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Structural characterization of novel chitin-binding lectins from the genus Artocarpus and their antifungal activity
Biochimica et Biophysica Acta 1764 (2006) 146 – 152 http://www.elsevier.com/locate/bba
Structural characterization of novel chitin-binding lectins from the genus Artocarpus and their antifungal activity Melissa B. Trindade a, Jose´ L.S. Lopes a, Andre´a Soares-Costa b, Ana Cristina Monteiro-Moreira c, Renato A. Moreira d, Maria Luiza V. Oliva e, Leila M. Beltramini a,* a
Institute of Physics of Sa˜o Carlos, University of Sa˜o Paulo, Sa˜o Paulo, Brazil b Federal University of Sa˜o Carlos, Sa˜o Paulo, Brazil c University of Fortaleza, Ceara´, Brazil d Federal University of Ceara´, Ceara´, Brazil e Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil
Received 27 July 2005; received in revised form 16 September 2005; accepted 22 September 2005 Available online 11 October 2005
Abstract Two novel chitin-binding lectins from seeds of Artocarpus genus were described in this paper, one from A. integrifolia (jackfruit) and one from A. incisa (breadfruit). They were purified from saline crude extract of seeds using affinity chromatography on chitin column, size-exclusion chromatography and reverse-phase chromatography on the C-18 column. Both are 14 kDa proteins, made up of 3 chains linked by disulfide bonds. The partial amino acid sequences of the two lectins showed they are homologous to each other but not to other plant chitin-binding proteins. Thus, they cannot be classified in any known plant chitin-binding protein family, particularly because of their inter-chain covalent bonds. Their circular dichroism spectra and deconvolution showed a secondary structure content of h-sheet and unordered elements. The lectins were thermally stable until 80 -C and structural changes were observed below pH 6. Both lectins inhibited the growth of Fusarium moniliforme and Saccharomyces cerevisiae, and presented hemagglutination activity against human and rabbit erythrocytes. These lectins were denoted jackin (from jackfruit) and frutackin (from breadfruit). D 2005 Elsevier B.V. All rights reserved. Keywords: Chitin-binding lectin; Lectin from Artocarpus; Fusarium moniliforme; Saccharomyces cerevisiae; Antifungal activity
1. Introduction The extraordinary ability of carbohydrates to establish a high-density coding system has broadened their role to be involved not only in energy metabolism, but also as coding words for a range of events. In this context, lectins are the main actors in this new paradigm, making it necessary to improve Abbreviations: CD, circular dichroism; DTT, dithiothreitol; EDTA, ethylenediaminotetraacetic acid disodium salt; LC/ESI/MS, liquid chromatography/electronspray ionization/mass spectrometry; GlcNAc, N-acetyl-glucosamine; PBS, phosphate buffered saline; PDM, potato dextrose medium; SEC, size exclusion chromatography; TFA, trifluoroacetic acid; HPLC, high performance liquid chromatography; UDA, Urtica dioica agglutinin; WGA, Wheat germ agglutinin * Corresponding author. Departamento de Fı´sica e Informa´tica, USP-Sa˜o Carlos, Av. Trabalhador Sa˜ocarlense, 400, CEP 13566-590, Sa˜o Carlos SP, Brazil. Fax: +55 16 3371 5381. E-mail address: [email protected] (L.M. Beltramini). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.09.011
our knowledge of this carbohydrate-binding protein class which has instigated experts to explore them, and that it has been widely known as mediating bioadhesion, transport, differentiation, and positive/negative growth control. Chitin-binding lectins have been isolated from diverse sources, including bacteria, insects, plants, and mammals [1– 6] and most of them have shown antifungal activity against phytopathogenic species, since chitin is the key component of the cell wall of these microorganisms. They have shown to affect fungal growth and development, disturbing the synthesis and/or deposition of chitin in the cell wall [7,8]. Plant lectins, in particular, have been extensively studied in a number of aspects, for instance, their antifungal and insecticide potential to protect the plants that produce them naturally or by introduction of genetic material coding for lectins harmless to humans [9,10]. The three-dimensional structures of protein –carbohydrate complexes have been investigated by X-ray crystallography, nuclear magnetic resonance and modeling [11– 14], and several authors
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have discussed the evolutionary relationship between chitinbinding domains of plants and invertebrates [15 –17]. To date, the results have shown that most plant chitin-binding lectins evolved from a common hevein ancestor, whose chitin-binding domains are characterized by conserved aromatic and cysteine amino acid residues. The former amino acids are involved in hydrophobic interactions with the glucosamine rings, enabling stabilization of complex. The hevein Ser19, through hydrogen bonding, also recognizes the acetamide moiety in GlcNAc. The high content of conserved cysteines is involved in several intrachain disulfide bonds, giving them rigidity and stability over a range of pH and temperature [11 – 13]. Structural studies of these lectins support the hypothesis that chitin-binding domains from plants and other organisms are not coancestral but share significant amino acid similarities and similar mechanisms of folding and saccharide binding [15,16]. Earlier studies of lectins from the Artocarpus genus have investigated their structural properties and their use as biotechnological tools [18 –23]. In the present work, we have gone further in pursuing other lectins present in the saline extract from seeds of Artocarpus spp. and here we describe two new chitinbinding lectins, denoted jackin (from jackfruit) and frutackin (from breadfruit), respectively. Several structural properties and their growth inhibition activity against Fusarium moniliforme, a phytopathogenic fungus that severely damages maize crops and causes huge economic losses in developing countries, and Saccharomyces cerevisiae, have also been investigated. 2. Materials and methods
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analyses of both lectins were performed by CD spectrum deconvolution using the Cluster program and Continll method [26,27]. For jackin pH experiments, the measurements also ranged from 198 to 250 nm. The lectin concentration was 0.28 mg/ml in each buffer, including the native conditions (PBS 150 mM NaCl, pH 7.4). The spectra were obtained after 20 min, 20 h and 40 h of incubation in 20 mM buffer, from pH 2.0 to 10.0 (KCl/HCl pH 2.0, citric acid/ NaOH pH 4.0 and 6.0, Tris/HCl pH 8.0, sodium borate/HCl pH 10.0). Another sample (0.5 mg/ml) was heated gradually in 5 -C steps, from 10 to 80 -C using a circulating water bath TC-100 (Jasco). At each temperature, the protein was incubated for 5 min and the spectrum was recorded from 198 to 250 nm.
2.3. Infrared spectroscopy The FT-IR spectra of jackin (1.2 mg/ml in 150 mM NaCl in PBS buffer pH 7.4) were obtained in a Magna-IR (Nicolet) spectrophotometer using an Attenuated Total Reflectance Kit (SpectraTech) with a 45- ZnSe crystal. The resulting spectrum was an average of 400 scans at a resolution of 4 cm 1 over the range of 400 to 4000 cm 1, collected under continual N2 purging. The buffer spectrum, recorded under identical conditions, was subtracted from the protein spectrum and the second derivative was performed using Origin 7.5 program.
