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Effect of amino acid substitutions on the candidacidal activity of LFampin 265–284
Peptides 26 (2005) 2093–2097
Effect of amino acid substitutions on the candidacidal activity of LFampin 265–284 Marieke I.A. van der Kraan, Christel van der Made, Kamran Nazmi, Wim van‘t Hof, Jasper Groenink, Enno C.I. Veerman, Jan G.M. Bolscher ∗ , Arie V. Nieuw Amerongen Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), Amsterdam, The Netherlands Received 21 December 2004; received in revised form 29 March 2005; accepted 30 March 2005 Available online 8 June 2005
Abstract LFampin 265–284, derived from bovine lactoferrin, has broad-spectrum antimicrobial activity against the yeast Candida albicans and several Gram-positive and Gram-negative bacteria. A glycine substitution scan was used to identify residues that are important for its candidacidal activity. Each single substitution of a positively charged residue led to considerable reduction in candidacidal activity, for each residue to a different extent. Substitution within the helix-facilitating N-terminal sequence DLIW had less severe effect; substitution of Ile and Trp led to a somewhat reduced potency. No substantial effects were found on the propensity to adopt a helical structure or to bind to C. albicans cells. © 2005 Elsevier Inc. All rights reserved. Keywords: Bovine lactoferrin; Lactoferrampin; LFampin; antimicrobial peptide; Candida albicans
1. Introduction Antimicrobial peptides are implicated in the innate immune systems of animals and plants (for recent reviews see [7,18]). Structural diversity among antimicrobial peptides is considerable, but most of them fold into an amphipathic ␣helical conformation upon interaction with the target organism. Antimicrobial amino acid stretches may appear as free peptides, but they can also be incorporated in the sequences of larger proteins. For instance bovine lactoferrin (bLF) possesses such an antimicrobial stretch: Lactoferricin B (LFcin B), a positively charged looped peptide encompassing residues 17–41, which is released by gastric pepsin cleavage [3,14,15]. It has more potent bactericidal and fungicidal activity than the parent protein [2,10,11,15,20], it has antitumor activity [12,23], it plays a regulatory role in the adaptive immune response and it has anti-inflammatory properties [6,16]. We have identified a second antimicrobial sequence within the N1-domain, designated lactoferrampin (LFampin) [21,22]. The corresponding peptide exhibited broad∗
Corresponding author. Tel.: +31 20 4448672; fax: +31 20 4448685. E-mail address: [email protected] (J.G.M. Bolscher).
0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.03.056
spectrum antimicrobial activity against the yeast Candida albicans and both Gram-positive and Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus mutans. In general, the activity of antimicrobial peptides is dependent on global structural parameters, such as a net positive charge and amphipathicity, rather than on the specific amino acid sequence. Nevertheless, in several antimicrobial peptides single critical residues have been identified [4,5,8,13]. In the present study we have conducted a glycine substitution scan of LFampin 265–284 to pinpoint residues that are critical for the candidacidal activity. The consequences of these substitutions for capacity to bind to C. albicans cells and for the tendency to adopt a helical structure are determined and discussed in relation to the according candidacidal activity.
2. Materials and methods 2.1. Synthesis and purification of peptides Peptides as listed in Table 1 were synthesized by Fmocchemistry with a MilliGen 9050 peptide synthesizer (Mil-
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Table 1 Properties of LFampin peptides with amino acid substitutions, together with the LC50 -values (M)a towards C. albicans
Peptides marked with an asterisk were also synthesized with an alanine substitution instead of a glycine substitution. These peptides (K273A, K277A and K280A) had less drastic effects on the antifungal activity; LC50 -values of alanine-substituted peptides were half that of glycine-substituted peptides (not shown). Double substitution resulting in the peptides K237/277A and K277/280A abolished the antifungal activity (LC50 -values of >100 M). a LC : the concentration of peptide (M) resulting in 50% reduction of the viable counts of the microorganism as measured by the viability assay. 50
