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First published online February 27, 2008; 10.1104/pp.107.111500 Plant Physiology 146:1909-1919 (2008) © 2008 American Society of Plant Biologists Proteinase Inhibitor from Ginkgo Seeds Is a Member of the Plant Nonspecific Lipid Transfer Protein Gene Family1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113–8657, Japan (Y.S., T.M., Y.M., M.T.); and Department of Biological Sciences, Faculty of Engineering, Gunma University, Kiryu, Gunma 376–8515, Japan (K.-i.H., H.K., H.Y.)
A 9-kD proteinase inhibitor was isolated from the seeds of ginkgo (Ginkgo biloba) and purified to homogeneity. This protein was revealed to partial-noncompetitively inhibit the aspartic acid proteinase pepsin and the cysteine proteinase papain (inhibition constant = 10–5–10–4 M). The cDNA of the inhibitor was revealed to contain a 357-bp open reading frame encoding a 119-amino acid protein with a potential signal peptide (27 residues), indicating that this protein is synthesized as a preprotein and secreted outside the cells. Semiquantitative reverse transcription-polymerase chain reaction revealed that this gene expresses only in seeds, not in stems, leaves, and roots, suggesting that the protein is involved in seed development and/or germination. The inhibitor showed about 40% sequence homology with type-I nonspecific lipid transfer protein (nsLTP1) from other plant species. Actually, this inhibitor exerted both lipid transfer activity and lipid-binding activity, while the protein did not show any antifungal and antibacterial activities. Furthermore, the site-directed mutagenesis study using a recombinant ginkgo nsLTP1 revealed that proline (Pro)-79 and phenylalanine-80 are important on phospholipid transfer activity and that Pro-79 and isoleucine-82 are essential for the binding activity toward cis-unsaturated fatty acids. On the other hand, the  -helical content of P79A and F80A mutants was significantly lower than that of the wild-type protein. It was noteworthy that the papain-inhibitory activity of P79A and F80A mutants was elevated twice as much as that of the wild-type protein. In summary, we concluded that Pro-79 plays a critical role in both the lipid transfer and binding activities of ginkgo nsLTP1.
Plants are exposed to a variety of potential threatening phytopathogens and pests. As a result, they have evolved various mechanisms for self-defense such as the production of secondary metabolites and defense-related proteins. In plant life cycle, seed germination is an especially vulnerable period for phytopathogen attack, because the rupture of a seed coat allows pathogens to invade the seed-storage tissues. Many phytopathogenic fungi are known to produce extracellular proteinases (Kalashnikova et al., 2003  ), and recent findings suggest that proteinases play an active role in the development of diseases (Sara and Heale, 1990). In a response to proteinase attacks by phytopathogens, plants synthesize inhibitory proteins that can suppress the enzyme activities (Ryan, 1990).
Ginkgo (Ginkgo biloba) is one of the oldest gymnosperm species and is called a living fossil. This plant shows a broad spectrum of resistance or tolerance to many phytopathogens; therefore, a large number of studies on this unique plant have been carried out. So far, a chitin-binding antimicrobial peptide has been isolated, and a jasmonate-dependent defensin gene has been cloned from gingko leaves (Huang et al., 2000
In this article, we report the purification of the other Cys proteinase inhibitor, characterization of the inhibitory activity, and cloning of its full-length cDNA sequence. As a result of gene cloning, this inhibitor was revealed to possess high homology with type-I nonspecific lipid transfer protein (nsLTP1), especially two consensus pentapeptides that have been proposed to be important for lipid catalysis or binding (Douliez et al., 2000
Purification of a Novel Proteinase Inhibitor from Ginkgo Seeds As shown in Figure 1A , the extracts from 775 g of shelled ginkgo seeds were fractionated on a Sephadex G-50 column to obtain a fraction with papain-inhibitory activity. The inhibitory fraction (0.52 g) was finally purified by ion-exchange chromatography (data not shown), and it appeared as a protein band with the molecular mass of approximately 10 kD on SDS-PAGE (Fig. 1B). The sample was estimated to be more than 95% pure and was identified with a molecular mass of 9,318.8 D by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the final yield from the starting material was about 83 mg. The NH2-terminal 28 amino acids were revealed to be APGCDTVDTDLAPCISYLQTGTGNPTVQ by direct protein sequencing.
