cterminal amide of 33 residues. The protocol used allowed a high yield (60%) synthesis of the correct peptide, as shown by mass analysis (4080.41 þ 0.57 Da vs a predicted mass of 4080.89), with few deletion products that were easily removed by RP-HPLC (not shown). Circular dichroism spectroscopy of the synthetic peptide indicates a transition from a random coil structure in aqueous solution to an K-helical conformation on addition of the helicogenic solvent TFE (Fig. 3), in keeping with secondary structure prediction. The estimated helical content was 18, 51 and 53% at 15, 30 and 45% TFE, and did not increase with higher concentrations of solvent (Fig. 3, inset). Under the conditions used, the helices appear to be monomeric, since the CD spectra do not change when measured at di¡erent peptide concentrations.
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Fig. 5. Kinetics of killing of E. coli ML-35 by BMAP-34 and BMAP-28 peptides. Bacteria, either untreated (R) or treated with 5 WM BMAP-34 (F) or 1 WM BMAP-28 (b), were diluted at the indicated time intervals, and then plated on Mueller-Hinton agar. The colony forming units were calculated by counting the plates after 16^18 h incubation at 37³C.
In addition, the existence of an isodicroic point at approximately 203 nm suggests the presence of just two conformations, i.e. helix and random coil, for each residue [16]. 3.3. Biological activity The antimicrobial activity of the synthetic BMAP34 peptide was determined as minimum inhibitory concentration (MIC) values, using a panel of Gram-negative and Gram-positive bacteria, and of the fungal species C. albicans. With the exception of P. aeruginosa, BMAP-34 completely inhibited the growth of all bacterial strains tested, including MRSA strains, with MIC values from 1.5 to 12 WM (Table 1). Conversely, BMAP-34 was inactive against C. albicans up to 96 WM concentration (Table 1), suggesting that eukaryotic cells are much less susceptible. This conclusion is also supported by the results of experiments aimed at evaluating the potential lytic activity on red blood cells. These experiments were performed by monitoring the hemoglobin release from human or bovine erythrocytes, following incubation of 10% cell suspensions with up to 50 WM peptide. The peptide did not a¡ect bovine erythrocytes at any concentration used, and showed a very modest activity when tested against human erythrocytes (3% hemolysis at 50 WM peptide, not shown). These data point to a good selectivity of BMAP-34 for prokaryotic cells, that may be related to the low
hydrophobicity of this peptide. This is suggested in particular by studies with model peptides indicating a strong correlation between hemolytic e¡ects and hydrophobicity [17]. A number of studies indicate that bacterial membranes are primary targets for cationic antimicrobial peptides [1^3]. Accordingly, the permeabilizing e¡ects of BMAP-34 on both the outer (OM) and inner (IM) membranes of E. coli ML-35 were determined. As shown in Fig. 4, 70 and 100% OM permeabilization were rapidly achieved at 1 and 5 WM peptide, respectively. The e¡ect on the IM was less pronounced, a 30% permeabilization being reached at 10 WM concentration after a 90 s lag time (Fig. 4). When compared with other cathelicidin-derived K-helical peptides such as bovine BMAP28 [18], pig PMAP-37 [19], and mouse CRAMP [20], this kinetics is much slower. In particular, BMAP-28 caused a rapid and complete permeabilization already at 1 WM concentration. We thus decided to further examine the antimicrobial activity of BMAP-34 by analyzing the kinetics of bacterial killing. A time course experiment was performed in Mueller^Hinton broth at 5 WM peptide using the E. coli ML-35 strain, which is equally susceptible to both BMAP-34 and BMAP-28 [18]. The results indicate that BMAP-34 inactivates about 60^70% of the bacteria by 10 min. However, a 2^3 log decrease in viable bacteria is attained only by 60^90 min, unlike BMAP-28, that causes a similar decrease in approximately 10 min (Fig. 5). This kinetics does not seem to be dependent on the ability of BMAP-34 to bind to the bacterial surface, as indicated by OM permeabilization (Fig. 4). Rather, these data parallel those of IM permeabilization, and indicate that a longer time is required for BMAP-34 to attain a productive interaction with this membrane, compared with BMAP-28. Whether this behavior depends on clustering at the membrane surface to cause a coopera-
Fig. 6. Alignment of the sequence of BMAP-34 peptide with those of PMAP-37 and CRAMP. The BMAP-34 sequence was deduced from the gene [7], those of PMAP-37 [19] and CRAMP [20] from the respective cDNAs. M and : indicate, respectively, identical and similar residues.
