Production and characterization of recombinant tachycitin, the Cys-rich chitin-binding protein

Tetsuya Suetake1,2, Tomoyasu Aizawa1, Nozomi Koganesawa1, Tsukasa Osaki3, Yoshihiro Kobashigawa1,2, Makoto Demura1, Shun-ichiro Kawabata3,4, Keiichi Kawano5, Sakae Tsuda2,6 and Katsutoshi Nitta1

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Tachycitin is an invertebrate chitin-binding protein with an amidated C-terminus, and possesses antimicrobial activity against both fungi and bacteria. The 1H-NMR-based tertiary structure of tachycitin was recently determined [Suetake et al. (2000)Go J. Biol. Chem., 275, 17929–17932]. In order to examine the structural and functional features of tachycitin more closely, we performed for the first time, gene expression, refolding, 15N-NMR-based characterizations, and antimicrobial activity measurements of a recombinant tachycitin (rTcn) that does not have the amide group at the C-terminus. The NMR analysis indicated that rTcn possesses the same structural construction as the native tachycitin. The backbone 15N relaxation measurements showed that the molecular motional correlation time of rTcn increases as its concentration increases, indicating that tachycitins have a tendency to aggregate with each other. rTcn exhibits antimicrobial activity against fungi but not against bacteria. The cell surface of fungi contains chitin as an essential constituent, but that of bacteria does not. These results suggest that not only the chitin-binding region but also the C-terminal amide group of tachycitin plays a significant role in its antimicrobial properties.

Keywords: amidation/antimicrobial activity/backbone dynamics/chitin binding/refolding


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antimicrobial substances are often small cationic peptides and extensively distributed in animals and plants, which have been known to participate in a host defense reaction (Boman, 1995Go; Broekaert et al., 1995Go; Dimarcq et al., 1998Go; Hancock and Diamond, 2000Go). Iwanaga and co-workers identified various cationic antimicrobial substances from the hemocyte granules of an aquatic invertebrate, Japanese horseshoe crab (Tachypleus tridentatus) (Nakamura et al., 1988Go; Shigenaga et al., 1993Go; Iwanaga et al., 1998Go). Among the substances, Kawabata et al. (Kawabata et al., 1996Go) isolated a cysteine-rich type of the peptide named tachycitin which shows a broad range of antimicrobial activity against both fungi and bacteria, and further exhibits agglutinating activity against bacteria. For tachycitin, we have succeeded in performing a 1H-NMR-based structural determination (Suetake et al., 2000Go), which revealed that tachycitin shares a significant tertiary structural similarity with several plant chitin-binding proteins with regard to the chitin-binding structural motif.

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., 1996Go)]. Tertiary structural information of the invertebrate chitin-binding proteins has only been provided from our NMR study (Suetake et al., 2000Go). 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 (H2N–Tyr1–Leu–Ala–Phe–Arg5– and –His69–Leu–Trp–Lys–Thr73–CONH2). Analyses of C-terminal threoninamide released after lysylendopeptidase digestion (Kawabata et al., 1996Go) 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., 1981Go), tachyplesins (Nakamura et al., 1988Go), Bombus pascuorum defensin (Rees et al., 1997Go) and penaeidins (Destoumieux et al., 1997Go)]. 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., 1991Go; Shen and Jacobs-Lorena, 1999Go; Suetake et al., 2000Go). 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., 1975Go; Broekaert et al., 1989Go, 1992Go; Parijs et al., 1991Go; Koo et al., 1998Go; Huang et al., 2000Go). 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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 References
 
Tachycitin expression vector preparation

The gene fragment encoding tachycitin (Kawabata et al., 1996Go) 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{alpha}. 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 0.8–1.0, and was induced with 100mg/l isopropyl-ß-D-thiogalactopyranoside (IPTG). After an additional 3–4 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 Tris–HCl, 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 Tris–HCl (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 Tris–HCl (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 (250–350 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 0–20% over 10 min followed by 20–40% over 15 min. The refolding conditions of rTcn were optimized as described later (Figure 1Go). Consequently, the refolding experiment was performed for 4 days at 4°C in the presence of 20 mM Tris–HCl (pH 7.5), 0.25 mM ß-mercaptoethanol, 3 mM EDTA and 50 mM NaCl.



