activity of this fragment but had minimal effect on the histamine-releasing activity. These findings ... quirements for expression of the biological activity of GNCPs.
INFECTION AND IMMUNITY, June 1995, p. 2344–2346 0019-9567/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 63, No. 6
Involvement of Cysteine Residues in the Biological Activity of the Active Fragments of Guinea Pig Neutrophil Cationic Peptides SHIN YOMOGIDA,* ISAO NAGAOKA,
AND
TATSUHISA YAMASHITA
Department of Biochemistry, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan Received 7 November 1994/Returned for modification 12 December 1994/Accepted 7 March 1995
Guinea pig neutrophil cationic peptides (GNCPs) are single-chain polypeptides with 31 amino acid residues containing six cysteine residues, which exhibit both antibacterial and histamine-releasing activities in vitro. In this study, the role of the sulfhydryl groups in defining the antibacterial and histamine-releasing activities of the active fragments of GNCP-1 (Arg-1 to Tyr-14 [Arg-1–Tyr-14] and Arg-15–Tyr-27 peptides) was examined by using peptides containing alkylated or nonalkylated sulfhydryl groups. Alkylation slightly increased the histamine-releasing activity of the Arg-15–Tyr-27 (RRLGTCIFQNRVY) peptide but abrogated the antibacterial activity. Alkylation of the Arg-1–Tyr-14 (RRCICTTRTCRFPY) peptide similarly reduced the antibacterial activity of this fragment but had minimal effect on the histamine-releasing activity. These findings suggest that cysteine residues with free sulfhydryl groups play an important role in the expression of the antibacterial activity of the active fragments of GNCP-1.
action (4, 9, 12). In this study, therefore, we examined the involvement of sulfhydryl groups in the biological activity of GNCPs using the two active fragments of GNCP-1. GNCPs were purified from guinea pig peritoneal exudate neutrophils as described previously (15). Neutrophils (purity of .95%) were suspended in ice-cold 0.34 M sucrose at a concentration of 2 3 108 cells per ml and disrupted by sonication on ice with four 10-s bursts at 25 W (Tomy ultrasonic disrupter model UD-201; Tominaga Works). The sonicate was centrifuged at 420 3 g for 12 min at 48C, and the resulting supernatant was further centrifuged at 8,200 3 g for 15 min at 48C. The resultant pellet (granule fraction) was suspended in 0.34 M sucrose and sonicated for 1 min in ice at 168 W for solubilization. Cationic peptides (GNCP-1 and GNCP-2) were isolated by acid-polyacrylamide gel electrophoresis (15); the solubilized granule samples were electrophoresed on 15% polyacrylamide slab gels in b-alanine buffer (pH 4.5) at 48C for 4 h at 150 V. Thereafter, polypeptide bands were visualized by immersing the slab gel in a solution of 0.25% eosin Y in 0.1 N NaOH for 30 s. The band containing GNCP-1 and GNCP-2 was excised, ground, and subjected to electrophoretic elution in 0.16% acetic acid. GNCP-1 was further separated from GNCP-2 by reverse-phase high-performance liquid chromatography (HPLC) on a Wakosil 5C8 column (7.5 by 300 mm; pore size, 12.0 nm; Wako Pure Chemical Industries). Water-acetonitrile gradients containing 0.1% trifluoroacetic acid were employed for elution. Peptides were synthesized on a model 430A peptide synthesizer (Perkin Elmer Applied Biosystems Division) by using alkylated cysteine with an acetoamidomethylated sulfhydryl group or nonalkylated cysteine with a free sulfhydryl group. The peptides were released from the solid-phase resin with trifluoroacetic acid containing 1 M trifluormethane-sulfonic acid and 5% 1,2-ethanedithiol. The purity of the synthetic peptides was confirmed by HPLC and by N-terminal sequencing in a model 477A pulsed liquid sequencer (Perkin Elmer Applied Biosystems Division). The bactericidal activities of the peptides were tested against Staphylococcus aureus (NIHJ JC-1) as described previously (15). Cells were grown in nutrient broth for 16 h at 378C with