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tive permeabilization (carpet e¡ect), rather than assembly of peptide monomers to form transmembrane channel, is not clear at the moment. 4. Discussion In this report, the product of the recently identi¢ed BMAP-34 gene has been localized to bovine neutrophil granules by Western analysis, and the structure and antimicrobial activity of the split product (BMAP-34 peptide) have been characterized. The peptide adds to a large group of K-helical antimicrobial peptides found in a variety of animal species [1]. When compared with known sequences, BMAP-34 exhibits the highest identity (82%) to the ovine homolog OaMAP-34 inferred from the respective gene [21], followed by such other cathelicidinderived K-helical peptides as pig PMAP-37 [19] and mouse CRAMP [20] (Fig. 6), which show an approximately 40% sequence identity. PMAP-37 and CRAMP also have in common with BMAP-34 the presence of several (three to six) negatively charged residues that cluster at the center of the polar face of the helix, with a charge distribution reminiscent of class A amphipathic helices [22]. In spite of these structural similarities, signi¢cant di¡erences, however, exist in the spectrum of activity of these peptides. Among the strains tested, both Gram-positive and Gram-negative organisms are susceptible to BMAP-34 at Wmolar concentrations, with the exception of P. aeruginosa. PMAP-37 and CRAMP, at comparable concentrations are active against Gramnegative bacteria, including P. aeruginosa, and show poor, or negligible activity against Gram-positive species, in particular staphylococcal strains. The antimicrobial action is in any case due to the ability of these peptides to disrupt bacterial membranes [19,20]. Their basic nature contributes to initial interaction with negatively charged membrane components, while their amphipathic character favors their incorporation into bacterial membranes. This general mechanism may be tuned by other structural features that may be responsible for diversi¢cation of the spectrum of activity. Studies with model peptides or analogs of natural peptides have identi¢ed several parameters (i.e. mean residue hydrophobicity (H), hydrophobic moment (W), helicity, and cationicity)
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that may in£uence the target cell speci¢city of antimicrobial peptides. Increase of H and W generally correlates with enhanced activity against Gram-positive bacteria and hemolysis, and is related to a reduction of peptide speci¢city for Gram-negative bacteria [17,23]. An increase of the positive charge often enhances the antibacterial e¡ects and has been shown to extend the spectrum of action to Gram-positive bacteria, without a parallel increase of the hemolytic activity [24,25]. This indeed seems to be the case for BMAP-34. Compared with PMAP-37 and CRAMP, the peptide shows a high net positive charge (+8, +2 and +5, respectively) that may explain the observed activity on Gram-positive strains. BMAP-34 is also assigned the highest hydrophilicity and amphipathicity scores, although a clear-cut correlation with the respective spectra of activity of the three peptides cannot be established based on these data alone. As demonstrated by a number of studies on structure/activity relationship, an unequivocal interpretation of di¡erences in the activity pro¢les of antimicrobial peptides is often di¤cult, due to the complexity of the elements involved in membrane interaction. Thus, the varied activity of the three peptides, as discussed in this study, likely is the result of a sensitive balance of structural motifs and subtle di¡erences in the target membrane composition. Acknowledgements We are grateful to Dr. A. Tossi from the Department of Biochemistry of the University of Trieste for critically reading the manuscript. This work was supported by grants from C.N.R. Target Project on Biotechnology, from the Italian Ministry for University and Research (ex MURST 60% and 40%), and from the Istituto Superiore Sanita©, Programma Nazionale di Ricerca sull'AIDS (Grant 50A.0.36).