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Fig. 1. The RP-HPLC pattern showing the amount of refolded rTcn after 4 days incubation under different sets of ß-mercaptoethanol concentrations and temperatures: (a) 0.5 mM and 22°C, (b) 0.25 mM and 22°C, (c) 0.5 mM and 4°C, and (d) 0.25 mM and 4°C. The highest peak observed in (a–d) each corresponds to the amount of an isomer of rTcn. The other buffer conditions are [Tris–HCl] = 20 mM, pH 7.5, [EDTA] = 3 mM and [NaCl] = 50 mM.

 
The solution of the peak containing rTcn was applied to a chitin affinity column (1.8x15 cm) previously equilibrated with Tris-EDTA (TE) buffer. After adsorption, the column was pre-washed with TE buffer containing 1 M NaCl. The elution of rTcn was carried out with 100 mM acetic acid and the peak fractions were collected and dialyzed against water, and then lyophilized. As for the expression of uniformly 15N-labeled rTcn, the cells were grown in M-9 minimal medium containing 15NH4Cl (1.0 g/l) and natural glucose (2.0 g/l). The enriched (>99%) 15N-labeled NH4Cl was purchased from Cambridge Isotope Laboratories (MA, USA). The NMR samples were prepared by dissolving rTcn in 0.3 ml of 90% H2O/10% D2O (v/v) whose pH was adjusted to 4.7 by additions of DCl and NaOD (final concentration of rTcn is 0.05~1 mM). It is noted that tachycitin in native form was purified from hemocyte debris of the horseshoe crab T.tridentatus as described (Kawabata et al., 1996Go). The purity of the final protein samples were analyzed by MALDI-TOF mass spectrometry (Voyager DE-PRO; PerSeptive Biosystems, MA, USA).

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 Tris–HCl (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 SDS–polyacrylamide gel electrophoresis (SDS–PAGE) was then performed to see whether rTcn is contained in the fractions of either the supernatant or precipitate (Kawabata et al., 1996Go).

Antimicrobial activity was measured by observation of rTcn’s growth inhibition activity against microorganisms as described by Saito et al. (Saito et al., 1995Go); 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., 1989Go), and 15N-edited nuclear Overhauser effect spectroscopy (NOESY) (Marion et al., 1989Go), as well as two-dimensional (2D) 1H-15N hetero nuclear single quantum coherence spectroscopy (HSQC) (Bax et al., 1983Go) 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., 1994Go). 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 ({tau}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., 1991Go). 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., 1994Go). 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., 1995Go), PIPP (Garret et al., 1991Go) and XEASY (Bartels et al., 1995Go) software.

The experimental data set of the 15N relaxation time was analyzed on the basis of the model-free approach (Lipari and Szabo, 1982aGo,bGo). 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., 1994Go). The value of averaged {tau}m was calculated by using the relaxation time of the residues whose NOE along the N–H bond vector is larger than 0.60 (Gagné et al., 1998Go). We estimated the order parameter (S2) and the segmental motion correlation time ({tau}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, {tau}e) by grid search, which minimized the calculation errors for each residue.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A key procedure to obtain a suitable amount of rTcn was to optimize the experimental condition for refolding of a denatured rTcn obtained from the cation exchange chromatography (SP-Sepharose FF column), which was applied against the solubilized inclusion body containing rTcn. Figure 1Go compares the amount of the refolding products observed as the RP-HPLC pattern. The highest peak observed in Figure1a–d each represents the amount of a major isomer of rTcn, and the other observed peaks are presumably ascribed to inappropriately disulfide-bonded forms of rTcn and/or other proteins. Before performing the HPLC, the four solutions (Figure 1a–dGo) were incubated for 4 days under different sets of ß-mercaptoethanol concentration and temperature in the presence of 20 mM Tris–HCl (pH 7.5), 3 mM EDTA and 50 mM NaCl. Consequently, the ß-mercaptoethanol concentration of 0.25 mM and temperature of 4°C (Figure 1dGo) were estimated as the optimal conditions to give the highest refolding efficiency of rTcn. The yield of the purified rTcn and the 15N-labeled rTcn per 1 l of the culture was ~1.0 and ~0.5 mg, respectively. Mass spectral analysis of the purified sample gave a molecular weight of 8486 ± 1, which is consistent with the calculated mass of the unamidated form of tachycitin. The purified rTcn was used for all the subsequent experiments.

Figure 2Go 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., 2000Go). The assignment is indicated for each cross-peak of Figure 2Go by amino acid type and residue number. No observation of the unassigned peak in Figure 2Go 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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Fig. 2. The 2D {1H-15N} HSQC spectrum of rTcn obtained at 30°C (pH 4.4). The whole resonance assignment was indicated for each cross-peak except for Tyr1 and Lys32. Horizontal lines connect the resonances of the side chain NH2-groups. Side chain resonances of two tryptophans are also indicated.