liGen/Biosearch, Bedford, MA) according to the manufacturer’s procedures. N-␣-Fmoc protected amino acids and preloaded PEG-PS-supports were obtained from Applied Biosystems (Foster City, CA). Peptides were labeled with fluorescein-5-isothiocyanate (FITC; Molecular Probes Europe BV, Leiden, The Netherlands) on an Fmoc-l-␥aminobutyric acid linker [21]. Purification was performed with RP-HPLC (Jasco Corporation, Tokyo, Japan) to a purity of at least 95% and the authenticity was confirmed by ion trap mass spectrometry with a LCQ Deca XP (Thermo Finnigan, San Jose, CA) [20]. The fluorescent-labeled peptides had the same candidacidal activities as the corresponding unlabeled peptides. 2.2. Strains and growth conditions C. albicans 315 (ATCC 10231) was cultured aerobically at 30 ◦ C in Sabouraud dextrose broth or on Sabouraud dextrose agar plates. The cells were cultured overnight and subcultured for 2–3 h to yield a mid-logarithmic growth culture at time of harvesting. Media were obtained from Difco (Becton Dickinson Microbiology systems, Sparks, MD). 2.3. Viability assay Two-fold serially diluted peptides (starting at a concentration of 100 M) in 1 mM potassium phosphate buffer, pH 7 (PPB), were incubated with C. albicans suspensions (approx. 7.5 × 106 CFU/ml) in 96-well U-bottom low affinity plates (Greiner, Recklinghausen, Germany). After 1 h of incubation at ambient temperature, cells were plated on Sabouraud dextrose agar plates. After 48 h, the percentage of viable cells was determined relative to incubation without added peptides
[21]. All experiments were repeated at least three times in duplicate. 2.4. FACScan analysis C. albicans cells were washed three times, set to a suspension of approximately 3.2 × 106 cells/ml and incubated with fluorescent-labeled peptides. Cell-associated fluorescence was measured at different time points with a FACScan (Fluorescence Associated Cell Scanner; Becton Dickinson, Franklin Lakes, NJ) equipped with a 15 mV Argon laser, at λexc = 488 nm and λem = 530 nm. Control experiments were carried out either without the addition of peptides, or with fluorescent-labeled inactive control peptide cystatin S 1–13, or with fluorescent dye. 2.5. Circular dichroism CD-spectra were recorded from 185 to 260 nm with a Jasco J-715 spectropolarimeter (Jasco Corporation), equipped with a Peltier temperature control system, at 20 ◦ C. Calibration was performed with an ammonium d-10camphorsulfonate solution, of which the concentration was checked spectro-photometrically, in milliQ water. Peptide concentrations used were 50 M in 1 mM phosphate buffer pH 7.0, in trifluoroethanol (TFE), or in mixtures of both solvents. A 0.1 cm cuvette for far-UV CD measurements was used. Ten scans were averaged for each sample. The response time was 0.25 s and the step size was set to 0.2 nm. Spectra were corrected for the background with an equally prepared sample without peptide and analyzed with the computer program CDNN.
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3. Results A glycine-scan of LFampin 265–284 peptide was performed (Table 1). Each of the residues Asp265 , Leu266 , Ile267 and Trp268 at the N-terminus and each of the charged residues were substituted. Their effect on the candidacidal activity was related to the binding to C. albicans and the tendency to adopt a helical structure. 3.1. Candidacidal activity of LFampin 265–284 variants Substitution of the negatively charged residues Asp265 and Glu276 had no or marginally stimulating effects on the candidacidal activity. On the other hand, replacement of each of the positively charged residues decreased the activity against C. albicans (Table 1). Substitution of Lys273 , Lys277 , Lys280 , Lys282 and Arg284 resulted in LC50 -values varying between 5 and 10 M in comparison with approx. 1 M for the parent peptide. Substitution of Lys269 decreased the killing activity only two-fold. The following order in activity was found: unsubstituted > Lys269 Lys282 > Arg284 > Lys280 > Lys277 > Lys273 . Substitution of the N-
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terminally situated Leu266 had no effect on the candidacidal activity, but substitution of the N-terminally situated Ile267 and Trp268 led to LC50 -values of 3.4 and 2.5, respectively. To see whether the effects of the different Lys substitutions are additive, double substituted peptides were synthesized. Multiple glycines would probably make the peptide too flexible, therefore double alanine-substituted (Lys273/277 and Lys277/280 ) peptides were analyzed as well as the respective single alanine-substitutions (Lys273 , Lys277 , and Lys280 ) to enable proper comparison with the glycine-variants. Substituting a single lysine for alanine had less severe effect on the candidacidal activity than substituting for glycine (approx. two-fold lower LC50 -value). Double substitution, resulting in peptides where Lys273/277 , or Lys277/280 were replaced by alanines, abolished the antifungal activity (LC50 -values of >100 M). 3.2. Binding of LFampin 265–284 variants to C. albicans To assess whether a decreased candidacidal activity was related to a decreased binding to C. albicans, a FACScan
Fig. 1. C. albicans cell-associated fluorescence of fluorescent-labeled LFampin peptides with single residue substitutions. Suspensions of 3.2 × 106 C. albicans cells/ml in 1 mM PPB were incubated with 5 M (A) LFampin 265–284, (B) LFampin 265–284 I267G, (C) LFampin 265–284 K269G and (D) LFampin 265–284 K273G. Cell-associated fluorescence was determined with a FACScan at the time points t = 1, 5 and 15 min.