2.4. Steady-state tryptophan fluorescence spectroscopy The fluorescence emission spectra of frutackin and jackin in native conditions (PBS, pH 7.4) at 0.07 mg/ml were measured after excitation at 280 and 295 nm. Jackin (0.6 mg/ml) stock solution was diluted in each buffer (20 mM sodium acetate/phosphate/borate, pH 4.0, 6.0, 8.0, 10.0, and PBS pH 7.4) to 0.07 mg/ml. Each sample was incubated for 40 min and the spectra were obtained with an ISS K2 spectrofluorimeter (ISS Fluorescence, Analytical and Biomedical Instruments—Illinois, USA) at 18 -C using a circulating water bath (Fisher Scientific) on a 1-cm pathlength rectangular quartz cuvette. The excitation wavelength was 295 nm and the emission spectra were recorded in the range of 310 to 450 nm. The reference spectra for each measurement and the buffer spectra were recorded and subtracted after each measurement.
2.1. Extraction and purification of jackin and frutackin 2.5. Partial amino acid sequencing The crude extracts from seeds were obtained as already described [23]. Crude extract (120 ml) was submitted to affinity chromatography in 12-ml aliquots on the Adenantera pavonina cross-linked galactomannan column, followed by a Sepharose d-Galactose column (Pierce) to deplete d-gal binding lectins. The void volume collected was then applied to a Sepharose dMannose column (Pierce) to deplete d-man binding lectins and subsequently passed through a chitin column (Sigma), equilibrated and washed with PBS. The retained proteins were eluted with 150 mM NaCl/HCl pH 2.6 and the pH of the eluate was immediately adjusted to neutrality. After dialofiltration on YM 3.5 membrane (Millipore) in PBS buffer, the proteins were 30-fold concentrated and loaded onto a Superdex 75 HR 10/30 column coupled to an ¨ KTA purifier system (Amersham Pharmacia Biotech). The column was preA equilibrated and eluted with PBS, at a flow rate of 0.5 ml/min, monitored by absorbance at 280 nm, and collected in 1-ml fractions. The lectins were submitted to reverse-phase chromatography on the C-18 column performed on a HPLC system (Bio-Rad) monitored at 220 nm, as described below for chain separations. The total protein content from the crude extract and the purified lectins were determined by the Lowry method [24], using BSA as standard. The molecular masses of lectins were determined using Tricine-SDS-PAGE [25], with and without h-mercaptoethanol, size-exclusion chromatography and electrospray ionization ion-trap mass spectrometry on a Waters LC/ESI/MS (this last one to jackin only).
2.2. Circular dichroism (CD) spectroscopy and Secondary structure estimate The CD spectra were recorded over the range of 198 – 250 nm, in native conditions, on a Jasco J-715 spectropolarimeter (Jasco Instruments, Tokyo, Japan) as an average of 8 scans using a 0.1-cm pathlength cylindrical quartz cuvette, with a protein concentration of 0.28 mg/ml. The secondary structure
The amino acid sequence of the three chains was performed after disulfide bridges were split by reduction and alkylation. Samples of previously lyophilized lectins were reduced by the Friedman reaction [28] with some modifications as follows: protein (0.4 mg) was dissolved in 200 Al 250 mM Tris – HCl pH 8.0, 6 M guanidine – HCl, 2 mM EDTA, 150 mM DTT and incubated for 4 h at 56 -C, in the dark, before N2 purging. The free sulphydryl groups of each chain were exposed to 6 Al 4-vinylpyridine and the reaction continued for more 2 h in the same initial conditions. The reaction was interrupted with 10% glacial acetic acid and the S-4-pyridylethyl derivative chains were desalted and separated on a reverse-phase C-18 column (250 4.6 mm, YMC Inc., Waters) performed on an HPLC system (Bio-Rad) monitored at 220 nm. The column was equilibrated with solvent A (0.1% TFA in H2O) and eluted using solvent B gradient 0 – 100% (90% acetonitrile: 10% H2O: 0.1% TFA). Three fractions from each lectin were obtained and submitted to N-terminal sequencing, performed as follows: 300 pmol of each chain purified in water and Sequa-Brene (Sigma-Aldrich) were applied to a special fibreglass disk (Wako, Osaka, Japan), previously treated with Sequa-Brene. The N-terminal sequence analysis was performed by automated Edman degradation in a Shimadzu PPSQ-23A protein sequencer, following the manufacturer’s recommendations.
2.6. Hemagglutination assay The assay was carried out on a 96-well polystyrene microplate using 50 Al of jackin or frutackin solution per well, initially at 0.56 mg/ml. Twofold serial dilution of lectin solution was added to an equal volume of a 2% suspension of washed rabbit and human erythrocytes. Wells containing PBS and red cells served as control. The hemagglutination activity was evaluated by naked eye after 2h incubation at room temperature (25 -C). After finding the minimum concentration of protein that agglutinates red cells, the following eight different
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monosaccharides were tested in the attempt to determine the carbohydrate specificity by inhibition of the activity: d-fructose, d-galactose, d-glucose, dmannose, l-fucose, N-methyl-mannosamine, N-acetyl-galactosamine and Nacetyl-d-glucosamine. 50 Al of jackin or frutackin at 0.33 mg/ml were mixed with every monosaccharide starting at concentration of 400 mM and then twofold serially diluted, and pre-incubated for 30 min. Then, 50 Al of 2% suspension of rabbit erythrocytes were added to each well. After 2-h incubation, the inhibition was evaluated visually, comparing with negative and positive controls.
2.7. Antifungal activity assay The antifungal activity was observed by incubating jackin in contact with spores of Aspergillus niger and Fusarium moniliforme. Inhibition assays with a Saccharomyces cerevisiae strain were carried out on the solid PDA culture medium. The growth inhibition bioassay of Aspergillus niger and Fusarium moniliforme were carried out as follows: the inhibition assay was performed by adding protein at 4.5 mg/ml to a solution with 1 107 spores per ml in 96-well microplate and PDA medium (final volume of 200 Al), followed by a twofold serially dilution. The microplate was incubated for 120 h at 28 -C and the mycelial growth inhibition was observed visually and compared with control microcultures containing only spores of fungi and medium. The antifungal activity was also evaluated using a hyphal-extension inhibition assay on PDA plates, where fungal mycelia were placed over the solid PDA. Sterile filter paper disks were distributed over the plate. Solutions of 2.25 mg/ml, the minimum inhibitory concentrations for fungal growth, 1.12 mg/ml and PBS buffer were applied on each disk and the activity was evaluated visually after 120-h incubation at 28 -C.
