1 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, 2 Structural Biology Group, Research Institute of Biological Resources, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, 3 Department of Biology, Kyushu University, Fukuoka 812-8581, 4 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Tokyo 101-0062 and 5 Department of Structural Biology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan
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Abstract |
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Keywords: amidation/antimicrobial activity/backbone dynamics/chitin binding/refolding
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Introduction |
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Tachycitin (73 residues, Mw = 8.5 kDa) is an invertebrate defense molecule which does not belong to any of the groups of antimicrobial peptide identified so far (e.g. insect defensin family), but shares a significant sequence homology with the cysteine-rich chitin-binding domain of the invertebrate chitin-binding proteins [e.g. the peritrophic matrix protein peritrophin-44 (Elvin et al., 1996)]. Tertiary structural information of the invertebrate chitin-binding proteins has only been provided from our NMR study (Suetake et al., 2000
). The structure of tachycitin mainly consists of a globular domain with a unique ß-sandwich fold, which is contributed by a three-stranded and a two-stranded ß-sheet, several turns, and five disulfide bridges. Additionally, a disordered structure was suggested for the N- and C-terminal pentapeptides segments of tachycitin (H2NTyr1LeuAlaPheArg5 and His69LeuTrpLysThr73CONH2). Analyses of C-terminal threoninamide released after lysylendopeptidase digestion (Kawabata et al., 1996
) indicates that the natural form of tachycitin possesses an amidated C-terminus, which is presumably a result of the post-translational modification as found for the other cationic antimicrobial peptides [e.g. cecropins (Steiner et al., 1981
), tachyplesins (Nakamura et al., 1988
), Bombus pascuorum defensin (Rees et al., 1997
) and penaeidins (Destoumieux et al., 1997
)]. For a segment Cys40 to Gly60 of tachycitin, a remarkable similarity in local structure with the plant chitin-binding proteins was identified (e.g. Cys12 to Gly32 of wheat germ agglutinin). In this region a chitin-binding site protrudes comprising of a ß-hairpin loop, in which the side chains of Trp and/or Tyr residues are involved. Based on this prominent local similarity, it was assumed that tachycitin and plant chitin-binding proteins are correlated with the convergent evolution process (Wright et al., 1991
; Shen and Jacobs-Lorena, 1999
; Suetake et al., 2000
). It has been demonstrated that the plant chitin-binding proteins exhibit antimicrobial activity against fungi, whose cell wall contains chitin as a vital constituent (Mirelman et al., 1975
; Broekaert et al., 1989
, 1992
; Parijs et al., 1991
; Koo et al., 1998
; Huang et al., 2000
). Hence, the similarity further allows us to assume that tachycitin has an antimicrobial function against fungi similar to the case of plant chitin-binding proteins. However, the antimicrobial function of tachycitin against bacteria is unclear, the cell surface of which does not contain chitin.
Here, we succeeded in performing a genetic expression and protein refolding of a recombinant antimicrobial protein tachycitin (rTcn) from inclusion bodies in Escherichia coli. A difference between native tachycitin and rTcn is that the latter does not have the amide group at the C-terminus, which appeared to depress the antimicrobial activity against bacteria, but not against fungi. The present characterization of the structure, the 15N-NMR dynamics property, and antimicrobial activity of the rTcn will help to understand more the details of the antimicrobial mechanism of tachycitin.