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Fig. 2. CD spectra of LFampin peptides. The CD spectra of LFampin peptides with amino acid substitutions were recorded in water/TFE mixtures for LFampin 265–284, LFampin 265–284 I267G, LFampin 265–284 K269G and LFampin 265–284 K273G. Water/TFE ratio of 100/0 ( ), 25/75 ( ) and 50/50 (—).
analysis was conducted of the following variants: LFampin 265–284 K269G, in which Lys269 was substituted for glycine, K273G and I267G. Fluorescent peptides were incubated with C. albicans and cell-associated fluorescence was measured. After 5 min incubation with LFampin 265–284 I267G, cellassociated fluorescence already reached its maximum values (Fig. 1B), which is similar to the fluorescence of the parent peptide (Fig. 1A). LFampin 265–284 K269G (Fig. 1C) and K273G (Fig. 1D) showed lower fluorescence compared to the parent peptide. At t = 1 min, for both peptides lower fluorescence was found than for the control peptide. Differences in fluorescence were diminished after 5 and 15 min. Consequently, the difference in binding was negligible. 3.3. CD-spectra of LFampin 265–284 variants In pure water, the spectra of all peptides showed a minimum molar ellipticity at 200 nm (Fig. 2), which is characteristic for random coil conformation. At increasing TFE content of the solvent, ␣-helical conformations were clearly inducible in all peptides, indicated by a maximum molar ellipticity below 200 nm and by two negative CD bands between 206–208 nm and 218 to the parent 222 nm, respectively. No substantial differences were found in the CD-spectra thus the single-substituted peptides posses comparable propensity to adopt helical conformations. 4. Discussion The effects of single residue substitutions in LFampin 265–284 on antimicrobial activity, C. albicans-binding
capacity and the propensity to adopt a helical conformation were investigated. The most conspicuous difference between LFampin 265–284 and other cationic amphipathic peptides is the presence of a negatively charged amphipathic helix turn at its N-terminus. Although positively charged residues are essential for the membrane-disrupting action, there is no direct and simple correlation between the net positive charge and the potency of an antimicrobial peptide. It has been suggested that, when the net-positive charge becomes too high, the membrane activity may decrease [9,17]. The replacement of each of the negatively charged residues Asp265 and Glu276 at its turn for glycine, with a +1 increase in net-positive charge, barely had any effect on the candidacidal activity. This indicates that the contribution of Asp265 to the net-positive charge does not play an important role in the difference between LFampin 265–284 [22] and less active LFampin 268–284 lacking the residues Asp265 -Leu266 -Ile267 [21]. Instead, we found that these residues may enhance antimicrobial activity by facilitating helix formation; whereas removal of Asp265 , or Asp265 -Leu266 had no effect on the helix formation, removal of the stretch Asp265 -Leu266 -Ile267 led to reduced helicity [22]. According to our glycine-replacement study, Ile267 was the only one of the three residues which had effect on the candidacidal activity, however, none of the three substitutions led to substantial decreased propensity to form a helical conformation (Fig. 2). This seems contradictory to our previous finding that the residues Asp265 -Leu266 -Ile267 are important for adopting a helical conformation. Apparently, the effect on helix-formation of elongating LFampin 268–284 with these three residues reflects the existence of a critical length rather than the occurrence of critical residues. Moreover, helix formation is a cooperative process involving motifs rather than individual amino acids, thus removal of a residue resulting in a shift of the helix-capping motif [1] usually has more impact than substitution of that residue. For instance the presence of Asp265 induced importance for Ile267 in the theoretical N-terminal helix-capping motif. The fourth residue, Trp268 , is also of particular interest. Tryptophan is considered as an important amino acid in antimicrobial peptides due to its alleged propensity to insert in lipid bilayers [19]. In LFcin B and derived peptides, the combination of Trp and Arg residues is important for strong binding and membrane perturbation. Substitution of Arg for Lys resulted in a strong reduction in potency [13]. The same effect has been found in other Trp/Arg-rich peptides [4,5]. In LFampin 265–284, Trp268 is not surrounded by Arg residues, but flanked by the positively charged Lys269 . Nevertheless, it displayed a high candidacidal potency that was only marginally affected by substitution for glycine. In contrast, substitution of the Trp268 in LFampin 268–284, which is Nterminal in this peptide, resulted in a strong reduction of the killing activity (unpublished results). Furthermore, the substitution of Arg284 did not have more influence on the potency than replacement of most of the Lys residues. Therefore, we conclude that this peptide can bind to C. albicans cells and