Partial Amino Acid Sequencing of the Proteinase Inhibitor The oxidized inhibitor was digested by trypsin, and the resulting peptides were separated by HPLC. As shown in Figure 2 , the elution profile of HPLC showed four major peaks at retention times of 12.5, 14.1, 16.6, and 18.8 min. To characterize all the peaks, we performed both the MALDI-TOF MS and NH2-terminal peptide sequencing. As a consequence, the peaks at retention times of 16.6 and 18.8 min were revealed as the peptides of APGCDTVDTDLAPCISYL and SLPGLCSVTLPFPISIATDCN, respectively (Fig. 2). The other peaks at retention times of 12.5 and 14.1 min exhibited a column artifact and a mixture of di- or tripeptides digested, respectively (data not shown).
Molecular Cloning of the Gene Encoding the Proteinase Inhibitor We first designed degenerate primers by using the partial amino acid sequences of the inhibitor and cloned an initial 267-bp cDNA by reverse transcription (RT)-PCR with the primers. Based on the DNA sequence obtained, we synthesized the gene-specific primers of GBs1 and GBs2 for 3' RACE and GBa3, GBa4, GBa5, GBa6, GBa7, and GBa8 for 5' RACE. Using the RACE method, we obtained the full-length cDNA sequence of the inhibitor gene, which was 700 bp long and contained a 357-bp open reading frame encoding a 119-amino acid protein (GenBank accession no. DQ836633). An untranslated 5' region was 46 bp upstream from the start codon, and the coding region was followed by an untranslated 3' region that was 294 bp long downstream from the stop codon. Two possible polyadenylation signals of AATAAA were found at the 62- and 194-bp positions downstream from the stop codon. It is probable that more than one polyadenylation signal site provides a high degree of flexibility.
The deduced amino acid sequence consisted of 119 residues, including eight Cys residues (positions 4, 14, 29, 30, 50, 52, 74, and 88). A search for the full-length precursor protein in carefully prepared extracts of the fresh seeds was unsuccessful; therefore, the NH2-terminal 27 residues of MMKISWQLWLLVAFAVMVCVWTPLSTA were considered to be a propeptide. Actually, the programs SignalP-3.0 (Bendtsen et al., 2004
A search of the National Center for Biotechnology Information (NCBI) database, using BlastP2.2.13, showed that the deduced inhibitory protein has high sequence homology with a lot of plant nsLTP. Figure 3
shows the sequence alignment of the inhibitor with gymnosperm and angiosperm nsLTP1. The plant nsLTP family can be divided into two subfamilies according to their molecular masses, namely, type-I nsLTP (nsLTP1, molecular mass about 9 kD) and type II (nsLTP2, about 7 kD; Kalla et al., 1994
Comparison of Gb-nsLTP1 with Other Homologous Proteins
The deduced sequence of Gb-nsLTP1 was 36% to 46% identical to other plant nsLTP1 sequences (Fig. 3). Gb-nsLTP1 also has two consensus pentapeptides (T/S-X-X-D-R/K and P-Y-X-I-S) that have been proposed to be important for lipid catalysis or binding (Douliez et al., 2000
To better understand the evolutionary relationships among plant nsLTP1, we constructed an unrooted phylogenetic tree of aligned amino acid sequences by using the neighbor-joining method. As shown in Figure 4 , Gb-nsLTP1 shared a common evolutionary origin and had a closer relationship with the gymnosperm species. The monocotyledonous and dicotyledonous species also formed a cluster, respectively. Tobacco (Nicotiana tabacum) plant possessed two types of nsLTP1; for instance, tobacco-2 nsLTP1 sequence was 35% and 91% identical to tobacco-1 and tobacco-3 sequences, respectively (Fig. 3). It is noteworthy that tobacco-2 and tobacco-3 nsLTP1 were grouped into a cluster with Gb-nsLTP1, indicating that ginkgo has a closer relationship with tobacco-2 and tobacco-3 than the gymnosperm species (Fig. 4). It is likely that tobacco-2, tobacco-3, and ginkgo nsLTP1 in vivo possess a unique function different from tobacco-1 and the other angiosperms.
