BBRC Biochemical and Biophysical Research Communications 295 (2002) 826–827 www.academicpress.com
The complete amino acid sequence of the pediocin-like antimicrobial peptide leucocin C Gunnar Fimland,a,* Knut Sletten,a,b and Jon Nissen-Meyera a
Department of Biochemistry, University of Oslo, Post Box 1041, Blindern, Oslo 0316, Norway b The Biotechnology Centre, University of Oslo, Oslo, Norway Received 15 June 2002
Abstract The pediocin-like antimicrobial peptide leucocin C produced by a strain of Leuconostoc mesenteroides has been purified using a recently developed rapid two-step procedure. The complete and corrected amino acid sequence of the peptide has been determined by Edman degradation of the intact peptide and a C-terminal fragment generated by cleavage with Asp-N endoprotease. Leucocin C contained 43 residues with the following sequence: KNYGNGVHCTKKGCSVDWGYAWTNIANNSVMNGLTGGNAGWHN. The molecular weight of leucocin C as determined by mass spectrometry was 4595, which is consistent with the theoretical molecular weight of 4596 calculated from the sequence. Moreover, the molecular weights of the two fragments generated by cleavage with AspN were also consistent with the determined sequence. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Bacteriocins; Antimicrobial peptides; Lactic acid bacteria; Amino acid sequence
The antimicrobial peptide leucocin C was recently isolated from Leuconostoc mesenteroides strains and identified as a pediocin-like peptide (i.e., bacteriocins that belong to class IIa) [1,2]. The pediocin-like peptides (bacteriocins) constitute a dominant and well-studied group of antimicrobial peptides produced by lactic acid bacteria (LAB). The peptides are of considerable interest because of their anti-listerial activity. The group contains at least 20 members, all of which are cationic, contain between 37 and 48 amino acid residues, permeabilize target-cell membranes, and all of them have very similar primary structures [3]. The earlier reported amino acid sequence (36 sequenced residues) of leucocin C was not complete, since its molecular weight as determined by mass spectrometry (4597–4598) was not consistent with the theoretical molecular weight as calculated from the sequence [1,2]. We report here the purification of leucocin C to homogeneity using a recently developed rapid two-step procedure and the determination of its complete and corrected amino acid sequence. *
Corresponding author. Fax: +47-22-85-44-43. E-mail address: gunnar.fi
[email protected] (G. Fimland).
Materials and methods Purification of leucocin C. Leucocin C was produced by L. mesenteroides 6 [1] grown in MRS (Oxoid) at 30 °C and purified using a rapid two-step procedure that was recently developed [4]. In the first step, 200 ml of an overnight culture of L. mesenteroides 6 were applied directly on a 6-ml SP Sepharose Fast Flow (Amersham Biosciences) cation exchange column. Bacteria and anionic compounds passed through the column, whereas cationic peptides such as leucocin C bound to the column and were eluted with 1 M NaCl after first washing the column with 0.15 M NaCl. In the second step, the 1 M NaCl fraction from the cation exchanger was applied at a high flow rate on a low-pressure Resource reverse-phase column (Amersham Biosciences). Leucocin C bound to the column and was eluted with a propanol gradient as described previously [4]. The fraction containing leucocin C was identified by measuring the antimicrobial activity and by determining the molecular weight of the peptide(s) in the fraction by mass spectrometry. Assay for antibacterial activity. The activity of leucocin C was measured by using a microtiter plate assay system, essentially as described previously [5]. Briefly, each well of a microtiter plate contained 200 ll of culture medium (MRS) with peptide fractions at twofold dilutions and an indicator strain at an optical density at 610 nm of about 0.01. The microtiter plate cultures were incubated overnight (12–16 h) at 30 °C, after which growth of the indicator strain (Lactobacillus sake NCDO 2714) was measured spectrophotometrically at 610 nm with a microtiter plate reader. One activity unit was defined as the amount of peptide that inhibited growth of the indicator strain by 50%.
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 7 6 9 - 6
G. Fimland et al. / Biochemical and Biophysical Research Communications 295 (2002) 826–827 Cleavage of leucocin C with Asp-N endoprotease. An aliquot of purified leucocin C was cleaved with Asp-N endoprotease (Pseudomonas fragi protease, Boehringer Mannheim; 0.1 lg enzyme to 20 lg peptide, incubated for 2 h at 30 °C). The two fragments that were generated were separated by reverse-phase chromatography by using a lRPC SC 2.1/10 C2 =C18 column (Amersham Biosciences) in the SMART chromatography system (Amersham Biosciences). The peptides were eluted from the column with a 15–35% (vol/vol) 2-propanol gradient in 0.1% (vol/vol) trifluoroacetic acid. Determination of molecular weight and amino acid sequence. The molecular weight of leucocin C and the two fragments generated by cleavage with Asp-N protease was determined with a Voyager-DE RP matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (Perseptive Biosystems); a-cyano-4-hydroxycinnamic acid was used as the matrix. The amino acid sequences were determined by Edman degradation with an Applied Biosystems 477A automatic sequencer with an on-line 120A phenylthiohydantoin amino acid analyzer.
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weight of 4597–4598 [1,2]. Moreover, the theoretical molecular weights of the N-terminal (residues 1–16) and C-terminal (residues 17–43) fragments are, respectively, 1693 and 2921, which are in good agreement with our measured values of 1692 and 2921. A comparison of the complete sequence that we have obtained for leucocin C with the earlier reported 36-residue partial sequence [1,2] reveals that the complete sequence has 7 additional C-terminal residues and that the alanine residue in position 22 of the earlier reported partial sequence should be a tryptophan residue.
Acknowledgment This work was supported by a grant from the Norwegian Research Council.
Results and discussion Leucocin C purified to homogeneity by cationic exchange and reverse phase chromatography was sequenced by Edman degradation of the intact peptide and of the C-terminal fragment obtained after cleavage with Asp-N protease (cleaves on the N-terminal side of Asp in position 17). Edman degradation of the first 28 residues of the intact peptide resulted in the sequence: KNYGNGVHCTKKGCSVDWGYAWTNIANN and Edman degradation of the entire C-terminal fragment generated by cleavage with Asp-N protease resulted in the sequence: DWGYAWTNIANNSVMNGLTGGNA GWHN. Combining these two sequences, which have an overlap of 12 residues, results in the following full length sequence for leucocin C: KNYGNGVHCTKKGCSVDWGYAWTNIANNSV MNGLTGGNAGWHN. The theoretical molecular weight of this sequence is 4596, which is in good agreement with our experimental value of 4595 for the molecular weight of the purified bacteriocin and with the earlier reported molecular
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