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kill them without the clustering of Trp and Arg residues which has been postulated as important for membrane anchoring. Most important for the activity of antimicrobial peptides are the positively charged residues, arranged in an amphipathic helix that can interact with the negatively charged phospholipid membrane of the target cell. Substitution of each of the positively charged residues for glycine led to a decrease in candidacidal potency, however, the extent of this decrease was dependent on the position within the stretch. Substitution of Lys273 gave the strongest decrease, suggesting that the presence of a charged residue at this position is important for candidacidal activity. Substitution of two Lys residues abolished this activity completely. As mentioned above, the nature of the positively charged residue, Arg or Lys, did not seem to play a role. Acknowledgements The authors thank Ms. A. Teeken for skillfully technical assistance, and A.H. Westphal and S. Nabuurs, Laboratory of Biochemistry, Wageningen University, Wageningen, for their help with CD-measurements. This research was financially supported by the Dutch Technology Foundation, STW, grant VTH5180, the Dutch Dairy association and DMV International. References [1] Aurora R, Rose GD. Helix capping. Protein Sci 1998;7:21–38. [2] Bellamy W, Takase M, Wakabayashi H, Kawase K, Tomita M. Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. J Appl Bacteriol 1992;73:472–9. [3] Bellamy W, Takase M, Yamauchi K, Wakabayashi H, Kawase K, Tomita M. Identification of the bactericidal domain of lactoferrin. Biochim Biophys Acta 1992;1121:130–6. [4] Blondelle SE, Perez-Paya E, Houghten RA. Synthetic combinatorial libraries: novel discovery strategy for identification of antimicrobial agents. Antimicrob Agents Chemother 1996;40:1067–71. [5] Blondelle SE, Takahashi E, Dinh KT, Houghten RA. The antimicrobial activity of hexapeptides derived from synthetic combinatorial libraries. J Appl Bacteriol 1995;78:39–46. [6] Brock J. Lactoferrin: a multifunctional immunoregulatory protein? Immunol Today 1995;16:417–9. [7] Brogden KA, Ackermann M, McCray PB, Tack BF. Antimicrobial peptides in animals and their role in host defences. Int J Antimicrob Agents 2003;22:465–78.
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[8] Chen P-W, Shyu C-L, Mao FC. Antibacterial activity of short hydrophobic and basic-rich peptides. Am J Vet Res 2003;64:1088– 92. [9] Dathe M, Wieprecht T. Structural features of helical antimicrobial peptides: their potential to modulate activity on model membranes and biological cells. Biochim Biophys Acta 1999;1462:71– 87. [10] Elass-Rochard E, Roseanu A, Legrand D, Trif M, Salmon V, Motas C, et al. Lactoferrin–lipopolysaccharide interaction: involvement of the 28–34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide. Biochem J 1995;312:839–45. [11] Hwang PM, Zhou N, Shan X, Arrowsmith CH, Vogel HJ. Three-dimensional solution structure of lactoferricin B, an antimicrobial peptide derived from bovine lactoferrin. Biochemistry 1998;37:4288–98. [12] Iigo M, Kuhara T, Ushida Y, Sekine K, Moore MA, Tsuda H. Inhibitory effects of bovine lactoferrin on colon carcinoma 26 lung metastasis in mice. Clin Exp Metastasis 1999;17:35–40. [13] Kang JH, Lee MK, Kim KL, Hahm K-S. Structure–biological activity relationships of 11-residue highly basic peptide segment of bovine lactoferrin. Int J Pept Protein Res 1996;48:357–63. [14] Kuwata H, Yi T-T, Tomita M, Hutchens TW. Direct evidence of the generation in human stomach of an antimicrobial peptide domain (lactoferricin) from ingested lactoferrin. Biochim Biophys Acta 1998;1429:129–41. [15] Kuwata H, Yi T-T, Yamauchi K, Teraguchi S, Hayasawa H, Tomita M, et al. The survival of ingested lactoferrin in the gastrointestinal tract of adult mice. Biochem J 1998;334:321–3. [16] Levay PF, Viljoen M. Lactoferrin: a general review. Haematologica 1995;80:252–67. [17] Matsuzaki K. Why and how are peptide–lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim Biophys Acta 1999;1462:1–10. [18] Okunade AL, Elvin-Lewis MP, Lewis WH. Natural antimycobacterial metabolites: current status. Phytochemistry 2004;65:1017– 32. [19] Schiffer M, Chang CH, Stevens FJ. The functions of tryptophan residues in membrane proteins. Protein Eng 1992;5:213–4. [20] Tomita M, Takase M, Bellamy W, Shimamura S. A review: the active peptide of lactoferrin. Acta Paediatr Jpn 1994;36:585–91. [21] Van der Kraan MIA, Groenink J, Nazmi K, Veerman ECI, Bolscher JGM, Nieuw Amerongen AV. Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin. Peptides 2004;25:177–83. [22] Van der Kraan MIA, Nazmi K, Teeken A, Groenink J, van’t Hof W, Veerman ECI, et al. Lactoferrampin, an antimicrobial peptide of bovine lactoferrin, exhibits its candidacidal activity by a cluster of positively charged residues at the C-terminus in combination with a helix facilitating N-terminal part. Biol Chem 2005;386:137–42. [23] Yoo YC, Watanabe S, Watanabe R, Hata K, Shimazaki K, Azuma I. Bovine lactoferrin and lactoferricin, a peptide derived from bovine lactoferrin, inhibit tumor metastasis in mice. Jpn J Cancer Res 1997;88:184–90.
Effect of amino acid substitutions on the candidacidal activity of LFampin 265–284 Marieke I.A. van der Kraan, Christel van der Made, Kamran Nazmi, Wim van‘t Hof, Jasper Groenink, Enno C.I. Veerman, Jan G.M. Bolscher ∗ , Arie V. Nieuw Amerongen Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), Amsterdam, The Netherlands Received 21 December 2004; received in revised form 29 March 2005; accepted 30 March 2005 Available online 8 June 2005
Abstract LFampin 265–284, derived from bovine lactoferrin, has broad-spectrum antimicrobial activity against the yeast Candida albicans and several Gram-positive and Gram-negative bacteria. A glycine substitution scan was used to identify residues that are important for its candidacidal activity. Each single substitution of a positively charged residue led to considerable reduction in candidacidal activity, for each residue to a different extent. Substitution within the helix-facilitating N-terminal sequence DLIW had less severe effect; substitution of Ile and Trp led to a somewhat reduced potency. No substantial effects were found on the propensity to adopt a helical structure or to bind to C. albicans cells. © 2005 Elsevier Inc. All rights reserved. Keywords: Bovine lactoferrin; Lactoferrampin; LFampin; antimicrobial peptide; Candida albicans
1. Introduction Antimicrobial peptides are implicated in the innate immune systems of animals and plants (for recent reviews see [7,18]). Structural diversity among antimicrobial peptides is considerable, but most of them fold into an amphipathic ␣helical conformation upon interaction with the target organism. Antimicrobial amino acid stretches may appear as free peptides, but they can also be incorporated in the sequences of larger proteins. For instance bovine lactoferrin (bLF) possesses such an antimicrobial stretch: Lactoferricin B (LFcin B), a positively charged looped peptide encompassing residues 17–41, which is released by gastric pepsin cleavage [3,14,15]. It has more potent bactericidal and fungicidal activity than the parent protein [2,10,11,15,20], it has antitumor activity [12,23], it plays a regulatory role in the adaptive immune response and it has anti-inflammatory properties [6,16]. We have identified a second antimicrobial sequence within the N1-domain, designated lactoferrampin (LFampin) [21,22]. The corresponding peptide exhibited broad∗
Corresponding author. Tel.: +31 20 4448672; fax: +31 20 4448685. E-mail address: [email protected] (J.G.M. Bolscher).
0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.03.056
spectrum antimicrobial activity against the yeast Candida albicans and both Gram-positive and Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus mutans. In general, the activity of antimicrobial peptides is dependent on global structural parameters, such as a net positive charge and amphipathicity, rather than on the specific amino acid sequence. Nevertheless, in several antimicrobial peptides single critical residues have been identified [4,5,8,13]. In the present study we have conducted a glycine substitution scan of LFampin 265–284 to pinpoint residues that are critical for the candidacidal activity. The consequences of these substitutions for capacity to bind to C. albicans cells and for the tendency to adopt a helical structure are determined and discussed in relation to the according candidacidal activity.