3. Results and discussion Jackin and frutackin were isolated by affinity chromatography on the chitin column, after depletion of the d-galactose and d-mannose binding lectins from the seed saline crude extract of jackfruit and breadfruit, respectively. Both were filtered on the Superdex-75 gel filtration column, submitted to reverse-phase chromatography and tricine-SDS-PAGE, demonstrating the same elution profile and apparent molecular weight. The yield from the total protein content of crude extracts was 2% jackin and 1% frutackin, obtained by the Lowry method. Fig. 1 illustrates the size-exclusion chromatography (SEC) of the jackin sample (from chitin affinity chromatography) in which two peaks can be seen. The lectins were eluted at 13 ml, corresponding to an apparent molecular weight around 12 kDa,
Fig. 1. Gel filtration of the chitin column eluate on Superdex-75 HR10/30 equilibrated and eluted with PBS buffer 150 mM NaCl pH 7.4 at 0.5 ml/min collected on 1-ml fractions. The eluate absorbance was monitored at 280 nm.
confirmed by tricine-SDS-PAGE. The first peak eluted before jackin showed several bands in the tricine SDS-PAGE and did not present hemagglutination activity. On the C-18 column both lectins were eluted about 25% of acetonitrile (figure not shown). The molecular mass of jackin determined by mass spectrometry was 14,225 Da. The mass spectrometry of frutackin was not accomplished because we did not have enough material from the same batch of seeds. The reduction and alkylation reactions of jackin and frutackin were performed using DTT, since h-mercaptoethanol was not efficient in reducing cysteine bridges. After desalting on the C-18 column, three main peaks were obtained, assigned to the three chains that constitute each lectin. These chains were submitted to the N-terminal analysis by automated Edman degradation reaction. 3.1. Structural analysis Each chain was submitted to N-terminal sequencing and the resulting analysis is shown in Fig. 2. Based on the jackin molecular mass (14,225 Da), the N-terminal sequence of its three chains represents about 70% of the total primary sequence. From these results, one can see that jackin and frutackin have 62% of identity of primary sequence between one another (at 70% of their amino acid sequence). However, when their sequences are compared to other proteins, namely hevein-like chitin-binding lectins, cysteine-rich proteins involved in plant defense, such as defensins, thionins and pathogenic-related proteins [7], it was found that jackin has 28% of identity to the pokeweed lectin PL-C, which is a hevein-like lectin. On the other hand, they cannot be grouped with the hevein family lectins as they do not present the main features of a hevein domain, like the conserved cysteines, as it is known, forming intrachain disulfide linkages. In fact, the eight cysteines found in the hevein domain are in the same polypeptide chain, while jackin in particular, besides having three polypeptide chains, the cysteine residues on the second or any other chains do not align with PL-C cysteines as it is shown in Fig. 2. The same was observed when the alignment of the aromatic amino acids was analyzed. The CD of jackin and frutackin, measured in the range of 198– 250 nm, are shown in Fig. 3 under native conditions. Both spectra are similar in shape, presenting a broad positive band around 230 nm for jackin and 229 nm for frutackin and a negative band around 214 nm for jackin and 212 nm for frutackin. The band around 230 nm is due to contributions of aromatic side chains Trp and Tyr and disulfide bonds, as has been described to ribonuclease A [29,30]. The band at 214 nm in the jackin and 212 nm in the frutackin spectra are assigned to h-sheet, strongly distorted by unordered structures [31]. The tertiary structure classification of these proteins, determined by the program CLUSTER, assigned jackin and frutackin to the all-beta protein group. The secondary structure fractions of the lectins were estimated by CONTINLL method, using a protein reference set selected by CLUSTER program: 62% h-sheet, 15% h-turns and 23% unordered structures for jackin and 49% h-sheet, 24% h-turns and 27% unordered structures for frutackin, both RMSD were 1%. The FT-IR spectroscopy
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Fig. 2. (a) Amino acid sequence alignment of jackin and frutackin. (b) Amino acid sequence alignment of lectin-C from Phytolacca americana (PL-C) and jackin. The alignments were performed using ClustalW program; ch1jackin: jackin chain 1; ch1frutac: frutackin chain 1; ch2jackin: jackin chain 2; ch2frutac: frutackin chain 2; ch3 jackin: jackin chain 3; ch3frutac: frutackin chain 3. The conserved amino acids are highlighted in gray and the conserved substitutions are in bold.
was also employed to confirm the main secondary structure components. The jackin’s second derivative FT-IR spectrum showed vibrational bands at 1633 (the main component) and 1627 cm 1, related to h-sheets. Unordered conformation could be assigned to the band at 1650 cm 1, although this component is very difficult to distinguish from the a-helix one because of the small frequency difference in the amide I C-0 stretch between them (figure not shown) [32 –34]. There is a discrepancy in the amplitude of the CD spectra at 215 and 225 –230 nm that do not reflect the differences observed in the values of secondary structure fractions of jackin and
Fig. 3. CD spectra of jackin (dashed line) and frutackin (solid line) PBS, at 25 -C in the range of 198 – 250 nm. The concentrations of jackin and frutackin were 0.28 mg/ml.
frutakin. As already mentioned above, the spectra around 215 nm are strongly distorted by unordered structures, particularly in frutackin, as the aromatic residue contributions were investigated by fluorescence emission spectroscopy and showed very similar results for both lectins. When excited at 280 and 295 nm, their emission spectra were very close in intensity as well as in the emission maximum: 344 nm for jackin and 338 nm for frutackin (figure not shown). Besides, an additional contribution of disulfide bridges in jackin spectrum may also be responsible for the observed difference in amplitude at 225– 230 nm when compared with the frutackin one. The thermal and pH stability of jackin were studied by exposing it to heat and to a range of pH values and recording the CD and fluorescence spectra. CD spectroscopy showed the remarkable thermal stability of the secondary structure of jackin as its native structural features were preserved up to 80 -C. The structural changes observed as the pH varied (from 2.0 to 10.0) only demonstrated pronounced effects at 214 nm with a blue shift of 5 nm (209 nm) at pH 2.0. From pH 4.0 to 10.0, subtle changes were observed as can be seen in Fig. 4a. Native jackin showed a tryptophan fluorescence emission maximum at 344 nm, typical of highly exposed tryptophan residues [35]. This result was compatible with the presence of tryptophan residues at the N-terminus that can be exposed in this protein structure as shown by the amino acid sequences. A slight blue shift (about 5 nm) was observed below pH 6.0 and an increase in fluorescence intensity occurred above pH 7.4, as seen in Fig. 4b. These spectroscopic experiments showed that jackin is particularly vulnerable to pH change below 6.0 and such behavior, together with the increase of fluorescence intensity at
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jackin and frutackin only recognizes chitin or N-acetyl-dglucosamine oligosaccharides. The antifungal activity of jackin was quantitatively tested on the filamentous fungi Aspergillus niger and Fusarium moniliforme. Jackin was put in contact with 1 107 spores/ml and the lectin inhibited the germination of F. moniliforme at a concentration of 2.25 mg/ml, as seen in Fig. 5. The lectin had no effect on the growth of A. niger up to 4.5 mg/ml. In the inhibition assay performed on agar plates, jackin hindered the normal development of hyphae by preventing the mycelia from producing spores, resulting in sterile fungus around the disk at the same protein concentration that inhibited the germination in the first assay. Frutackin antifungal assay was performed only for qualitative tests and its antifungal activity was the same to the jackin. Since jackin and frutackin are chitin-binding lectins, their fungistatic action must be associated with the binding of the lectins on the hyphal cell wall, preventing it from complete development [9,36]. A noteworthy inhibition of Saccharomyces cerevisiae growth by jackin and frutackin was also observed. Other biotechnological applications for these lectins, particularly against other phytopathogenic fungi, are being investigated, together with a strategy for obtaining them as recombinant proteins due to their low contents in the seeds. 4. Conclusions
Fig. 4. (a) CD of jackin as a function of pH. The spectra were recorded in the range of 198 – 250 nm, from 2.0 to 10.0 after 20 min of incubation. (b) Jackin fluorescence emission spectra as a function of pH (from 4.0 to 10.0). Jackin concentration was 0.07 mg/ml; the samples were excited at 295 nm and were recorded from 300 to 450 nm.