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Materials and methods |
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The gene fragment encoding tachycitin (Kawabata et al., 1996) was used as a template for the polymerase chain reaction (PCR). The primers containing NdeI and EcoRI sites were used for the PCR strategy, and their sequences were as follows: 5'-AACTGCAGCTCGACCATATGTACCTGGCTT-TTCGTTGCGG-3' for the 5' primer and 5'-AACTGCA-GGAGCTCGAATTCTTATGTTTTCCAAAGATGACATTC-3' for the 3' primer. The primers were designed to eliminate N- and C-terminal putative signal sequences. Pyrococcus furiosus DNA polymerase (Pfu polymerase; Stratagene, CA, USA) was used. The condition for one PCR cycle was as follows: denaturation for 45 s at 95°C, annealing for 45 s at 55°C, and extension for 1 min at 72°C. This cycle was repeated 30 times. The PCR product was purified with GFX PCR DNA and the Gel Band Purification Kit (Amersham, NJ, USA). The purified PCR product and the parent vector, pET-22b(+) (Novagen, WI, USA) were digested with NdeI and EcoRI, and then ligated using T4 DNA ligase (DNA Ligation Kit; Takara, Tokyo, Japan) overnight at 4°C. The ligated vector was transformed into E.coli, DH5
. The amplified vector was purified and its sequence was confirmed using a DNA sequencer (Hitachi SQ5500).
Expression, refolding and purification of rTcn
Escherichia coli BL21 (DE3)/pLysS competent cells were transformed with the vector and cultured overnight in LB broth supplemented with 50 mg/l ampicillin at 37°C. In order to inoculate the final culture, the overnight culture was resuspended into the expression media. The culture was incubated at 37°C in shake-flasks until the optical density (600 nm) reached 0.81.0, and was induced with 100mg/l isopropyl-ß-D-thiogalactopyranoside (IPTG). After an additional 34 h of incubation, the cells were harvested by centrifugation. The harvested cell paste was disrupted by an ultrasonicator (Insonator 201M; Kubota, Tokyo, Japan) in a cold lysis buffer (20 mM TrisHCl, pH 7.5, 3 mM EDTA and 0.5% Triton X-100). From the lysed cell suspension, inclusion bodies were isolated by centrifugation at 12 000 g for 20 min at 4°C and were washed with 0.5% Triton X-100 and distilled water, then stored at 20°C. The inclusion bodies solubilized with 8 M urea, 20 mM TrisHCl (pH 7.5), 3 mM EDTA and 200 mM ß-mercaptoethanol, were then incubated for 3 h at 37°C to reduce any disulfide bonds. After removal of insoluble material by centrifugation for 1 h at 4°C, the solution was applied to a SP-Sepharose FF column (Pharmacia Biotech, Uppsala, Sweden) pre-equilibrated with 6 M urea, 20 mM TrisHCl (pH 7.5), 3 mM EDTA and 3 mM ß-mercaptoethanol. The column was washed with 6 M urea and eluted with 0.3 M NaCl. The refolding reaction was initiated by rapid dilution. Reduced rTcn (250350 µg/ml) in 4 M urea was 10-fold diluted into a refolding buffer. The amount of the rTcn isomer was monitored after 1, 2, 3 and 4 days of refolding experiments using reverse-phase HPLC (RP-HPLC) on a YMC-Pack C4-AP column (250x4.6 mm). The solvent systems used for HPLC were 0.1% (v/v) aq. trifluoroacetic acid (TFA) in water, and 0.1% (v/v) aq. TFA in acetonitrile. The fraction containing rTcn was eluted using a flow rate of 0.5 ml/min and acetonitrile gradients of 020% over 10 min followed by 2040% over 15 min. The refolding conditions of rTcn were optimized as described later (Figure 1). Consequently, the refolding experiment was performed for 4 days at 4°C in the presence of 20 mM TrisHCl (pH 7.5), 0.25 mM ß-mercaptoethanol, 3 mM EDTA and 50 mM NaCl.
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Biological assays
For the measurements of chitin-binding activity of rTcn, the excess amount of powdered chitin (Chitin EX; Funakoshi Co., Ltd, Tokyo, Japan) was mixed with rTcn dissolved in 100 ml of 20 mM TrisHCl (pH 7.5), 150 mM NaCl and 2 mM CaCl2, and then incubated at room temperature for 15 min. After centrifugation (15 000 r.p.m. 32,200 g) of the mixture for 2 min, the precipitate that mainly comprises chitin powder was washed with 1 M NaCl and 10% acetic acid to detach the chitin-bound protein. The SDSpolyacrylamide gel electrophoresis (SDSPAGE) was then performed to see whether rTcn is contained in the fractions of either the supernatant or precipitate (Kawabata et al., 1996).