2. Materials and methods 2.1. Synthesis and purification of peptides Peptides as listed in Table 1 were synthesized by Fmocchemistry with a MilliGen 9050 peptide synthesizer (Mil-
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Table 1 Properties of LFampin peptides with amino acid substitutions, together with the LC50 -values (M)a towards C. albicans
Peptides marked with an asterisk were also synthesized with an alanine substitution instead of a glycine substitution. These peptides (K273A, K277A and K280A) had less drastic effects on the antifungal activity; LC50 -values of alanine-substituted peptides were half that of glycine-substituted peptides (not shown). Double substitution resulting in the peptides K237/277A and K277/280A abolished the antifungal activity (LC50 -values of >100 M). a LC : the concentration of peptide (M) resulting in 50% reduction of the viable counts of the microorganism as measured by the viability assay. 50
liGen/Biosearch, Bedford, MA) according to the manufacturer’s procedures. N-␣-Fmoc protected amino acids and preloaded PEG-PS-supports were obtained from Applied Biosystems (Foster City, CA). Peptides were labeled with fluorescein-5-isothiocyanate (FITC; Molecular Probes Europe BV, Leiden, The Netherlands) on an Fmoc-l-␥aminobutyric acid linker [21]. Purification was performed with RP-HPLC (Jasco Corporation, Tokyo, Japan) to a purity of at least 95% and the authenticity was confirmed by ion trap mass spectrometry with a LCQ Deca XP (Thermo Finnigan, San Jose, CA) [20]. The fluorescent-labeled peptides had the same candidacidal activities as the corresponding unlabeled peptides. 2.2. Strains and growth conditions C. albicans 315 (ATCC 10231) was cultured aerobically at 30 ◦ C in Sabouraud dextrose broth or on Sabouraud dextrose agar plates. The cells were cultured overnight and subcultured for 2–3 h to yield a mid-logarithmic growth culture at time of harvesting. Media were obtained from Difco (Becton Dickinson Microbiology systems, Sparks, MD). 2.3. Viability assay Two-fold serially diluted peptides (starting at a concentration of 100 M) in 1 mM potassium phosphate buffer, pH 7 (PPB), were incubated with C. albicans suspensions (approx. 7.5 × 106 CFU/ml) in 96-well U-bottom low affinity plates (Greiner, Recklinghausen, Germany). After 1 h of incubation at ambient temperature, cells were plated on Sabouraud dextrose agar plates. After 48 h, the percentage of viable cells was determined relative to incubation without added peptides
[21]. All experiments were repeated at least three times in duplicate. 2.4. FACScan analysis C. albicans cells were washed three times, set to a suspension of approximately 3.2 × 106 cells/ml and incubated with fluorescent-labeled peptides. Cell-associated fluorescence was measured at different time points with a FACScan (Fluorescence Associated Cell Scanner; Becton Dickinson, Franklin Lakes, NJ) equipped with a 15 mV Argon laser, at λexc = 488 nm and λem = 530 nm. Control experiments were carried out either without the addition of peptides, or with fluorescent-labeled inactive control peptide cystatin S 1–13, or with fluorescent dye. 2.5. Circular dichroism CD-spectra were recorded from 185 to 260 nm with a Jasco J-715 spectropolarimeter (Jasco Corporation), equipped with a Peltier temperature control system, at 20 ◦ C. Calibration was performed with an ammonium d-10camphorsulfonate solution, of which the concentration was checked spectro-photometrically, in milliQ water. Peptide concentrations used were 50 M in 1 mM phosphate buffer pH 7.0, in trifluoroethanol (TFE), or in mixtures of both solvents. A 0.1 cm cuvette for far-UV CD measurements was used. Ten scans were averaged for each sample. The response time was 0.25 s and the step size was set to 0.2 nm. Spectra were corrected for the background with an equally prepared sample without peptide and analyzed with the computer program CDNN.