basic pH, can be explained by the basic nature of jackin, since the majority of residues among the polar ones identified up to this point in the primary sequence of jackin are of basic character, namely 15 lysines and 4 arginines [35]. The same spectroscopic investigations were not performed with frutackin because we did not have more samples from the same batch of seeds.
We have isolated new chitin-binding lectins from the seeds of A. integrifolia, jackin, and A. incisa, frutackin, which promoted hemagglutination and growth inhibition against fungi F. moniliforme and S. cerevisiae. Jackin and frutackin are homologous to each other, in terms of their molecular mass, secondary structure, and primary sequence. An interesting aspect is that they seem to be different from the most common chitin-binding lectins already described in the literature, among plants and invertebrates. The highest sequence identity with other chitin-binding lectins was 28%, obtained with a plant
3.2. Biological activities The lectins’ hemagglutination activities were employed to find the minimal concentration of protein required to agglutinate rabbit and human red blood cells and also to investigate their specificities towards monosaccharides. Lectins’ hemagglutination activities, as described in the methods section, were evaluated by naked eye after 2-h incubation at 25 -C, and the minimal concentration required to agglutinate both erythrocytes was 0.15 mg/ml. This concentration was used in the assay of agglutination inhibition by monosaccharides. Lectins were pre-incubated with the saccharides d-fructose, d-galactose, dglucose, d-mannose, l-fucose, N-methyl-mannosamine, Nacetyl-galactosamine and N-acetyl-d-glucosamine, and none of them was able to inhibit the agglutination activities, hinting that
Fig. 5. Antifungal activity of purified jackin against F. moniliforme in vitro. Left: germination inhibition assay using twofold serially diluted protein at concentrations of 2.25, 1.12, 0.56, 0.28, 0.14, 0.07, 0.035 mg/ml and control sample (wells 1 to 8). Jackin inhibited the hyphal growth at 2.25 mg/ml (first well). Right: mycelial growth on Petri dishes; disk A: jackin at 2.25 mg/ml; disk B: jackin at 1.25 mg/ml; disk C: PBS buffer 150 mM NaCl, pH 7.4 (disks A, B and C are indicated by dashed circles).
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hevein-like lectin, PL-C [12]. Besides, the most important amino acid residues related to chitin recognition and binding on PL-C are not found in the corresponding positions in jackin, showing that they definitely are not homologous. However, jackin and thereby frutackin, do show similarities to heveinlike lectins, such as the numerous cysteine, aromatic and serine amino acid residues, these last ones, being the key amino acids involved in the interaction with chitin in hevein-like lectins [13]. In addition, the fluorescence spectrum of native jackin and frutackin showed tryptophan residues exposed to the medium, indicating they could be available to interact with the chitin column via hydrophobic interaction between the typtophan side chain and glucosamine rings. The jackin hemagglutination activity was not inhibited by the most common monosaccharides found on organisms, indicating that jackin can preferentially binds to chitin, as already seen with WGA, UDA and other hevein-like lectins. The secondary structure of jackin and frutackin showed they are all h-rich proteins as with the majority of hevein-like lectins. In light of these results, we suggest that these new lectins denoted jackin and frutackin could constitute a new branch of the chitinbinding lectin family, as a result of their primary constitution (number and arrangement of the chains) to form a chitinbinding domain. Acknowledgements We are thankful to Dr. Antoˆnio Miranda from Federal University of Sa˜o Paulo (UNIFESP) for the mass spectrometry measurements, to Prof. Jose´ Odair Pereira from Federal University of Amazonas for having kindly given the fungi strains of F. moniliforme and A. niger and Prof. Jose´ Tadeu de Oliveira for performing the S. cerevisiae assay. We also acknowledge Prof. Dra. Heloisa S. Araujo for productive discussion. Supported by Brazilian financial agencies: FAPESP and CNPq. References [1] R. Campos-Olivas, I. Ho¨rr, C. Bormann, B. Jung, A.M. Gronenborn, Solution structure, backbone dynamics and chitin-binding of the anti¨ 901, J. Mol. Biol. 308 (2001) fungal protein from Streptomyces tendae TU 765 – 782. [2] T. Vuocolo, C.H. Eisemann, R.D. Pearson, P. Willadsen, E.L. Tellam, Identification and molecular characterization of a peritrophin gene, peritrophin-48 from the myiasis fly Chrysomya bezziana, Insect Biochem. Mol. 31 (2001) 919 – 932. [3] K.P.B. Van den Bergh, P. Proost, J. Van Damme, J. Coosemans, E.J.M. Van Damme, W.J. Peumans, Five disulfide bridges stabilize a hevein-type antimicrobial peptide from the bark of spindle tree (Euonymus europaeus L.), FEBS Lett. 530 (2002) 181 – 185. [4] K. Van Dellen, S.K. Ghosh, P.W. Robbins, B. Loftus, J. Samuelson, Entamoeba histolytica lectins contain unique 6-Cys or 8-Cys chitinbinding domains, Infect. Immun. 70 (2002) 3259 – 3263. [5] J.E. Rebers, J.H. Willis, A conserved domain in arthropod cuticular proteins binds chitin, Insect Biochem. Mol. 31 (2001) 1083 – 1093. [6] M. Suzuki, M. Morimatsu, B. Syuto, The identification and properties of chitin-binding protein b01 (CBPb01), J. Vet. Med. Sci. 64 (2002) 477 – 481.