Neutrophils serve to protect mammalian hosts against microbial infections by intracellular killing of ingested organisms through oxygen-dependent and/or oxygen-independent mechanisms (3, 6–8, 13). Neutrophil granules contain antimicrobial proteins and peptides that contribute to the oxygen-independent mechanism. The most abundant granular antimicrobial components are low-molecular-weight cationic peptides (defensins). We previously purified the two structurally homologous cationic peptides, guinea pig neutrophil cationic peptides 1 and 2 (GNCP-1 and GNCP-2). GNCP-1 and GNCP-2 are single polypeptides containing 31 amino acid residues, which differ only by the substitution of an isoleucine (GNCP-1) for a leucine (GNCP-2) at position 21 (15). GNCP-1 and GNCP-2 have both antibacterial and histamine-releasing activities (15), suggesting that GNCPs not only contribute to the oxygenindependent antimicrobial system of phagocytes but also act to recruit mast cells into participating in acute inflammatory reactions. Recently, we have begun to examine structural requirements for expression of the biological activity of GNCPs. Because GNCP-1 and GNCP-2 display almost the same biological activities and GNCP-1 is more abundant than GNCP-2 in neutrophils (15), GNCP-1 was subjected to chemical and proteolytic treatment to obtain active fragments: Arg-1 to Tyr-14 (Arg-1–Tyr-14) (RRCICTTRTCRFPY) and Arg-15– Tyr-27 (RRLGTCIFQNRVY) (16). These fragments have the Arg-Arg sequence at the N terminus, suggesting that the arginine residues may be involved in the biological activities of GNCPs. In fact, the two synthetic peptides corresponding to Arg-1–Tyr-14 and Arg-15–Tyr-27 exerted antibacterial and histamine-releasing activities, and both activities were reduced and abrogated through the successive omission of the two Arg residues. The former peptide has three cysteine residues, and the latter has one cysteine residue. The cysteine residues in these peptides were all alkylated to protect sulfhydryl groups from oxidation in the synthetic process (16). Sulfhydryl groups are known to be important in enzyme activity and hormone
* Corresponding author. Mailing address: Department of Biochemistry, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyoku, Tokyo 113, Japan. Phone: 81 3-5802-1033. Fax: 81 3-3814-9300. 2344
VOL. 63, 1995
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FIG. 1. Effect of alkylation of sulfhydryl group on histamine-releasing and antibacterial activities of Arg-15–Tyr-27 peptide. (A) Mast cells were incubated with various concentrations of GNCP-1 (E) or alkylated (å) and nonalkylated (Ç) Arg-15–Tyr-27 peptides in Tris-ACM (pH 7.4) for 30 min at 378C. Histamine release is corrected for spontaneous release and expressed as the percentage of total histamine. (B) Bacteria (S. aureus) were incubated with various concentrations of GNCP-1 (E) or alkylated (å) and nonalkylated (Ç) Arg-15–Tyr-27 peptides in 10 mM sodium phosphate buffer (pH 7.4) for 20 min at 378C, diluted, and then plated. Antibacterial activity is expressed as killing percentage. Data are the means 6 standard deviations for three separate experiments.
shaking, washed twice in 10 mM phosphate buffer (pH 7.4) with centrifugation (6,000 3 g for 10 min), and diluted in the same buffer to give approximately 2 3 106 CFU/ml. Bacterial suspensions were incubated with various concentrations of the peptides in a total volume of 0.2 ml of 10 mM phosphate buffer for 20 min at 378C. In control experiments, the cells were incubated with the solvent of the peptides (0.01% acetic acid). After 1,000-fold dilution of the mixture with 10 mM phosphate buffer, 0.1-ml aliquots were spread on nutrient agar plates and incubated for 18 to 20 h to allow full colony development. The resulting colonies were counted, and the antibacterial activity was expressed as the percentage of colonies killed, determined by the following formula: killing (%) 5 [12 (number of colonies in the presence of the peptide/number of colonies in the absence of the peptide)] 3 100. Mast cells (purity, .85%) were obtained from SpragueDawley rats, and histamine release was assayed as described previously (15). Histamine release was initiated by the addition of 0.05 ml of peptides in 0.01% acetic acid to mast cells (2 3 104/ml) in a total volume of 1.5 ml Tris-ACM buffer (119 mM NaCl, 5 mM KCl, 0.6 mM CaCl2, 0.03% bovine serum albumin, 31 mM Tris-HCl [pH 7.4]), and then mast cells were incubated at 378C for 30 min. Histamine release was terminated by placing test tubes in an ice-water bath, followed by centrifugation at 220 3 g for 10 min at 48C. The histamine concentration was determined by the o-phthaldialdehyde spectrophotofluorometric procedure (10, 14). Total cellular histamine was determined by using the cell samples which had been incubated at 378C with peptides for 30 min and then sonicated for 30 s. Histamine