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3. Results A glycine-scan of LFampin 265–284 peptide was performed (Table 1). Each of the residues Asp265 , Leu266 , Ile267 and Trp268 at the N-terminus and each of the charged residues were substituted. Their effect on the candidacidal activity was related to the binding to C. albicans and the tendency to adopt a helical structure. 3.1. Candidacidal activity of LFampin 265–284 variants Substitution of the negatively charged residues Asp265 and Glu276 had no or marginally stimulating effects on the candidacidal activity. On the other hand, replacement of each of the positively charged residues decreased the activity against C. albicans (Table 1). Substitution of Lys273 , Lys277 , Lys280 , Lys282 and Arg284 resulted in LC50 -values varying between 5 and 10 M in comparison with approx. 1 M for the parent peptide. Substitution of Lys269 decreased the killing activity only two-fold. The following order in activity was found: unsubstituted > Lys269 Lys282 > Arg284 > Lys280 > Lys277 > Lys273 . Substitution of the N-
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terminally situated Leu266 had no effect on the candidacidal activity, but substitution of the N-terminally situated Ile267 and Trp268 led to LC50 -values of 3.4 and 2.5, respectively. To see whether the effects of the different Lys substitutions are additive, double substituted peptides were synthesized. Multiple glycines would probably make the peptide too flexible, therefore double alanine-substituted (Lys273/277 and Lys277/280 ) peptides were analyzed as well as the respective single alanine-substitutions (Lys273 , Lys277 , and Lys280 ) to enable proper comparison with the glycine-variants. Substituting a single lysine for alanine had less severe effect on the candidacidal activity than substituting for glycine (approx. two-fold lower LC50 -value). Double substitution, resulting in peptides where Lys273/277 , or Lys277/280 were replaced by alanines, abolished the antifungal activity (LC50 -values of >100 M). 3.2. Binding of LFampin 265–284 variants to C. albicans To assess whether a decreased candidacidal activity was related to a decreased binding to C. albicans, a FACScan
Fig. 1. C. albicans cell-associated fluorescence of fluorescent-labeled LFampin peptides with single residue substitutions. Suspensions of 3.2 × 106 C. albicans cells/ml in 1 mM PPB were incubated with 5 M (A) LFampin 265–284, (B) LFampin 265–284 I267G, (C) LFampin 265–284 K269G and (D) LFampin 265–284 K273G. Cell-associated fluorescence was determined with a FACScan at the time points t = 1, 5 and 15 min.
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Fig. 2. CD spectra of LFampin peptides. The CD spectra of LFampin peptides with amino acid substitutions were recorded in water/TFE mixtures for LFampin 265–284, LFampin 265–284 I267G, LFampin 265–284 K269G and LFampin 265–284 K273G. Water/TFE ratio of 100/0 ( ), 25/75 ( ) and 50/50 (—).
analysis was conducted of the following variants: LFampin 265–284 K269G, in which Lys269 was substituted for glycine, K273G and I267G. Fluorescent peptides were incubated with C. albicans and cell-associated fluorescence was measured. After 5 min incubation with LFampin 265–284 I267G, cellassociated fluorescence already reached its maximum values (Fig. 1B), which is similar to the fluorescence of the parent peptide (Fig. 1A). LFampin 265–284 K269G (Fig. 1C) and K273G (Fig. 1D) showed lower fluorescence compared to the parent peptide. At t = 1 min, for both peptides lower fluorescence was found than for the control peptide. Differences in fluorescence were diminished after 5 and 15 min. Consequently, the difference in binding was negligible. 3.3. CD-spectra of LFampin 265–284 variants In pure water, the spectra of all peptides showed a minimum molar ellipticity at 200 nm (Fig. 2), which is characteristic for random coil conformation. At increasing TFE content of the solvent, ␣-helical conformations were clearly inducible in all peptides, indicated by a maximum molar ellipticity below 200 nm and by two negative CD bands between 206–208 nm and 218 to the parent 222 nm, respectively. No substantial differences were found in the CD-spectra thus the single-substituted peptides posses comparable propensity to adopt helical conformations. 4. Discussion The effects of single residue substitutions in LFampin 265–284 on antimicrobial activity, C. albicans-binding