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Structural characterization of novel chitin-binding lectins from the genus Artocarpus and their antifungal activity Melissa B. Trindade a, Jose´ L.S. Lopes a, Andre´a Soares-Costa b, Ana Cristina Monteiro-Moreira c, Renato A. Moreira d, Maria Luiza V. Oliva e, Leila M. Beltramini a,* a
Institute of Physics of Sa˜o Carlos, University of Sa˜o Paulo, Sa˜o Paulo, Brazil b Federal University of Sa˜o Carlos, Sa˜o Paulo, Brazil c University of Fortaleza, Ceara´, Brazil d Federal University of Ceara´, Ceara´, Brazil e Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil
Received 27 July 2005; received in revised form 16 September 2005; accepted 22 September 2005 Available online 11 October 2005
Abstract Two novel chitin-binding lectins from seeds of Artocarpus genus were described in this paper, one from A. integrifolia (jackfruit) and one from A. incisa (breadfruit). They were purified from saline crude extract of seeds using affinity chromatography on chitin column, size-exclusion chromatography and reverse-phase chromatography on the C-18 column. Both are 14 kDa proteins, made up of 3 chains linked by disulfide bonds. The partial amino acid sequences of the two lectins showed they are homologous to each other but not to other plant chitin-binding proteins. Thus, they cannot be classified in any known plant chitin-binding protein family, particularly because of their inter-chain covalent bonds. Their circular dichroism spectra and deconvolution showed a secondary structure content of h-sheet and unordered elements. The lectins were thermally stable until 80 -C and structural changes were observed below pH 6. Both lectins inhibited the growth of Fusarium moniliforme and Saccharomyces cerevisiae, and presented hemagglutination activity against human and rabbit erythrocytes. These lectins were denoted jackin (from jackfruit) and frutackin (from breadfruit). D 2005 Elsevier B.V. All rights reserved. Keywords: Chitin-binding lectin; Lectin from Artocarpus; Fusarium moniliforme; Saccharomyces cerevisiae; Antifungal activity
1. Introduction The extraordinary ability of carbohydrates to establish a high-density coding system has broadened their role to be involved not only in energy metabolism, but also as coding words for a range of events. In this context, lectins are the main actors in this new paradigm, making it necessary to improve Abbreviations: CD, circular dichroism; DTT, dithiothreitol; EDTA, ethylenediaminotetraacetic acid disodium salt; LC/ESI/MS, liquid chromatography/electronspray ionization/mass spectrometry; GlcNAc, N-acetyl-glucosamine; PBS, phosphate buffered saline; PDM, potato dextrose medium; SEC, size exclusion chromatography; TFA, trifluoroacetic acid; HPLC, high performance liquid chromatography; UDA, Urtica dioica agglutinin; WGA, Wheat germ agglutinin * Corresponding author. Departamento de Fı´sica e Informa´tica, USP-Sa˜o Carlos, Av. Trabalhador Sa˜ocarlense, 400, CEP 13566-590, Sa˜o Carlos SP, Brazil. Fax: +55 16 3371 5381. E-mail address: [email protected] (L.M. Beltramini). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.09.011
our knowledge of this carbohydrate-binding protein class which has instigated experts to explore them, and that it has been widely known as mediating bioadhesion, transport, differentiation, and positive/negative growth control. Chitin-binding lectins have been isolated from diverse sources, including bacteria, insects, plants, and mammals [1– 6] and most of them have shown antifungal activity against phytopathogenic species, since chitin is the key component of the cell wall of these microorganisms. They have shown to affect fungal growth and development, disturbing the synthesis and/or deposition of chitin in the cell wall [7,8]. Plant lectins, in particular, have been extensively studied in a number of aspects, for instance, their antifungal and insecticide potential to protect the plants that produce them naturally or by introduction of genetic material coding for lectins harmless to humans [9,10]. The three-dimensional structures of protein –carbohydrate complexes have been investigated by X-ray crystallography, nuclear magnetic resonance and modeling [11– 14], and several authors
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have discussed the evolutionary relationship between chitinbinding domains of plants and invertebrates [15 –17]. To date, the results have shown that most plant chitin-binding lectins evolved from a common hevein ancestor, whose chitin-binding domains are characterized by conserved aromatic and cysteine amino acid residues. The former amino acids are involved in hydrophobic interactions with the glucosamine rings, enabling stabilization of complex. The hevein Ser19, through hydrogen bonding, also recognizes the acetamide moiety in GlcNAc. The high content of conserved cysteines is involved in several intrachain disulfide bonds, giving them rigidity and stability over a range of pH and temperature [11 – 13]. Structural studies of these lectins support the hypothesis that chitin-binding domains from plants and other organisms are not coancestral but share significant amino acid similarities and similar mechanisms of folding and saccharide binding [15,16]. Earlier studies of lectins from the Artocarpus genus have investigated their structural properties and their use as biotechnological tools [18 –23]. In the present work, we have gone further in pursuing other lectins present in the saline extract from seeds of Artocarpus spp. and here we describe two new chitinbinding lectins, denoted jackin (from jackfruit) and frutackin (from breadfruit), respectively. Several structural properties and their growth inhibition activity against Fusarium moniliforme, a phytopathogenic fungus that severely damages maize crops and causes huge economic losses in developing countries, and Saccharomyces cerevisiae, have also been investigated. 2. Materials and methods
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analyses of both lectins were performed by CD spectrum deconvolution using the Cluster program and Continll method [26,27]. For jackin pH experiments, the measurements also ranged from 198 to 250 nm. The lectin concentration was 0.28 mg/ml in each buffer, including the native conditions (PBS 150 mM NaCl, pH 7.4). The spectra were obtained after 20 min, 20 h and 40 h of incubation in 20 mM buffer, from pH 2.0 to 10.0 (KCl/HCl pH 2.0, citric acid/ NaOH pH 4.0 and 6.0, Tris/HCl pH 8.0, sodium borate/HCl pH 10.0). Another sample (0.5 mg/ml) was heated gradually in 5 -C steps, from 10 to 80 -C using a circulating water bath TC-100 (Jasco). At each temperature, the protein was incubated for 5 min and the spectrum was recorded from 198 to 250 nm.
2.3. Infrared spectroscopy The FT-IR spectra of jackin (1.2 mg/ml in 150 mM NaCl in PBS buffer pH 7.4) were obtained in a Magna-IR (Nicolet) spectrophotometer using an Attenuated Total Reflectance Kit (SpectraTech) with a 45- ZnSe crystal. The resulting spectrum was an average of 400 scans at a resolution of 4 cm 1 over the range of 400 to 4000 cm 1, collected under continual N2 purging. The buffer spectrum, recorded under identical conditions, was subtracted from the protein spectrum and the second derivative was performed using Origin 7.5 program.