Kinetic Properties and pH Dependence of the Proteinase Inhibitory Activities We examined whether Gb-nsLTP1 inhibits Asp-, Ser-, Cys-, or metallo-proteinases. As a consequence, this inhibitor was found to inhibit papain (74%), pepsin (43%), and the Ser proteinase trypsin (25%) under the conditions described in "Materials and Methods." On the other hand, Gb-nsLTP1 showed no inhibitory activity against the metallo-proteinase thermolysin. The Lineweaver-Burk plots toward papain and pepsin are shown in Figure 5, A and B , respectively. These plots indicate that Gb-nsLTP1 partial-noncompetitively inhibited the activities of papain and pepsin, consistent with these Dixon plots, whose intersection points also did not exist on the y axis (data not shown). The obtained inhibition constant values against papain and pepsin were 2.40 x 10–5 and 8.50 x 10–5 M, respectively. The optimal-inhibitory pH toward papain was observed around 5 (Fig. 6 ). We also examined the substrate inhibition effect toward papain and trypsin by using a 60 mg mL–1 bovine serum albumin solution. As a result, the substrate inhibitions were observed to be 4% for papain and 1% for trypsin, indicating that the effect was negligible for the trypsin and papain assays.
Assays of the Antifungal, Antibacterial, Lipid Transfer, and Lipid-Binding Activities Gb-nsLTP1 did not exhibit antifungal activity against Fusarium oxysporum, Fusarium culmorum, Trichoderma reesei, Aspargillus fumigatus, or Mucor spinescens (data not shown). Similarly, this protein did not show antibacterial activity against E. coli (data not shown). In general, the positively-charged face of plant defensive proteins is believed to bind to the negatively-charged membrane surface of microorganisms; accordingly, Gb-nsLTP1 with the neutral pI may be unable to bind to the membrane fully. The lipid transfer activity of Gb-nsLTP1 was investigated with fluorescence spectroscopy by monitoring the increase of the fluorescence due to transferring pyrene moieties from quenched donor vesicles to unquenched acceptor vesicles. The fluorescence intensity increased as the protein catalyzed the shuffling of lipid molecules between the vesicles. The fluorescence intensity reached a plateau when the fluorescent lipid molecules were equally distributed between the vesicles (Fig. 7 ). The lipid transfer rate of Gb-nsLTP1 was revealed to be 2.44 nmol pyrene phospholipid min–1 mg–1.
The lipid-binding assay revealed that Gb-nsLTP1 could not associate with most saturated fatty acids (C8:0–C18:0) and an unsaturated fatty acid with a trans-bond (C18:1, trans-9), because the observed fluorescence was 76% to 121% under several amounts of each fatty acid (Fig. 8 ). In contrast, 50 µM cis-unsaturated fatty acids, palmitoleic acid (C16:1, cis-9), oleic acid (C18:1, cis-9), linoleic acid (C18:2, cis-9,12), and linolenic acid (C18:3, cis-9,12,15) more efficiently displaced 2-p-toluidinonaphtalene-6-sulfonate (TNS), a fluorescent probe (24%–34% of the control fluorescence; Fig. 8). These results suggest that Gb-nsLTP1 has high lipid-binding activity toward cis-unsaturated fatty acids, and the substrate specificity was similar to that of tobacco nsLTP1 (Buhot et al., 2004 ).
Expression Profile of Gb-nsLTP1 Semiquantitative RT-PCR was carried out to investigate the expression pattern of Gb-nsLTP1 in different tissues. The result showed that there was high Gb-nsLTP1 expression in the immature and mature seeds, while no expression could be detected in the stems, leaves, and roots (Fig. 9 ), indicating that Gb-nsLTP1 is a seed-specific expressing gene.