capacity and the propensity to adopt a helical conformation were investigated. The most conspicuous difference between LFampin 265–284 and other cationic amphipathic peptides is the presence of a negatively charged amphipathic helix turn at its N-terminus. Although positively charged residues are essential for the membrane-disrupting action, there is no direct and simple correlation between the net positive charge and the potency of an antimicrobial peptide. It has been suggested that, when the net-positive charge becomes too high, the membrane activity may decrease [9,17]. The replacement of each of the negatively charged residues Asp265 and Glu276 at its turn for glycine, with a +1 increase in net-positive charge, barely had any effect on the candidacidal activity. This indicates that the contribution of Asp265 to the net-positive charge does not play an important role in the difference between LFampin 265–284 [22] and less active LFampin 268–284 lacking the residues Asp265 -Leu266 -Ile267 [21]. Instead, we found that these residues may enhance antimicrobial activity by facilitating helix formation; whereas removal of Asp265 , or Asp265 -Leu266 had no effect on the helix formation, removal of the stretch Asp265 -Leu266 -Ile267 led to reduced helicity [22]. According to our glycine-replacement study, Ile267 was the only one of the three residues which had effect on the candidacidal activity, however, none of the three substitutions led to substantial decreased propensity to form a helical conformation (Fig. 2). This seems contradictory to our previous finding that the residues Asp265 -Leu266 -Ile267 are important for adopting a helical conformation. Apparently, the effect on helix-formation of elongating LFampin 268–284 with these three residues reflects the existence of a critical length rather than the occurrence of critical residues. Moreover, helix formation is a cooperative process involving motifs rather than individual amino acids, thus removal of a residue resulting in a shift of the helix-capping motif [1] usually has more impact than substitution of that residue. For instance the presence of Asp265 induced importance for Ile267 in the theoretical N-terminal helix-capping motif. The fourth residue, Trp268 , is also of particular interest. Tryptophan is considered as an important amino acid in antimicrobial peptides due to its alleged propensity to insert in lipid bilayers [19]. In LFcin B and derived peptides, the combination of Trp and Arg residues is important for strong binding and membrane perturbation. Substitution of Arg for Lys resulted in a strong reduction in potency [13]. The same effect has been found in other Trp/Arg-rich peptides [4,5]. In LFampin 265–284, Trp268 is not surrounded by Arg residues, but flanked by the positively charged Lys269 . Nevertheless, it displayed a high candidacidal potency that was only marginally affected by substitution for glycine. In contrast, substitution of the Trp268 in LFampin 268–284, which is Nterminal in this peptide, resulted in a strong reduction of the killing activity (unpublished results). Furthermore, the substitution of Arg284 did not have more influence on the potency than replacement of most of the Lys residues. Therefore, we conclude that this peptide can bind to C. albicans cells and
M.I.A. van der Kraan et al. / Peptides 26 (2005) 2093–2097
kill them without the clustering of Trp and Arg residues which has been postulated as important for membrane anchoring. Most important for the activity of antimicrobial peptides are the positively charged residues, arranged in an amphipathic helix that can interact with the negatively charged phospholipid membrane of the target cell. Substitution of each of the positively charged residues for glycine led to a decrease in candidacidal potency, however, the extent of this decrease was dependent on the position within the stretch. Substitution of Lys273 gave the strongest decrease, suggesting that the presence of a charged residue at this position is important for candidacidal activity. Substitution of two Lys residues abolished this activity completely. As mentioned above, the nature of the positively charged residue, Arg or Lys, did not seem to play a role. Acknowledgements The authors thank Ms. A. Teeken for skillfully technical assistance, and A.H. Westphal and S. Nabuurs, Laboratory of Biochemistry, Wageningen University, Wageningen, for their help with CD-measurements. This research was financially supported by the Dutch Technology Foundation, STW, grant VTH5180, the Dutch Dairy association and DMV International. References [1] Aurora R, Rose GD. Helix capping. Protein Sci 1998;7:21–38. [2] Bellamy W, Takase M, Wakabayashi H, Kawase K, Tomita M. Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. J Appl Bacteriol 1992;73:472–9. [3] Bellamy W, Takase M, Yamauchi K, Wakabayashi H, Kawase K, Tomita M. Identification of the bactericidal domain of lactoferrin. Biochim Biophys Acta 1992;1121:130–6. [4] Blondelle SE, Perez-Paya E, Houghten RA. Synthetic combinatorial libraries: novel discovery strategy for identification of antimicrobial agents. Antimicrob Agents Chemother 1996;40:1067–71. [5] Blondelle SE, Takahashi E, Dinh KT, Houghten RA. The antimicrobial activity of hexapeptides derived from synthetic combinatorial libraries. J Appl Bacteriol 1995;78:39–46. [6] Brock J. Lactoferrin: a multifunctional immunoregulatory protein? Immunol Today 1995;16:417–9. [7] Brogden KA, Ackermann M, McCray PB, Tack BF. Antimicrobial peptides in animals and their role in host defences. Int J Antimicrob Agents 2003;22:465–78.
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