Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds
-
Yoriko Sawano
, Takuya Miyakawa , Hiroshi Yamazaki , Masaru Tanokura und Ken-ichi Hatano
Abstract
A novel basic protein with antifungal activity was isolated from the seeds of Ginkgo biloba and purified to homogeneity. The protein inhibited the growth of some fungi (Fusarium oxysporum, Trichoderma reesei, and Candida albicans) but did not exhibit antibacterial action against Escherichia coli. Furthermore, this protein showed weak inhibitory activity against the aspartic protease pepsin. To design primers for gene amplification, the NH2-terminal and partial internal amino acid sequences were determined using peptides obtained from a tryptic digest of the oxidized protein. The full-length cDNA of the antifungal protein was cloned and sequenced by RT-PCR and rapid amplification of cDNA ends (RACE). The cDNA contained a 402-bp open reading frame encoding a 134-aa protein with a potential signal peptide (26 residues), suggesting that this protein is synthesized as a preprotein and secreted outside the cells. The antifungal protein shows approximately 85% identity with embryo-abundant proteins from Picea abies and Picea glauca at the amino acid level; however, there is no homology between this protein and other plant antifungal proteins, such as defensin, and cyclophilin-, miraculin- and thaumatin-like proteins.
References
Bendtsen, J.D., Nielsen, H., von Heijne, G., and Brunak, S. (2004). Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol.340, 783–795.10.1016/j.jmb.2004.05.028Suche in Google Scholar
Broekaert, W.F., Terras, F.R., Cammue, B.P., and Osborn, R.W. (1995). Plant defensins: novel antimicrobial peptides as components of the host defense system. Plant Physiol.108, 1353–1358.10.1104/pp.108.4.1353Suche in Google Scholar
Bulet, P., Stöcklin, R., and Menin, L. (2004). Anti-microbial peptides: from invertebrates to vertebrates. Immunol. Rev.198, 169–184.10.1111/j.0105-2896.2004.0124.xSuche in Google Scholar
Campos, F., Zamudio, F., and Covarrubias, A.A. (2006). Two different late embryogenesis abundant proteins from Arabidopsis thaliana contain specific domains that inhibit Escherichia coli growth. Biochem. Biophys. Res. Commun.342, 406–413.10.1016/j.bbrc.2006.01.151Suche in Google Scholar
Chen, Y.H., Yang, J.T., and Martinez, H.M. (1972). Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry11, 4120–4131.10.1021/bi00772a015Suche in Google Scholar
Chen, Z. (2001). A superfamily of proteins with novel cysteine-rich repeats. Plant Physiol.126, 473–476.10.1104/pp.126.2.473Suche in Google Scholar
Czernic, P., Visser, B., Sun, W., Savoure, A., Deslandes, L., Marco, Y., Van Montagu, M., and Verbruggen, N. (1999). Characterization of an Arabidopsis thaliana receptor-like protein kinase gene activated by oxidative stress and pathogen attack. Plant J.18, 321–327.10.1046/j.1365-313X.1999.00447.xSuche in Google Scholar
Dash, C., Ahmad, A., Nath, D., and Rao, M. (2001). Novel bifunctional inhibitor of xylanase and aspartic protease: implications for inhibition of fungal growth. Antimicrob. Agents Chemother.45, 2008–2017.10.1128/AAC.45.7.2008-2017.2001Suche in Google Scholar
De Lucca, A.J., Cleveland, T.E., and Wedge, D.E. (2005). Plant-derived antifungal proteins and peptides. Can. J. Microbiol.51, 1001–1014.10.1139/w05-063Suche in Google Scholar
Diamond, B.J., Shiflett, S.C., Feiwel, N., Matheis, R.J., Noskin, O., Richards, J.A., and Schoenberger, N.E. (2000). Ginkgo biloba extract: mechanisms and clinical indications. Arch. Phys. Med. Rehab.81, 668–678.10.1016/S0003-9993(00)90052-2Suche in Google Scholar