Expression and Purification of the Recombinant Proteins The recombinant Gb-nsLTP1 was expressed as a thioredoxin (Trx) fusion protein (Trx-Gb-nsLTP1; Mr = 25,400) in the soluble fraction of the cell lysate (Fig. 10 , lane 2), and the Trx-Gb-nsLTP1 protein was purified by Ni-NTA chromatography (Fig. 10, lane 3). The fusion protein was cleaved by an HRV3C protease, resulting in the production of the recombinant protein (Mr = 9,600; Fig. 10, lane 4). Finally, the protein was purified by Mono-S chromatography to give a single band on SDS-PAGE (Fig. 10, lane 5), and the purity of the sample was estimated to be more than 95% by MALDI-TOF MS (data not shown). Almost all yields of the recombinant proteins were approximately 5 mg L–1 of bacterial culture, while the yield of the mutant P79A was less than 1 mg L–1. MS analysis gave an average molecular mass of 9,586.9 D for the wild-type recombinant (Table I ) and 9,318.8 D for native Gb-nsLTP1. The difference indicates that the recombinant protein contained an additional peptide Gly-Pro derived from the cloning vector. The MS analysis of the mutant proteins revealed that they were consistent with the amino acid substitutions and properly formed four disulfide bonds (Table I).
Characterization of Gb-nsLTP1 Mutants To clarify the structure-activity relationship of Gb-nsLTP1, it is very important to examine the biological activity of the mutant proteins, for instance, lipid transfer ability or papain inhibition. Especially, the biological function of plant nsLTP1 is related to the capacity of binding and/or transporting amphiphilic lipids. Therefore, we introduced four independent mutations (R46A, P79A, F80A, and I82A) into the highly conserved lipid-binding sites as known among plant nsLTP1 (Fig. 3). As a result, the phospholipid transfer activity of the mutants P79A and F80A was much lower than the wild-type protein (Table I). The activity of the R46A mutant showed 88%; on the other hand, the I82A mutant did not lose any lipid transfer activity (101%). Moreover, we measured the binding activity of the mutant proteins toward cis-unsaturated fatty acids, palmitoleic acid, oleic acid, linoleic acid, and linolenic acid. The mutants P79A and I82A showed lower binding activity against most of these fatty acids than the wild-type protein; on the other hand, the mutants R46A and F80A did not lose any lipid-binding activity (Fig. 11 ). It is interesting to note that the papain-inhibitory activity of the mutants P79A and F80A was elevated twice as much as the wild-type protein (Table I).
CD Measurements
Figure 12
shows CD spectra of Gb-nsLTP1 at four pH values between 2 and 7. As shown in the inset, this protein appears to be stable and rich (20%–30%) in helical structure under the above conditions. This stability is likely due to the presence of the four disulfide bonds. Analysis of the spectrum at pH 5.0 by the CONTIN program indicated that Gb-nsLTP1 is composed of
In this study, we isolated a novel proteinase inhibitor (Gb-nsLTP1) from ginkgo seeds, and its full-length cDNA was identified by RT-PCR and RACE based on the sequence information from the partial peptide sequencing. As shown in Figure 3, the deduced amino acid sequence had approximately 40% similarity to nsLTP1 from the gymnosperm and angiosperm species. Predictably, this protein had a great potential of lipid transfer activity (Fig. 7) and exhibited a unique binding ability to only unsaturated fatty acids with cis-bond(s) (Fig. 8). This indicates that linear-chain fatty acids are not easily accommodated in the hydrophobic binding site of Gb-nsLTP1 and that cis-unsaturated fatty acids exhibit more compacted structures and therefore show a higher affinity for this protein. Interestingly, P79A and F80A mutants caused a significant loss of the lipid transfer activity (Table I). Here, one may consider whether their lower transfer capabilities are due to the inability to bind either any lipid (a fatty acid as well as a phospholipid) or just a phospholipid with two acyl chains (if the peptide cavity is reduced). The binding assay using the mutant proteins revealed that the binding activity of the mutant F79A and I82A were lower than the wild-type protein (Fig. 11); therefore, it was concluded that their lower transfer capability is due to the inability to bind to any lipid.