Antimicrobial activity was measured by observation of rTcns growth inhibition activity against microorganisms as described by Saito et al. (Saito et al., 1995); i.e. a microorganism suspension and rTcn solution mixture was incubated for 1 h at 37°C, and then plated on agar plates so as to count the numbers of surviving colonies after 24 h. The same microorganism suspension without rTcn was also plated, and the colony numbers were counted after 24 h as a reference. Here we define the activity as the percentage of the surviving colonies: i.e. [(surviving colony numbers) / (total colony numbers)]x100 (%). These experiments were performed against different concentrations of rTcn (0
100 µg/ml), and the same set of experiments was also carried out for native tachycitin for comparison. The microorganisms used for this antimicrobial assay were Staphylococcus aureus 209P (Gram-positive bacteria), E.coli B (Gram-negative bacteria) and Pichia pastoris GS115 (fungi).
NMR spectroscopy
NMR experiments were performed on a Varian UNITY Inova 500 spectrometer equipped with a triple-resonance pulse-field x, y, and z gradient probe heads. For 15N-labeled tachycitin, three-dimensional (3D) 15N-edited total correlation spectroscopy (TOCSY) (Kay et al., 1989), and 15N-edited nuclear Overhauser effect spectroscopy (NOESY) (Marion et al., 1989
), as well as two-dimensional (2D) 1H-15N hetero nuclear single quantum coherence spectroscopy (HSQC) (Bax et al., 1983
) were performed. The 15N-T1, 15N-T2, and {15N-1H}-NOE experiments were performed using the pulse sequences from Farrow et al. (Farrow et al., 1994
). The backbone amide 15N relaxation data were collected at four different concentrations (0.26, 0.34, 0.52 and 1.00 mM) in order to calibrate the dependence of the overall correlation time (
m) on the rTcn concentration. The 15N-NMR relaxation delays of 10, 20, 40, 80, 160, 320 and 640 ms were used for T1 measurements, and those of 10, 30, 50, 70, 90, 110, 130 and 150 ms for T2 measurements. For the T2 measurements, we chose 2.8 s of relaxation delay between transients to lower the sample heating. The number of transients per complex t1 point was 16. The decays of cross-peak intensities with time T in the 15N-T1 and -T2 experiments were fitted to a single exponential by a non-linear least-squares method. All the 1H-15N 2D spectra were peak-picked using PIPP and STAPP programs (Garret et al., 1991
). The {15N-1H} steady-state NOE values were obtained from the ratios of the intensities of experiments recorded with and without proton saturation. The uncertainty in the NOE values was estimated from the baseline noise levels as described previously (Farrow et al., 1994
). The chemical shifts were referenced to the internal standard, 2,2,3,3-tetradeutero-3-(trimethylsilyl) propionic acid sodium salt (0.00 p.p.m.). All NMR data were processed using NMRPipe (Delaglio et al., 1995
), PIPP (Garret et al., 1991
) and XEASY (Bartels et al., 1995
) software.
The experimental data set of the 15N relaxation time was analyzed on the basis of the model-free approach (Lipari and Szabo, 1982a,b
). Theoretical expressions for the T1, T2 and NOE relaxation parameters of an amide 15N nucleus have been well established. The practical calculation of these parameters was carried out in the same manner as Farrow et al. (Farrow et al., 1994
). The value of averaged
m was calculated by using the relaxation time of the residues whose NOE along the NH bond vector is larger than 0.60 (Gagné et al., 1998
). We estimated the order parameter (S2) and the segmental motion correlation time (
e) using the model-free approach on the basis of an assumption that tachycitin undergoes the isotropic tumbling motion. We found a set of the best-fit parameters (S2,
e) by grid search, which minimized the calculation errors for each residue.