2.4. Steady-state tryptophan fluorescence spectroscopy The fluorescence emission spectra of frutackin and jackin in native conditions (PBS, pH 7.4) at 0.07 mg/ml were measured after excitation at 280 and 295 nm. Jackin (0.6 mg/ml) stock solution was diluted in each buffer (20 mM sodium acetate/phosphate/borate, pH 4.0, 6.0, 8.0, 10.0, and PBS pH 7.4) to 0.07 mg/ml. Each sample was incubated for 40 min and the spectra were obtained with an ISS K2 spectrofluorimeter (ISS Fluorescence, Analytical and Biomedical Instruments—Illinois, USA) at 18 -C using a circulating water bath (Fisher Scientific) on a 1-cm pathlength rectangular quartz cuvette. The excitation wavelength was 295 nm and the emission spectra were recorded in the range of 310 to 450 nm. The reference spectra for each measurement and the buffer spectra were recorded and subtracted after each measurement.
2.1. Extraction and purification of jackin and frutackin 2.5. Partial amino acid sequencing The crude extracts from seeds were obtained as already described [23]. Crude extract (120 ml) was submitted to affinity chromatography in 12-ml aliquots on the Adenantera pavonina cross-linked galactomannan column, followed by a Sepharose d-Galactose column (Pierce) to deplete d-gal binding lectins. The void volume collected was then applied to a Sepharose dMannose column (Pierce) to deplete d-man binding lectins and subsequently passed through a chitin column (Sigma), equilibrated and washed with PBS. The retained proteins were eluted with 150 mM NaCl/HCl pH 2.6 and the pH of the eluate was immediately adjusted to neutrality. After dialofiltration on YM 3.5 membrane (Millipore) in PBS buffer, the proteins were 30-fold concentrated and loaded onto a Superdex 75 HR 10/30 column coupled to an ¨ KTA purifier system (Amersham Pharmacia Biotech). The column was preA equilibrated and eluted with PBS, at a flow rate of 0.5 ml/min, monitored by absorbance at 280 nm, and collected in 1-ml fractions. The lectins were submitted to reverse-phase chromatography on the C-18 column performed on a HPLC system (Bio-Rad) monitored at 220 nm, as described below for chain separations. The total protein content from the crude extract and the purified lectins were determined by the Lowry method [24], using BSA as standard. The molecular masses of lectins were determined using Tricine-SDS-PAGE [25], with and without h-mercaptoethanol, size-exclusion chromatography and electrospray ionization ion-trap mass spectrometry on a Waters LC/ESI/MS (this last one to jackin only).
2.2. Circular dichroism (CD) spectroscopy and Secondary structure estimate The CD spectra were recorded over the range of 198 – 250 nm, in native conditions, on a Jasco J-715 spectropolarimeter (Jasco Instruments, Tokyo, Japan) as an average of 8 scans using a 0.1-cm pathlength cylindrical quartz cuvette, with a protein concentration of 0.28 mg/ml. The secondary structure
The amino acid sequence of the three chains was performed after disulfide bridges were split by reduction and alkylation. Samples of previously lyophilized lectins were reduced by the Friedman reaction [28] with some modifications as follows: protein (0.4 mg) was dissolved in 200 Al 250 mM Tris – HCl pH 8.0, 6 M guanidine – HCl, 2 mM EDTA, 150 mM DTT and incubated for 4 h at 56 -C, in the dark, before N2 purging. The free sulphydryl groups of each chain were exposed to 6 Al 4-vinylpyridine and the reaction continued for more 2 h in the same initial conditions. The reaction was interrupted with 10% glacial acetic acid and the S-4-pyridylethyl derivative chains were desalted and separated on a reverse-phase C-18 column (250 4.6 mm, YMC Inc., Waters) performed on an HPLC system (Bio-Rad) monitored at 220 nm. The column was equilibrated with solvent A (0.1% TFA in H2O) and eluted using solvent B gradient 0 – 100% (90% acetonitrile: 10% H2O: 0.1% TFA). Three fractions from each lectin were obtained and submitted to N-terminal sequencing, performed as follows: 300 pmol of each chain purified in water and Sequa-Brene (Sigma-Aldrich) were applied to a special fibreglass disk (Wako, Osaka, Japan), previously treated with Sequa-Brene. The N-terminal sequence analysis was performed by automated Edman degradation in a Shimadzu PPSQ-23A protein sequencer, following the manufacturer’s recommendations.
2.6. Hemagglutination assay The assay was carried out on a 96-well polystyrene microplate using 50 Al of jackin or frutackin solution per well, initially at 0.56 mg/ml. Twofold serial dilution of lectin solution was added to an equal volume of a 2% suspension of washed rabbit and human erythrocytes. Wells containing PBS and red cells served as control. The hemagglutination activity was evaluated by naked eye after 2h incubation at room temperature (25 -C). After finding the minimum concentration of protein that agglutinates red cells, the following eight different
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monosaccharides were tested in the attempt to determine the carbohydrate specificity by inhibition of the activity: d-fructose, d-galactose, d-glucose, dmannose, l-fucose, N-methyl-mannosamine, N-acetyl-galactosamine and Nacetyl-d-glucosamine. 50 Al of jackin or frutackin at 0.33 mg/ml were mixed with every monosaccharide starting at concentration of 400 mM and then twofold serially diluted, and pre-incubated for 30 min. Then, 50 Al of 2% suspension of rabbit erythrocytes were added to each well. After 2-h incubation, the inhibition was evaluated visually, comparing with negative and positive controls.
2.7. Antifungal activity assay The antifungal activity was observed by incubating jackin in contact with spores of Aspergillus niger and Fusarium moniliforme. Inhibition assays with a Saccharomyces cerevisiae strain were carried out on the solid PDA culture medium. The growth inhibition bioassay of Aspergillus niger and Fusarium moniliforme were carried out as follows: the inhibition assay was performed by adding protein at 4.5 mg/ml to a solution with 1 107 spores per ml in 96-well microplate and PDA medium (final volume of 200 Al), followed by a twofold serially dilution. The microplate was incubated for 120 h at 28 -C and the mycelial growth inhibition was observed visually and compared with control microcultures containing only spores of fungi and medium. The antifungal activity was also evaluated using a hyphal-extension inhibition assay on PDA plates, where fungal mycelia were placed over the solid PDA. Sterile filter paper disks were distributed over the plate. Solutions of 2.25 mg/ml, the minimum inhibitory concentrations for fungal growth, 1.12 mg/ml and PBS buffer were applied on each disk and the activity was evaluated visually after 120-h incubation at 28 -C.