In this article, Gb-nsLTP1 was revealed to possess inhibitory activities against the Asp and Cys proteinases. Therefore, it seems likely that Gb-nsLTP1 serves to keep inactive some proteinases that are synthesized during seed development until the plant needs them. Apparently, such proteinases are the most important enzymes involved in transforming the large ginkgo storage proteins into small nitrogenous compounds. Such proteins or peptides can easily be utilized by the growing plant in the following germination. Actually, ginkgo seeds contain several storage proteins such as a 30-kD storage glycoprotein (Kimura et al., 1999
On the other hand, it was predicted that the mature protein is secreted extracellularly due to the NH2-terminal propeptide, which conflicts with the above hypothesis that the inhibitor localizes intracellularly with the target proteinases. Plant nsLTP1 are found primarily on the cell walls of aerial organs and seeds; therefore, nsLTP1 could be involved indirectly in plant defense by creating a mechanical barrier of cutin and might be involved directly due to their intrinsic antibiotic properties (Douliez et al., 2000
Several three-dimensional structures of nsLTP1-lipid complexes have revealed that the hydrophobic ligands are inserted into the internal cavity of nsLTP1 (e.g. Shin et al., 1995
In the solution structure of nsLTP1 from mung bean (Vigna radiata), Mb-nsLTP1, Arg-44 and Tyr-79 are located near the entrance of the tunnel-like hydrophobic cavity and interact with the polar headgroup of lipid molecules (Lin et al., 2005
Materials Mature ginkgo (Ginkgo biloba) seeds were obtained from the garden of our university in September, 2004. The immature seeds, leaves, and stems were collected there in May. The roots were obtained from the seedlings kindly gifted by Japan Ginkgo Farm (Fukushima, Japan). All the tissues were immediately frozen in liquid nitrogen and were stored at –80°C. Purified proteinases and the synthetic substrates were from Sigma-Aldrich. Oligonucleotide primers were synthesized by Sigma Genosys Japan KK and ThermoElectron GmbH Molecular Biology. All other reagents were of the highest grade commercially available.
Unless otherwise stated, all purification steps were carried out at 4°C. The mature ginkgo seeds were shelled and fully homogenized with 2 to 3 volumes of an extraction buffer containing 50 mM Tris-HCl buffer at pH 7.5, 10 mM KCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 5 mM iodoacetic acid. After stirring overnight, the homogenate was centrifuged at 33,570g for 10 min at 4°C, and the clear supernatant was freeze-dried. The resulting powder was resuspended in distilled water and dialyzed against the extraction buffer by using a 1,000-Mr cutoff membrane. The dialyzed solution was then applied onto a Sephadex G-50 gel-filtration column (2.2 x 96 cm; GE Healthcare Life Sciences) equilibrated with 50 mM Tris-HCl buffer, pH 7.5, containing 0.1 M NaCl.
The resulting sample was finally purified on an SP-Sepharose high-performance column (1.8 x 15 cm; GE Healthcare Life Sciences) equilibrated with 10 mM ammonium acetate buffer at pH 4.3. The adsorbed protein with the papain-inhibitory activity was eluted using a linear NaCl gradient (0–1 M). The identification and purity of the sample were confirmed by both MALDI-TOF MS and SDS-PAGE (15% gels) without dithiothreitol (DTT; Laemmli, 1970
The sample was oxidized with performic acid (Hirs, 1967
The cDNA encoding the proteinase inhibitor was isolated based on a PCR cloning strategy. Total RNA was extracted from the mature ginkgo seeds by using Plant RNA Isolation Reagent (Invitrogen). To isolate the cDNA, RT-PCR was performed after DNase treatment using a TURBO DNA-free kit (Ambion). The first-strand cDNA was synthesized from total RNA with AccuScript reverse transcriptase (Stratagene) by using oligo(dT)12-18 primer (Invitrogen). PCR was carried out using AmpliTaq Gold DNA polymerase (Applied Biosystems). The degenerate primers for amplification were 5'-GCICCIGGITGYGAYACIGT-3' (GBf1) and 5'-RTTRCARTCIGTIGCDAT-3' (GBr2; R = A/G, Y = C/T, D = G/A/T, I = inosine), which were respectively designed based on the NH2-terminal (APGCDTV) and internal amino acid sequences (IATDCN). PCR was performed using the first-strand cDNA as templates and with GBf1 and GBr4 as primers. The amplified DNA fragments were cloned into a pCR2.1-TOPO vector by using the TOPO TA Cloning kit (Invitrogen) and sequenced using a BigDye Terminator kit on an ABI Prism3700 DNA Analyzer (Applied Biosystems).