References [1] H.G. Boman, Peptide antibiotics and their role in innate immunity, Annu. Rev. Immunol. 13 (1995) 62^92. [2] R.E.W. Hancock, R. Lehrer, Cationic peptides: a new source of antibiotics, Tibtech 16 (1998) 82^88. [3] M. Zanetti, R. Gennaro, D. Romeo, Cathelicidins: a novel
BBAGEN 24681 14-10-98
368
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14] [15]
R. Gennaro et al. / Biochimica et Biophysica Acta 1425 (1998) 361^368 protein family with a common proregion and a variable Cterminal antimicrobial domain, FEBS Lett. 374 (1995) 1^ 5. M. Zanetti, L. Litteri, R. Gennaro, H. Horstmann, D. Romeo, Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor molecules stored in the large granules, J. Cell Biol. 111 (1990) 1363^1371. M. Scocchi, B. Skerlavaj, D. Romeo, R. Gennaro, Proteolytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins, Eur. J. Biochem. 209 (1992) 589^595. A. Panyutich, J. Shi, P.L. Boutz, C. Zhao, T. Ganz, Porcine polymorphonuclear leukocytes generate extracellular microbicidal activity by elastase-mediated activation of secreted proprotegrins, Infect. Immun. 65 (1997) 978^985. M. Scocchi, S. Wang, M. Zanetti, Structural organization of the bovine cathelicidin gene family and identi¢cation of a novel member, FEBS Lett. 417 (1997) 311^315. B. Rost, C. Sander, Prediction of protein secondary structure at better than 70% accuracy, J. Mol. Biol. 232 (1993) 584^ 599. L. Zhang, C. Goldammer, B. Henkel, G. Panhaus, F. Zu«hl, G. Jung and E. Bayer, 3rd Int. Symp. Innovations and Perspectives in Solid Phase Synthesis, Poster 82, 1993, Oxford, UK. Y.-H. Chen, J.T. Yang, H. Chau, Determination of the helix and L form of proteins in aqueous solution by circular dichroism, Biochemistry 13 (1974) 3350^3359. M.A. Buck, T.A. Olah, C.J. Weitzmann, B.S. Cooperman, Protein estimation by the product of integrated peak area and £ow rate, Anal. Biochem. 182 (1989) 295^299. R. Gennaro, B. Skerlavaj, D. Romeo, Puri¢cation, composition and activity of two bactenecins, antibacterial peptides of bovine neutrophils, Infect. Immun. 57 (1989) 3142^3146. B. Skerlavaj, D. Romeo, R. Gennaro, Rapid membrane permeabilization and inhibition of vital functions of Gram-negative bacteria by bactenecins, Infect. Immun. 58 (1990) 3724^3730. D. Eisenberg, Three dimensional structure of membrane and surface proteins, Annu. Rev. Biochem. 47 (1984) 595^623. A. Tossi, C. Tarantino, D. Romeo, Design of synthetic anti-
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
microbial peptides based on sequence analysis and amphipathicity, Eur. J. Biochem. 250 (1997) 549^558. S. Padmanabhan, S. Marqusee, T. Ridgeway, T.M. Lawe, R.L. Baldwin, Relative helix forming tendencies of nonpolar amino acids, Nature 344 (1990) 268^270. T. Wieprecht, M. Dathe, M. Beyermann, E. Krause, W.L. Maloy, D.L. MacDonald, M. Bienert, Peptide hydrophobicity controls the activity and selectivity of magainin 2 amide in interaction with membranes, Biochemistry 36 (1997) 6124^6132. B. Skerlavaj, R. Gennaro, L. Bagella, L. Merluzzi, A. Risso, M. Zanetti, Biological characterization of two novel cathelicidin-derived peptides and identi¢cation of structural requirements for their antimicrobial and cell lytic activities, J. Biol. Chem. 271 (1996) 28375^28381. A. Tossi, M. Scocchi, M. Zanetti, P. Storici, R. Gennaro, PMAP-37, a novel pig myeloid antibacterial peptide, Eur. J. Biochem. 228 (1995) 941^946. R.L. Gallo, K. Kim, M. Bern¢eld, C. Kozak, M. Zanetti, L. Merluzzi, R. Gennaro, Identi¢cation of CRAMP, a cathelin related antimicrobial peptide expressed in the embryonic and adult mouse, J. Biol. Chem. 272 (1997) 13088^13093. K.M. Huttner, M.R. Lambeth, H.R. Burkin, D.J. Burkin, T.E. Broad, Localization and genomic organization of sheep antimicrobial peptide genes, Gene 206 (1998) 85^91. J.P. Segrest, H. de Loof, J.G. Dohlman, C.G. Brouillette, G.M. Anantharamaiah, Amphipathic helix motif: classes and properties, Proteins 8 (1990) 103^117. M. Dathe, T. Wieprecht, H. Nikolenko, L. Handel, W.L. Maloy, D.L. MacDonald, M. Beyermann, M. Bienert, Hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides, FEBS Lett. 403 (1997) 208^212. K. Matsuzaki, K. Sugishita, M. Harada, N. Fujii, K. Miyajima, Interactions of an antimicrobial peptide, magainin 2, with outer and inner membranes of Gram-negative bacteria, Biochim. Biophys. Acta 1327 (1997) 119^130. W.L. Maloy, U.P. Kari, Structure^activity studies on magainins and other host-defense peptides, Biopolymers 37 (1995) 105^122.
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