release was corrected for spontaneous release (3.0% 6 0.4% [mean 6 standard deviation], n 5 3) and expressed as the percentage of total histamine. Figure 1 demonstrates the effect of alkylation of the sulfhydryl group on the ability of the Arg-15–Tyr-27 (RRLGTCI FQNRVY) peptide to release histamine from mast cells and to kill bacteria. The synthetic alkylated peptide showed somewhat higher levels of histamine-releasing activity than GNCP-1, whereas the synthetic nonalkylated peptide displayed lower levels of activity than GNCP-1 (Fig. 1A). The 50% effective dose (ED50) values were 0.3, 0.14, and 0.82 mM with GNCP-1, alkylated peptide, and nonalkylated peptide, respectively. Next, we examined the ability of Arg-15–Tyr-27 peptides to kill bacteria. The activities were greatly affected by whether cysteine residues were alkylated (Fig. 1B). The alkylated peptide showed a remarkable decrease in activity compared with GNCP-1, whereas the nonalkylated peptide showed almost the same dose-dependent pattern as GNCP-1. ED50 values were 0.68, 8.0, and 0.72 mM with GNCP-1, alkylated peptide, and nonalkylated peptide, respectively. Figure 2 shows the effect of alkylation of sulfhydryl groups on the biological activities of the Arg-1–Tyr-14 (RRCICTTRT CRFPY) peptide, which contains cysteine residues at positions 3, 5, and 10. The synthetic peptide in which all the cysteine residues were alkylated showed low levels of histamine-releasing activity compared with GNCP-1 (ED50 of 3.5 mM) (Fig. 2A). Synthetic peptides with an alkylated cysteine residue at position 10 and nonalkylated cysteine residues at positions 3 and 5 displayed almost the same histamine-releasing activity as
FIG. 2. Effect of alkylation of sulfhydryl group on the histamine-releasing and antibacterial activities of Arg-1–Tyr-14 peptide. (A) Mast cells were incubated with various concentrations of GNCP-1 (E) or alkylated (å) and nonalkylated (Ç) Arg-1–Tyr-14 peptides in Tris-ACM (pH 7.4) for 30 min at 378C. Histamine release is corrected for spontaneous release and expressed as the percentage of total histamine. (B) Bacteria (S. aureus) were incubated with various concentrations of GNCP-1 (E) or alkylated (å) and nonalkylated (Ç) Arg-1–Tyr-14 peptides in 10 mM sodium phosphate buffer (pH 7.4) for 20 min at 378C, diluted, and then plated. Antibacterial activity is expressed as killing percentage. The nonalkylated peptide contained an alkylated cysteine residue at position 10 and nonalkylated cysteine residues at positions 3 and 5. Data are the means 6 standard deviations for three separate experiments.
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the peptide with all alkylated cysteine residues (ED50 of 4.0 mM) (Fig. 2A). On the other hand, the antibacterial activity of the Arg-1–Tyr-14 peptide was affected by the alkylation of cysteine residues, as seen in Fig. 2B. The antibacterial activity of the synthetic peptide was remarkably decreased by alkylation of all cysteine residues compared with that of GNCP-1 (ED50 of 28 mM) but was not decreased by alkylation of the cysteine residue at position 10 (ED50 of 1.4 mM). In addition to GNCPs, cathepsin G (1), lactoferrin (2), bactericidal/permeability-increasing protein (11), and major basic protein (5) are known as antibacterial proteins. The active fragments of these proteins have been isolated, and the positively charged amino acids present in these molecules, such as arginine, are assumed to be important for the expression of the antibacterial activity (1, 2, 5, 11). We have recently shown that the biological activities of the active fragments of GNCP-1 are reduced and abrogated through the successive omission of the two N-terminal arginine residues (16). In addition, we have found that the antibacterial activity of the active fragment of the major basic protein is completely abolished by the conversion of arginine residues to the citrulline residues (5). On the other hand, the role of the cysteine residues of the antibacterial proteins has not been clearly identified, although sulfhydryl groups are known to be important in enzyme activity and hormone action (4, 9, 12). The results of the present study indicated that the alkylation of cysteine residues significantly reduced the antibacterial activity of the active fragments of GNCP-1. Therefore, not only arginine residues but also cysteine residues with free sulfhydryl groups are likely to play an important role in the expression of the antibacterial activity of the active fragments of GNCP-1. The mechanism by which cysteine residues participate in the antibacterial activity of these peptides remains to be elucidated. Our present findings may help the design of clinically useful antibacterial peptides in the future. This work was supported in part by the Science Research Promotion Fund from the Japan Private School Promotion Foundation.
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