3. Results and discussion Jackin and frutackin were isolated by affinity chromatography on the chitin column, after depletion of the d-galactose and d-mannose binding lectins from the seed saline crude extract of jackfruit and breadfruit, respectively. Both were filtered on the Superdex-75 gel filtration column, submitted to reverse-phase chromatography and tricine-SDS-PAGE, demonstrating the same elution profile and apparent molecular weight. The yield from the total protein content of crude extracts was 2% jackin and 1% frutackin, obtained by the Lowry method. Fig. 1 illustrates the size-exclusion chromatography (SEC) of the jackin sample (from chitin affinity chromatography) in which two peaks can be seen. The lectins were eluted at 13 ml, corresponding to an apparent molecular weight around 12 kDa,
Fig. 1. Gel filtration of the chitin column eluate on Superdex-75 HR10/30 equilibrated and eluted with PBS buffer 150 mM NaCl pH 7.4 at 0.5 ml/min collected on 1-ml fractions. The eluate absorbance was monitored at 280 nm.
confirmed by tricine-SDS-PAGE. The first peak eluted before jackin showed several bands in the tricine SDS-PAGE and did not present hemagglutination activity. On the C-18 column both lectins were eluted about 25% of acetonitrile (figure not shown). The molecular mass of jackin determined by mass spectrometry was 14,225 Da. The mass spectrometry of frutackin was not accomplished because we did not have enough material from the same batch of seeds. The reduction and alkylation reactions of jackin and frutackin were performed using DTT, since h-mercaptoethanol was not efficient in reducing cysteine bridges. After desalting on the C-18 column, three main peaks were obtained, assigned to the three chains that constitute each lectin. These chains were submitted to the N-terminal analysis by automated Edman degradation reaction. 3.1. Structural analysis Each chain was submitted to N-terminal sequencing and the resulting analysis is shown in Fig. 2. Based on the jackin molecular mass (14,225 Da), the N-terminal sequence of its three chains represents about 70% of the total primary sequence. From these results, one can see that jackin and frutackin have 62% of identity of primary sequence between one another (at 70% of their amino acid sequence). However, when their sequences are compared to other proteins, namely hevein-like chitin-binding lectins, cysteine-rich proteins involved in plant defense, such as defensins, thionins and pathogenic-related proteins [7], it was found that jackin has 28% of identity to the pokeweed lectin PL-C, which is a hevein-like lectin. On the other hand, they cannot be grouped with the hevein family lectins as they do not present the main features of a hevein domain, like the conserved cysteines, as it is known, forming intrachain disulfide linkages. In fact, the eight cysteines found in the hevein domain are in the same polypeptide chain, while jackin in particular, besides having three polypeptide chains, the cysteine residues on the second or any other chains do not align with PL-C cysteines as it is shown in Fig. 2. The same was observed when the alignment of the aromatic amino acids was analyzed. The CD of jackin and frutackin, measured in the range of 198– 250 nm, are shown in Fig. 3 under native conditions. Both spectra are similar in shape, presenting a broad positive band around 230 nm for jackin and 229 nm for frutackin and a negative band around 214 nm for jackin and 212 nm for frutackin. The band around 230 nm is due to contributions of aromatic side chains Trp and Tyr and disulfide bonds, as has been described to ribonuclease A [29,30]. The band at 214 nm in the jackin and 212 nm in the frutackin spectra are assigned to h-sheet, strongly distorted by unordered structures [31]. The tertiary structure classification of these proteins, determined by the program CLUSTER, assigned jackin and frutackin to the all-beta protein group. The secondary structure fractions of the lectins were estimated by CONTINLL method, using a protein reference set selected by CLUSTER program: 62% h-sheet, 15% h-turns and 23% unordered structures for jackin and 49% h-sheet, 24% h-turns and 27% unordered structures for frutackin, both RMSD were 1%. The FT-IR spectroscopy
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Fig. 2. (a) Amino acid sequence alignment of jackin and frutackin. (b) Amino acid sequence alignment of lectin-C from Phytolacca americana (PL-C) and jackin. The alignments were performed using ClustalW program; ch1jackin: jackin chain 1; ch1frutac: frutackin chain 1; ch2jackin: jackin chain 2; ch2frutac: frutackin chain 2; ch3 jackin: jackin chain 3; ch3frutac: frutackin chain 3. The conserved amino acids are highlighted in gray and the conserved substitutions are in bold.
was also employed to confirm the main secondary structure components. The jackin’s second derivative FT-IR spectrum showed vibrational bands at 1633 (the main component) and 1627 cm 1, related to h-sheets. Unordered conformation could be assigned to the band at 1650 cm 1, although this component is very difficult to distinguish from the a-helix one because of the small frequency difference in the amide I C-0 stretch between them (figure not shown) [32 –34]. There is a discrepancy in the amplitude of the CD spectra at 215 and 225 –230 nm that do not reflect the differences observed in the values of secondary structure fractions of jackin and
Fig. 3. CD spectra of jackin (dashed line) and frutackin (solid line) PBS, at 25 -C in the range of 198 – 250 nm. The concentrations of jackin and frutackin were 0.28 mg/ml.
frutakin. As already mentioned above, the spectra around 215 nm are strongly distorted by unordered structures, particularly in frutackin, as the aromatic residue contributions were investigated by fluorescence emission spectroscopy and showed very similar results for both lectins. When excited at 280 and 295 nm, their emission spectra were very close in intensity as well as in the emission maximum: 344 nm for jackin and 338 nm for frutackin (figure not shown). Besides, an additional contribution of disulfide bridges in jackin spectrum may also be responsible for the observed difference in amplitude at 225– 230 nm when compared with the frutackin one. The thermal and pH stability of jackin were studied by exposing it to heat and to a range of pH values and recording the CD and fluorescence spectra. CD spectroscopy showed the remarkable thermal stability of the secondary structure of jackin as its native structural features were preserved up to 80 -C. The structural changes observed as the pH varied (from 2.0 to 10.0) only demonstrated pronounced effects at 214 nm with a blue shift of 5 nm (209 nm) at pH 2.0. From pH 4.0 to 10.0, subtle changes were observed as can be seen in Fig. 4a. Native jackin showed a tryptophan fluorescence emission maximum at 344 nm, typical of highly exposed tryptophan residues [35]. This result was compatible with the presence of tryptophan residues at the N-terminus that can be exposed in this protein structure as shown by the amino acid sequences. A slight blue shift (about 5 nm) was observed below pH 6.0 and an increase in fluorescence intensity occurred above pH 7.4, as seen in Fig. 4b. These spectroscopic experiments showed that jackin is particularly vulnerable to pH change below 6.0 and such behavior, together with the increase of fluorescence intensity at
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jackin and frutackin only recognizes chitin or N-acetyl-dglucosamine oligosaccharides. The antifungal activity of jackin was quantitatively tested on the filamentous fungi Aspergillus niger and Fusarium moniliforme. Jackin was put in contact with 1 107 spores/ml and the lectin inhibited the germination of F. moniliforme at a concentration of 2.25 mg/ml, as seen in Fig. 5. The lectin had no effect on the growth of A. niger up to 4.5 mg/ml. In the inhibition assay performed on agar plates, jackin hindered the normal development of hyphae by preventing the mycelia from producing spores, resulting in sterile fungus around the disk at the same protein concentration that inhibited the germination in the first assay. Frutackin antifungal assay was performed only for qualitative tests and its antifungal activity was the same to the jackin. Since jackin and frutackin are chitin-binding lectins, their fungistatic action must be associated with the binding of the lectins on the hyphal cell wall, preventing it from complete development [9,36]. A noteworthy inhibition of Saccharomyces cerevisiae growth by jackin and frutackin was also observed. Other biotechnological applications for these lectins, particularly against other phytopathogenic fungi, are being investigated, together with a strategy for obtaining them as recombinant proteins due to their low contents in the seeds. 4. Conclusions
Fig. 4. (a) CD of jackin as a function of pH. The spectra were recorded in the range of 198 – 250 nm, from 2.0 to 10.0 after 20 min of incubation. (b) Jackin fluorescence emission spectra as a function of pH (from 4.0 to 10.0). Jackin concentration was 0.07 mg/ml; the samples were excited at 295 nm and were recorded from 300 to 450 nm.