 
The 1H-NMR spectra of rTcn and native tachycitin appear to be almost identical. As an example, Figure 3aGo compares the high-field shifted methyl region between rTcn and native tachycitin purified from horseshoe crab hemocytes. The observations of the extremely high-field shifted methyl groups of Leu37, Leu44 and Val52 at identical positions in the two spectra typically indicates that the two molecules construct the same hydrophobic core. All the Hß chemical shifts of Cys residues were also identical between the native tachycitin and rTcn except for Cys33 resonances that were not observed due to line broadening. Furthermore, the long-range NOEs were observed around the five pairs of cysteines, each of which construct a disulfide bond (Cys40 Hß–Cys53 Hß, Cys6 Hß–Ser10 Hß, Cys30 HN–Pro17 H{alpha}, Cys40 HN–Ser26 H{alpha}, and Cys53 H{alpha}–Tyr46 H{alpha}). These data clearly indicate that the pairs of disulfide bonds in rTcn are similarly constructed in the native tachycitin. In Figure 3bGo, deviations of the H{alpha} chemical shifts from the ‘random coil’ value [{Delta}{delta}obs (ppm)] are compared between rTcn and native tachycitin. The secondary structural assignments (Suetake et al., 2000Go) are also indicated in Figure 3bGo. The deviations of the H{alpha} chemical shift has been known to be highly correlated with the formation of secondary structure (Wishart et al., 1992Go), so that the identical profiles of Figure 3bGo imply that two molecules construct the same structural fold. Presumably, rTcn constructs a 3D structure almost identical to the native tachycitin, comprising of a three-stranded and a two-stranded ß-sheet following several turns (Figure 4Go) (Suetake et al., 2000Go).



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Fig. 3. Comparisons of the 1H-NMR data between rTcn and native tachycitin: (a) comparison of the high-field shifted methyl regions; (b) comparison of the chemical shift deviation of the 1H{alpha}-resonances from random coil positions plotted against the residue numbers. The secondary structural constructions and disulfide bond formations are illustrated at the top.

 


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Fig. 4. Amphipathic structure of tachycitin. Ribbon representation of tachycitin shows that it forms a distorted ß-sandwich fold with a three-stranded ß-sheet (ß1, ß2 and ß3) and a two-stranded ß-sheet (ß4 and ß5) followed by a helical turn ({alpha}1). The various solid lines for the N- and C-terminal regions indicate the range of movement of these regions suggested by their few NMR-derived constraints. Several side chains are labeled to indicate the amphipathicity of the protein; basic groups (H31, K32, R36) and hydrophobic groups (L13, L21, Y22, V64) are located at the opposite side of the central ß-sheet (ß1–ß3).

 
In order to characterize the backbone dynamics of tachycitin, we have performed the 15N relaxation measurements on the 15N-labeled rTcn. The relaxation parameters of R1 (=1/T1), R2 (=1/T2) and heteronuclear {15N-1H} NOE were obtained for 66 backbone amide resonances of tachycitin out of 68. For Lys32 and Cys33, the relaxation parameters were not measured because of their significant line broadening. The obtained backbone amide 15N relaxation data for rTcn are shown in Figure 5a–cGo. On the basis of the obtained parameters, the overall tumbling correlation time ({tau}m) was first calculated for rTcn under various protein concentrations. As shown in Figure 5Go, it appeared that dilution of the protein solution significantly affects the relaxation rates, R1 and R2, the former increases while the latter decreases (Figure 5a and bGo). These results suggest that the tumbling motion of rTcn becomes slower with increasing concentration. It should be noted that the chemical shift positions of the 1H- and 15N-NMR of rTcn are not affected by the change in protein concentration. The value of overall rotational correlation time ({tau}m) was found to increase as the protein concentration is increased: 5.8 ns (0.26 mM), 6.1 ns (0.34 mM), 6.8 ns (0.52 mM) and 11.0 ns (1.00 mM). These results indicate that tachycitins have a tendency to aggregate non-specifically with each other when the protein concentration is larger than ~0.2 mM. Such a concentration dependence of {tau}m was identified for several other proteins, e.g. dynamin pleckstrin homology (PH) domain (Fushman et al., 1997Go) and the DNA-binding domain of v-Myc (Fieber et al., 2001Go).