To obtain the full-length cDNA, 3' RACE and 5' RACE were performed (Frohman et al., 1988 For 5' RACE, first-strand cDNA synthesis was performed with the antisense primer of 5'-GAAAGGCAGAGTAACGCTACACAGTCCCG-3' (GBa3). A poly(A) tail was added to the 3' ends of the first-strand cDNA with terminal transferase (Roche Diagnostics) after RNase-H treatment. The poly(A)-tailed cDNA was used as a template for the first round of PCR with the adaptor primer and the nested antisense primer of 5'-AGGCTTTTGACGGCGTTCGCAACC-3' (GBa4). The resulting product was subjected to nested PCR with the adaptor primer and another nested primer located at the 5' end of GBa4, 5'-ACTCGAATAGCGGCCGTCTTTATACATTCAC-3' (GBa5). The fragments amplified by 3' RACE and 5' RACE were cloned into HincII-digested pUC19 vector via blunt-end ligation and were sequenced. To obtain further 5' sequences, a second round of 5' RACE was performed as follows: the first-strand cDNA synthesis was done with the antisense primer of 5'-GCAGTTCCGGCCAGCGTTTTGACACC-3' (GBa6) and was tailed at the 3' end. Primary PCR was performed using the poly(A)-tailed cDNA as a template with the adaptor primer and the nested antisense primer of 5'-AGCATTGTACAGTTGGGTTGCCGGTTCC-3' (GBa7), followed by nested PCR with the adaptor primer and another nested primer specific for the new 5' end, 5'-GTGTCTACAGTGTCACATCCGGGTGCTGC-3' (GBa8).
The obtained sequences were analyzed using bioinformatics tools at Web sites (www.ncbi.nlm.nih.gov, www.expasy.org). Before analysis, the putative and experimentally verified signal sequences were added. The phylogenetic analysis of several plant nsLTP1 were aligned with ClustalW (Thompson et al., 1994
Inhibitory activity toward pepsin was determined by the method of Anson (1938)
In the case of papain, we thoroughly mixed 16 µL of about 0.1 mg mL–1 enzyme solution, 16 µL of a sample solution (final concentration of 147 µM), and 652 µL of 50 mM sodium acetate buffer, pH 5.0, containing 2 mM EDTA and 1 mM DTT in a 1-cm path length quartz cuvette. In the case of trypsin, we used about 0.01 mg mL–1 enzyme solution and 20 mM sodium phosphate buffer, pH 6.3, containing 0.6 mM CaCl2. The cuvette was placed in the thermostatted cell compartment (25°C) of a Beckman DU-640 spectrophotometer. After at least 2 min of preincubation, 16 µL of the substrate p-nitrophenyl benzyloxycarbonyl-L-lysinate (1.1 mg mL–1) was added to the tip of a flattened glass rod; the solution was then stirred for 10 s. The initial velocity (v0) was calculated from an increase in A340 of the released p-nitrophenol. The percent inhibition was calculated by the following equation: Inhibition (%) = [1 – (v0 with sample)/(v0 without sample)] x 100. The inhibition constant values for pepsin and papain were calculated by a Dixon plot analysis (Dixon, 1953 In the case of thermolysin, the buffer used was 50 mM Tris-HCl buffer, pH 7.5, containing 10 mM CaCl2 and 100 mM NaCl; N-(3-[2-furyl]acryloyl)-Gly-Leu amide (1.0 mg mL–1) was used as a substrate for the enzyme. Fifty microliters of about 0.01 mg mL–1 enzyme solution, 16 µL of a sample solution (final concentration of 49 