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Results and discussion |
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Figure 2 shows the 1H-15N HSQC spectrum of the 15N-labeled rTcn at 30°C. The sequence-specific assignment for rTcn was completed by identification of the sequential NOEs using the 15N-edited NOESY and the 15N-edited TOCSY spectra as well as the previous 1H-NMR assignments for the native tachycitin (BioMagResBank accession no. 4290) (Suetake et al., 2000
). The assignment is indicated for each cross-peak of Figure 2
by amino acid type and residue number. No observation of the unassigned peak in Figure 2
indicates the successful recombination and purification of this protein. It should be noted that the resonance of the NH-group of Lys32 does not appear to be due to a significant line broadening, while it is observed at higher temperatures (>35°C). The backbone resonance of Tyr1 is not observed at any temperature due to a rapid exchange with the solvent.
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The chitin-binding activity of rTcn was examined by SDSPAGE for the supernatant and precipitate obtained after centrifugation against the suspension containing the excess amount of chitin mixed with rTcn. In the electrophoretogram, no rTcn band was observed for the supernatant, while it was observed for the precipitate (the bound protein was eluted with 10% acetic acid after washing with 1 M NaCl). This result clearly shows that rTcn has the ability to bind to chitin; i.e. the chitin-binding activity is not changed by recombination. Because rTcn does not have the amide group at the C-terminal end, the data further suggests that the C-terminal amidation is not essential for the chitin-binding activity. It is noted again that the NMR structural study (Suetake et al., 2000) suggested that a segment from Cys40 to Cys60 of tachycitin is an essential site for chitin binding. The antimicrobial activity was further examined for native tachycitin and rTcn by observation of their growth inhibition efficiencies against the three representative microorganisms: S.aureus 209P (Gram-positive bacteria), E.coli B (Gram-negative bacteria) and P.pastoris GS115 (fungi). Figure 6
plots the percentage of the surviving colonies of these microorganisms under various protein concentrations of native tachycitin and rTcn. The reduction of the percentage indicates the increased efficiency of the antimicrobial activity of tachycitin. As shown in Figure 6ac
, the native tachycitin significantly reduces the percentage of the surviving colonies of the Gram-positive and -negative bacteria, and fungi as well, showing that native tachycitin is active against both bacteria and fungi. However, no significant reduction in the percentage of the surviving colonies is observed for rTcns activity against Gram-positive and -negative bacteria (Figure 6a and b
). Only against fungi is the antimicrobial activity recognized for both native tachycitin and rTcn (Figure 6c
). These results suggest that rTcn, which does not have the amidated C-terminus, retains the antimicrobial activity against fungi but is not active against bacteria. It should be noted that fungi contain chitin as an essential constituent of the cell surface but bacteria do not. Therefore, this disappearance of the antimicrobial activity against bacteria is presumably ascribed to the deletion of the C-terminal amide group due to recombination. The reduction of the antimicrobial activity was also reported for non-amidated forms of sarcotoxin IA (Nakajima et al., 1987
) and cecropin A (Callaway et al., 1993
). For sarcotoxin IA, Nakajima et al. (Nakajima et al., 1987
) assumed that the C-terminal amidation raises the electropositivity of the peptide, which might have a significant contribution to the electrostatic interaction between the peptide and cell membrane. In addition, an amphipathic structure is significantly implicated in the interaction between bacterial surface and many antimicrobial peptides including cecropin A and sarcotoxin IA. Such amphipathicity is also identified in the tertiary structure of tachycitin shown in Figure 4
; a cluster of the hydrophobic residues (e.g. Leu13, Leu21, Tyr22 and Val64) and that of the hydrophilic residues (e.g. His31, Lys32 and Arg36) are located at the opposite side of the central ß-sheet (ß1ß3).
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Notes |
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Acknowledgments |
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References |
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Received April 23, 2002; revised May 23, 2002; accepted June 10, 2002.