Dong, J.Z. and Dunstan, D.I. (1999). Cloning and characterization of six embryogenesis-associated cDNAs from somatic embryos of Picea glauca and their comparative expression during zygotic embryogenesis. Plant Mol. Biol.39, 859–864.10.1023/A:1006146622614Suche in Google Scholar
Frohman, M.A., Dush, M.K., and Martin, G.R. (1988). Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA85, 8998–9002.10.1073/pnas.85.23.8998Suche in Google Scholar
Hirs, C.H.W. (1967). Performic acid oxidation. Methods Enzymol.11, 197–199.10.1016/S0076-6879(67)11021-5Suche in Google Scholar
Huang, X., Xie, W.-J., and Gong, Z.-Z. (2000). Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba. FEBS Lett.478, 123–126.10.1016/S0014-5793(00)01834-2Suche in Google Scholar
Kim, M.-H., Park, S.-C., Kim, J.-Y., Lee, S.Y., Lim, H.-T., Cheong, H., Hahm, K.-S., and Park, Y. (2006). Purification and characterization of a heat-stable serine protease inhibitor from the tubers of new potato variety ‘Golden Valley’. Biochem. Biophys. Res. Commun.346, 681–686.10.1016/j.bbrc.2006.05.186Suche in Google Scholar
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227, 680–685.10.1038/227680a0Suche in Google Scholar
McCubbin, W.D., Kay, C.M., and Lane, B.G. (1985). Hydrodynamic and optical properties of the wheat germ Em protein. Can. J. Biochem. Cell Biol.63, 803–811.10.1139/o85-102Suche in Google Scholar
Morris, E.R. and Walker, J.C. (2003). Receptor-like protein kinases: the keys to response. Curr. Opin. Plant Biol.6, 339–342.10.1016/S1369-5266(03)00055-4Suche in Google Scholar
Nakai, K. and Kanehisa, M. (1992). A knowledge base for predicting protein localization sites in eukaryotic cells. Genomics14, 897–911.10.1016/S0888-7543(05)80111-9Suche in Google Scholar
Provencher, S.W. and Glöckner, J. (1981). Estimation of globular protein secondary structure from circular dichroism. Biochemistry20, 33–37.10.1021/bi00504a006Suche in Google Scholar PubMed
Schägger, H. and von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem.166, 368–379.10.1016/0003-2697(87)90587-2Suche in Google Scholar
Shao, H.-B., Liang, Z.-S, and Shao, M.-A. (2005). LEA proteins in higher plants: structure, function, gene expression and regulation. Colloids Surf. B Biointerfaces45, 131–135.Suche in Google Scholar
Shen, G., Pang, Y., Wu, W., Deng, Z., Liu, X., Lin, J., Zhao, L., Sun, X., and Tang, K. (2005). Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba. J. Plant Physiol.43, 836–843.10.1016/j.jplph.2005.01.019Suche in Google Scholar
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner F.H., Provenzano, M.D., Fujimoto, E.K, Goeke, N.M., Olson, B.J., and Klenk, D.C. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem.150, 76–85.10.1016/0003-2697(85)90442-7Suche in Google Scholar
ten Have, A., Dekkers, E., Kay, J., Phylip, L.H., and van Kan, J.A. (2004). An aspartic protease gene family in the filamentous fungus Botrytis cinerea contains members with novel features. Microbiology150, 2475–2489.Suche in Google Scholar
Tsukuda, S., Gomi, K., Yamamoto, H., and Akimitsu, K. (2006). Characterization of cDNAs encoding two distinct miraculin-like proteins and stress-related modulation of the corresponding mRNAs in Citrus jambhiri Lush. Plant Mol. Biol.60, 125–136.10.1007/s11103-005-2941-4Suche in Google Scholar PubMed
Wang, H. and Ng, T.B. (2000). Ginkbilobin, a novel antifungal protein from Ginkgo biloba seeds with sequence similarity to embryo-abundant protein. Biochem. Biophys. Res. Commun.279, 407–411.10.1006/bbrc.2000.3929Suche in Google Scholar PubMed
Wurtele, E.S., Wang, H., Durgerian, S., Nikolau, B.J., and Ulrich, T.H. (1993). Characterization of a gene that is expressed early in somatic embryogenesis of Daucus carota. Plant Physiol.102, 303–312.10.1104/pp.102.1.303Suche in Google Scholar PubMed PubMed Central