µM), and 584 µL of the buffer were mixed thoroughly in the quartz cuvette. After at least 2 min of preincubation at 37°C, 50 µL of the substrate was added to the tip of a flattened glass rod; the solution was then stirred for 10 s. Hydrolysis of the substrate by thermolysin was measured by a decrease in A345. The pH dependence of the inhibitory activity toward papain was determined using a solution of 50 mM buffer, 2 mM EDTA, and 1 mM DTT over the pH range of 2.0 to 7.5. The buffers used were Gly-HCl, pH 2.0 to 3.0; sodium citrate, pH 3.0 to 5.5; and sodium phosphate, pH 5.5 to 7.5. A sample solution (final concentration of 145 µM) and about 0.1 mg mL–1 enzyme solution were mixed in the appropriate buffer. The other methods were the same as described above.
We carried out antifungal activity assay against several fungi, including plant and human pathogens Fusarium oxysporum, Fusarium culmorum, Trichoderma reesei, Aspargillus fumigatus, and Mucor spinescens, in 100- x 15-mm petri plates containing 20 mL of potato dextrose agar. Sterile blank paper discs (0.6 cm in diameter) were placed at a distance of 0.5 cm from the rim of mycelial colony. Thirty microliters of a sample solution (25 mg mL–1) was added to a disc. The plates were incubated at 24°C for 72 h until mycelial growth had enveloped discs containing the control and formed crescents of inhibition around discs containing antifungal samples. The assay for antibacterial activity was conducted using sterile petri plates (100 x 15 mm) containing 10 mL of Luria-Bertani agar (1.5% agar). Three milliliters of warm nutrient agar (0.7%) containing Escherichia coli was poured into the plates. The sterile blank paper discs were placed on the agar; to one of the discs was then added a sample solution (20 µL) in 10 mM Tris-HCl buffer at pH 7.4. Only the same buffer was added to the control disc. The plate was incubated at 30°C for 20 to 24 h. A transparent ring around the paper disc signifies antibacterial activity.
Lipid transfer activity was assayed using fluorescence spectroscopy as described previously (Samuel et al., 2002
The lipid-binding assay of Gb-nsLTP1 was carried out using TNS. Fluorescence experiments were performed at 25°C in the fluorescence spectrophotometer as described previously (Buhot et al., 2004
Total RNA was extracted separately from different tissues (stem, leaf, root, immature seed, and mature seed). An aliquot of 1 µg of total RNA was used as a template in a single-step RT-PCR analysis using PrimeScript One Step RT-PCR kit version 2 (TaKaRa) with the primers of 5'-ATGATGAAGATATCGTGGCAGCTC-3' and 5'-TTAAACGATCTTGTTACAGTCGGTGG-3'. The template was reversely transcribed at 50°C for 30 min and denatured at 94°C for 2 min, followed by 25 cycles of amplification (94°C for 30 s, 60°C for 30 s, and 72°C for 45 s) and by extension at 72°C for 10 min. As a control, RT-PCR reaction for the housekeeping gene (18S rRNA gene) was performed using the specific primers of 5'-ATGATAACTCGACGGATCGC-3' and 5'-CTTGGATGTGGTAGCCGTTT-3'. The PCR products were separated on 2% (w/v) agarose gel stained with SYBR Safe DNA gel stain (Invitrogen).