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Fig. 5. The backbone amide 15N-NMR relaxation data of rTcn. The 15N-NMR relaxation parameters, (a) R1 (=1/T1), (b) R2 (=1/T2), and (c) the steady-state {15N-1H} NOE, were measured at the 1H Lamour frequency of 500 MHz and are shown with error bars. The data in (a–c) were measured for two different concentrations of 15N-labeled rTcn; 0.26 mM (•) and 1.00 mM ({triangleup}). The apparent order parameter (S2) in the 0.26 mM sample was calculated for each residue as shown in (d). The locations of the secondary structure and disulfide bonds are also indicated at the top.

 
Our previous NMR study reported that the 3D structure of the N- and C-terminal regions of tachycitin each possess a disordered structure (Figure 4Go). The chemical shifts of whole non-exchangeable protons were estimated to be the random coil values for the residues 2–5 (within 0.23 p.p.m.) and the residues 69–73 (within 0.19 p.p.m.), with the exception of the His69 H{delta}2 proton that is sensitive to the solvent pH. Disordered structure was also evidenced by the large temperature coefficient values (>5 p.p.b./K) obtained for the residues 1–5 and 69–73. In the present study, the smaller values of the R1, R2 and 1H-15N NOEs were obtained for the N- and C-terminal regions (Figure 5a–cGo). These results indicate that the N- and C-terminal region’s backbone motions are considerably higher in the pico- to nanosecond time-scale. Figure 5dGo shows the apparent values of the backbone motion order parameter (S2) for the 0.26 mM sample calculated according to the model-free approach (Lipari et al., 1982a,b). The data is not significantly informative regarding the correlation between the S2 profile and the structural characteristics of tachycitin including its putative chitin-binding site (Cys40–Cys60). It is noted that the lower order parameter (below 0.7) suggesting a lower molecular motional restriction was calculated for Gly7, Ser10 (loop), Gly43 (loop) and Cys53 (C-terminal of ß-hairpin).

The chitin-binding activity of rTcn was examined by SDS–PAGE 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., 2000Go) 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 6Go 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 6a–cGo, 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 rTcn’s activity against Gram-positive and -negative bacteria (Figure 6a and bGo). Only against fungi is the antimicrobial activity recognized for both native tachycitin and rTcn (Figure 6cGo). 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., 1987Go) and cecropin A (Callaway et al., 1993Go). For sarcotoxin IA, Nakajima et al. (Nakajima et al., 1987Go) 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 4Go; 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 (ß1–ß3).



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Fig. 6. Comparison of the growth inhibition activity between rTcn ({circ}) and native tachycitin (•) against (a) Gram-positive bacteria (S.aureus), (b) Gram-negative bacteria (E.coli B) and (c) fungi (P.pastoris). The activity was plotted as the percentage of the surviving colonies: [(surviving colony numbers) / (total colony numbers)]x100 (%) (see the text for details).

 
The present 15N-NMR relaxation study indicates that tachycitin undergoes a self-oligomerization at higher concentrations (Figure 5Go). It was demonstrated that a dimeric antimicrobial protein named wheat germ agglutinin (WGA) agglutinates certain bacteria through the interaction with N-acetylglucosamine (Lotan et al., 1975Go). Taking into account the similarity with WGA, the self-oligomerization property of tachycitin is assumed to be related to its bacterial agglutinating activity (Kawabata et al., 1996Go). As shown in Figure 4Go, the ordered conformation has not been defined for the N- and C-terminal pentapeptides segments (Figure 4Go, residues 1–5, 69–73), for which high segmental mobility was also indicated from the backbone dynamics data. The agglutinating function of tachycitin might be ascribed to these mobile segments. Of course, it is necessary to obtain more experimental data before a final conclusion can be drawn; we need to examine whether tachycitin undergoes a self-oligomerization at the bacterial surface and to perform a larger number of antimicrobial tests. To summarize, here we have presented the first heteronuclear NMR-based characterization and the data of antimicrobial assays of a disulfide-rich recombinant protein of tachycitin. The availability of this recombinant protein will make it possible to carry out further experiments, providing a more detailed insight into the role of this chitin-binding protein in the host defense system.


    Notes
 
6 To whom correspondence should be addressed. E-mail: sakae.tsuda{at}aist.go.jp Back


    Acknowledgments
 
The authors are grateful to Ai Miura for keeping the NMR spectrometer at optimum performance. This study was partly supported by the Program for Promotion for Basic Research Activities for Innovative Biosciences, Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received April 23, 2002; revised May 23, 2002; accepted June 10, 2002.





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