Ye, X.Y. and Ng, T.B. (2000). Mungin, a novel cyclophilin-like antifungal protein from the mung bean. Biochem. Biophys. Res. Commun.273, 1111–1115.10.1006/bbrc.2000.3067Suche in Google Scholar PubMed
Ye, X.Y., Wang, H.X., and Ng, T.B. (1999). First chromatographic isolation of an antifungal thaumatin-like protein from French bean legumes and demonstration of its antifungal activity. Biochem. Biophys. Res. Commun.263, 130–134.10.1006/bbrc.1999.1166Suche in Google Scholar PubMed
©2007 by Walter de Gruyter Berlin New York
Artikel in diesem Heft
- Supplementary material to the paper “Evolutionary selection pressure and family relationships among connexin genes”
- Evolutionary selection pressure and family relationships among connexin genes
- Characterization of the large subunit of EcoHK31I methyltransferase by structural modeling and mutagenesis
- Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds
- Maximal Ca2+i stimulation of cardiac Na+/Ca2+ exchange requires simultaneous alkalinization and binding of PtdIns-4,5-P2 to the exchanger
- A highly conserved protein secreted by the prostate cancer cell line PC-3 is expressed in benign and malignant prostate tissue
- Properties and partial purification of sialate-O-acetyltransferase from bovine submandibular glands
- Raft association and lipid droplet targeting of flotillins are independent of caveolin
- On the presence of C2-ceramide in mammalian tissues: possible relationship to etherphospholipids and phosphorylation by ceramide kinase
- Specific inhibition of interleukin-13 activity by a recombinant human single-chain immunoglobulin domain directed against the IL-13 receptor α1 chain
- Effects of disease-modifying anti-rheumatic drugs (DMARDs) on the activities of rheumatoid arthritis-associated cathepsins K and S
- Compartmentalised expression of meprin in small intestinal mucosa: enhanced expression in lamina propria in coeliac disease
- Human dipeptidyl peptidase III acts as a post-proline-cleaving enzyme on endomorphins
- Transgenic mouse brains for the evaluation and quality control of BSE tests
Artikel in diesem Heft
- Supplementary material to the paper “Evolutionary selection pressure and family relationships among connexin genes”
- Evolutionary selection pressure and family relationships among connexin genes
- Characterization of the large subunit of EcoHK31I methyltransferase by structural modeling and mutagenesis
- Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds
- Maximal Ca2+i stimulation of cardiac Na+/Ca2+ exchange requires simultaneous alkalinization and binding of PtdIns-4,5-P2 to the exchanger
- A highly conserved protein secreted by the prostate cancer cell line PC-3 is expressed in benign and malignant prostate tissue
- Properties and partial purification of sialate-O-acetyltransferase from bovine submandibular glands
- Raft association and lipid droplet targeting of flotillins are independent of caveolin
- On the presence of C2-ceramide in mammalian tissues: possible relationship to etherphospholipids and phosphorylation by ceramide kinase
- Specific inhibition of interleukin-13 activity by a recombinant human single-chain immunoglobulin domain directed against the IL-13 receptor α1 chain
- Effects of disease-modifying anti-rheumatic drugs (DMARDs) on the activities of rheumatoid arthritis-associated cathepsins K and S
- Compartmentalised expression of meprin in small intestinal mucosa: enhanced expression in lamina propria in coeliac disease
- Human dipeptidyl peptidase III acts as a post-proline-cleaving enzyme on endomorphins
- Transgenic mouse brains for the evaluation and quality control of BSE tests