The DNA sequence encoding Gb-nsLTP1 was amplified from the first-strand cDNA mixture using PfuUltra Hotstart DNA polymerase (Stratagene). The forward primer was 5'-GCACCCGGATGTGACACTGTAGAC-3', and the reverse was 5'-CGGGGATCCTTATTAAACGATCTTGTTACAGTCGG-3', designed to contain an additional stop codon and a BamHI site. The PCR product was phosphorylated by T4 polynucleotide kinase (TaKaRa), digested with BamHI, and ligated into a SmaI/BamHI-digested expression vector of pET48 (Novagen) driven by a T7 promoter. The resulting vector of pET48-Gb-nsLTP1 encodes a Trx-tag, a (His)6-tag, an HRV3C protease recognition site, and Gb-nsLTP1 in this order. The expression vectors for the mutants were constructed from a pET48-Gb-nsLTP1 vector by using the QuikChange mutagenesis kit (Stratagene).
E. coli Origami2(DE3) cells (Novagen) were transformed with pET48-Gb-nsLTP1. The cells were cultured at 37°C by shaking in a Luria-Bertani medium containing kanamycin (30 µg mL–1) and tetracycline (10 µg mL–1). The production of the recombinant proteins was induced at an optical density at 600 nm of 0.5 by addition of isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.5 mM. After induction at 25°C for 16 h, the cells were harvested by centrifugation, washed once with 0.5% (w/v) NaCl, and stored at –80°C. The frozen cells were resuspended in a binding buffer (50 mM sodium phosphate buffer at pH 8.0, 300 mM NaCl, and 10 mM imidazole) containing 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 80 nM aprotinin, 2 µM leupeptin, 4 µM bestatin, 1.5 µM pepstatin A, and 1.4 µM E-64; the resulting suspension was lysed by ultrasonication. The soluble fraction was separated from the insoluble by centrifugation (40,000g at 4°C for 30 min), and the supernatant was added onto a Ni-NTA-agarose column (Qiagen) equilibrated with the binding buffer. After washing with the buffer, the sample was eluted with 50 mM sodium phosphate buffer, pH 8.0, containing 300 mM NaCl and 300 mM imidazole and was dialyzed against a 20 mM Tris-HCl buffer at pH 7.5. The regions of Trx-tag and (His)6-tag were digested with HRV3C protease (Novagen) according to the manufacturer's instructions. The sample was applied onto a Mono-S 10/10 column (GE Healthcare Life Sciences) equilibrated with 20 mM MES buffer at pH 5.5 and was eluted with a linear gradient of 0 to 0.2 M NaCl in the MES buffer. The resulting sample was pooled, dialyzed against 10 mM MES buffer at pH 6.0, and concentrated by ultrafiltration using a 3,000-Mr cutoff Vivaspin (Sartorius). The purity and identification of the sample were checked by SDS-PAGE and MALDI-TOF MS as described above. The mutant proteins were expressed and purified as described above.
All CD spectra were recorded at room temperature with a Jasco J-600 spectropolarimeter (JASCO International). Gb-nsLTP1 was adjusted to pH 2.0, 4.0, 5.0, or 7.0 with protein concentrations of approximately 70 µM, while the recombinant Gb-nsLTP1 proteins were only to pH 7.0. Individual CD spectra were averaged over two scans with 0.2-nm increments using a 0.5-mm path length cuvette. The spectra thus obtained were submitted to a smoothing procedure and expressed in terms of mean residue ellipticity [ Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number DQ836633. Received October 25, 2007; accepted February 19, 2008; published February 27, 2008.
1 This work was supported by three national projects from the Ministry of Education, Culture, Sports, Science and Technology of Japan: Protein Structure and Functional Analyses, the Targeted Proteins Research Program, and the 21st Century Center of Excellence Program. 
2 These authors contributed equally to the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ken-ichi Hatano (hatano{at}chem-bio.gunma-u.ac.jp). www.plantphysiol.org/cgi/doi/10.1104/pp.107.111500 * Corresponding author; e-mail hatano{at}chem-bio.gunma-u.ac.jp.
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] in degree cm2 dmol–1. The fraction of helical content (fH) was calculated using the following equation (Chen et al., 1972
xylose-containing N-glycans. Biosci Biotechnol Biochem 63: 463–467