basic pH, can be explained by the basic nature of jackin, since the majority of residues among the polar ones identified up to this point in the primary sequence of jackin are of basic character, namely 15 lysines and 4 arginines [35]. The same spectroscopic investigations were not performed with frutackin because we did not have more samples from the same batch of seeds.
We have isolated new chitin-binding lectins from the seeds of A. integrifolia, jackin, and A. incisa, frutackin, which promoted hemagglutination and growth inhibition against fungi F. moniliforme and S. cerevisiae. Jackin and frutackin are homologous to each other, in terms of their molecular mass, secondary structure, and primary sequence. An interesting aspect is that they seem to be different from the most common chitin-binding lectins already described in the literature, among plants and invertebrates. The highest sequence identity with other chitin-binding lectins was 28%, obtained with a plant
3.2. Biological activities The lectins’ hemagglutination activities were employed to find the minimal concentration of protein required to agglutinate rabbit and human red blood cells and also to investigate their specificities towards monosaccharides. Lectins’ hemagglutination activities, as described in the methods section, were evaluated by naked eye after 2-h incubation at 25 -C, and the minimal concentration required to agglutinate both erythrocytes was 0.15 mg/ml. This concentration was used in the assay of agglutination inhibition by monosaccharides. Lectins were pre-incubated with the saccharides d-fructose, d-galactose, dglucose, d-mannose, l-fucose, N-methyl-mannosamine, Nacetyl-galactosamine and N-acetyl-d-glucosamine, and none of them was able to inhibit the agglutination activities, hinting that
Fig. 5. Antifungal activity of purified jackin against F. moniliforme in vitro. Left: germination inhibition assay using twofold serially diluted protein at concentrations of 2.25, 1.12, 0.56, 0.28, 0.14, 0.07, 0.035 mg/ml and control sample (wells 1 to 8). Jackin inhibited the hyphal growth at 2.25 mg/ml (first well). Right: mycelial growth on Petri dishes; disk A: jackin at 2.25 mg/ml; disk B: jackin at 1.25 mg/ml; disk C: PBS buffer 150 mM NaCl, pH 7.4 (disks A, B and C are indicated by dashed circles).
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hevein-like lectin, PL-C [12]. Besides, the most important amino acid residues related to chitin recognition and binding on PL-C are not found in the corresponding positions in jackin, showing that they definitely are not homologous. However, jackin and thereby frutackin, do show similarities to heveinlike lectins, such as the numerous cysteine, aromatic and serine amino acid residues, these last ones, being the key amino acids involved in the interaction with chitin in hevein-like lectins [13]. In addition, the fluorescence spectrum of native jackin and frutackin showed tryptophan residues exposed to the medium, indicating they could be available to interact with the chitin column via hydrophobic interaction between the typtophan side chain and glucosamine rings. The jackin hemagglutination activity was not inhibited by the most common monosaccharides found on organisms, indicating that jackin can preferentially binds to chitin, as already seen with WGA, UDA and other hevein-like lectins. The secondary structure of jackin and frutackin showed they are all h-rich proteins as with the majority of hevein-like lectins. In light of these results, we suggest that these new lectins denoted jackin and frutackin could constitute a new branch of the chitinbinding lectin family, as a result of their primary constitution (number and arrangement of the chains) to form a chitinbinding domain. Acknowledgements We are thankful to Dr. Antoˆnio Miranda from Federal University of Sa˜o Paulo (UNIFESP) for the mass spectrometry measurements, to Prof. Jose´ Odair Pereira from Federal University of Amazonas for having kindly given the fungi strains of F. moniliforme and A. niger and Prof. Jose´ Tadeu de Oliveira for performing the S. cerevisiae assay. We also acknowledge Prof. Dra. Heloisa S. Araujo for productive discussion. Supported by Brazilian financial agencies: FAPESP and CNPq. References [1] R. Campos-Olivas, I. Ho¨rr, C. Bormann, B. Jung, A.M. Gronenborn, Solution structure, backbone dynamics and chitin-binding of the anti¨ 901, J. Mol. Biol. 308 (2001) fungal protein from Streptomyces tendae TU 765 – 782. [2] T. Vuocolo, C.H. Eisemann, R.D. Pearson, P. Willadsen, E.L. Tellam, Identification and molecular characterization of a peritrophin gene, peritrophin-48 from the myiasis fly Chrysomya bezziana, Insect Biochem. Mol. 31 (2001) 919 – 932. [3] K.P.B. Van den Bergh, P. Proost, J. Van Damme, J. Coosemans, E.J.M. Van Damme, W.J. Peumans, Five disulfide bridges stabilize a hevein-type antimicrobial peptide from the bark of spindle tree (Euonymus europaeus L.), FEBS Lett. 530 (2002) 181 – 185. [4] K. Van Dellen, S.K. Ghosh, P.W. Robbins, B. Loftus, J. Samuelson, Entamoeba histolytica lectins contain unique 6-Cys or 8-Cys chitinbinding domains, Infect. Immun. 70 (2002) 3259 – 3263. [5] J.E. Rebers, J.H. Willis, A conserved domain in arthropod cuticular proteins binds chitin, Insect Biochem. Mol. 31 (2001) 1083 – 1093. [6] M. Suzuki, M. Morimatsu, B. Syuto, The identification and properties of chitin-binding protein b01 (CBPb01), J. Vet. Med. Sci. 64 (2002) 477 – 481.
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