Valdirene M. Gomes

Plant defensins - prospects for the biological functions and biotechnological properties.

Plant defensins are a prominent family of cationic peptides in the plant kingdom. They are structurally and functionally related to defensins that have been previously characterized in mammals and insects. They present molecular masses between 5 and 7kDa and possess a pattern of eight conserved Cys residues. The three-dimensional structure of plant defensins is small and globular. It has three anti-parallel beta-sheets and one alpha-helix that is stabilized by a structural motif composed of disulfide bridges. This motif is found in other peptides with biological activity and is called the Cys stabilized alphabeta motif (CSalphabeta). Based on the growing knowledge on defensin structure, gene expression and regulation, and also their in vitro biological activity, it has become clear that plant defensins are complex and sophisticated peptides whose function extends beyond their role in defense of plants against microbial infection. This review discusses recent data and will present comprehensive information regarding the study of defensins.

Role of plant lipid transfer proteins in plant cell physiology-a concise review.

Plant lipid transfer proteins (LTP) are cationic peptides, subdivided into two families, which present molecular masses of around 7 and 10 kDa. The peptides were, thus, denominated due to their ability to reversibly bind and transport hydrophobic molecules in vitro. Both subfamilies possess conserved patterns of eight cysteine residues and the three-dimensional structure reveals an internal hydrophobic cavity that comprises the lipid binding site. Based on the growing knowledge regarding structure, gene expression and regulation and in vitro activity, LTPs are likely to play a role in key processes of plant physiology. Although the roles of plant LTPs have not yet been fully determined. This review aims to present comprehensive information of recent topics, cover new additional data, and present new perspectives on these families of peptides.

Isolation and partial characterization of a novel lectin from Talisia esculenta seeds that interferes with fungal growth

A novel plant lectin has been isolated from the seeds of Talisia esculenta and partially characterized. The purified lectin showed two protein bands in SDS-PAGE (20,000 and 40,000 kDa) and agglutinated human and animal erythrocytes. Of the various sugars tested, the lectin was best inhibited by mannose. A search of sequence databases showed that the N-terminal sequence had no homology to any known protein. The lectin inhibited the growth of the fungi Fusarium oxysporum, Colletotrichum lindemuthianum and Saccharomyces cerevisiae.

Purification, biochemical characterization and antifungal activity of a new lipid transfer protein (LTP) from Coffea canephora seeds with α-amylase inhibitor properties.

BACKGROUND A growing number of cysteine-rich antimicrobial peptides (AMPs) have been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides, which include lipid transfer proteins (LTPs), play an important role in the protection of plants against microbial infection. METHODS Peptides from Coffea canephora seeds were extracted in Tris-HCl buffer (pH 8.0), and chromatographic purification of LTP was performed by DEAE and reverse-phase HPLC. The purified peptide was submitted to amino acid sequence, antimicrobial activity and mammalian α-amylase inhibitory analyses. RESULTS The purified peptide of 9kDa had homology to LTPs isolated from different plants. Bidimensional electrophoresis of the 9kDa band showed the presence of two isoforms with pIs of 8.0 and 8.5. Cc-LTP(1) exhibited strong antifungal activity, against Candida albicans, and also promoted morphological changes including the formation of pseudohyphae on Candida tropicalis, as revealed by electron micrograph. Our results show that Cc-LTP(1) interfered in a dose-dependent manner with glucose-stimulated, H(+)-ATPase-dependent acidification of yeast medium and that the peptide permeabilized yeast plasma membranes to the dye SYTOX green, as verified by fluorescence microscopy. Interestingly, we also showed for the first time that the well characterized LTP(1) family, represented here by Cc-LTP(1), was also able to inhibit mammalian α-amylase activity in vitro. CONCLUSIONS AND GENERAL SIGNIFICANCE In this work we purified, characterized and evaluated the in vitro effect on yeast of a new peptide from coffee, named Cc-LPT1, which we also showed, for the first time, the ability to inhibit mammalian α-amylase activity.

A 2S albumin-homologous protein from passion fruit seeds inhibits the fungal growth and acidification of the medium by Fusarium oxysporum.

Antimicrobial proteins have been isolated from a wide range of plant species. More recently, it has become increasingly clear that these types of proteins play an important role in the protection of plants. In this study, we investigate the presence of defense-related proteins from passion fruit (Passiflora edulis f. flavicarpa) seeds. Initially, seed flour was extracted for 2h (at 4 degrees C) with phosphate buffer, pH 5.5. The precipitate obtained between 0 and 70% relative ammonium sulfate saturation was re-dissolved in distilled water and heated at 80 degrees C for 15 min. The resulting suspension was clarified by centrifugation and the supernatant (F/0-70) was extensively dialyzed. A Sephadex G-50 size exclusion column was employed for further separation of proteins. The fraction with antifungal activity was pooled and submitted to CM-Sepharose cation exchange. Two proteins, named Pf1 and Pf2, were eluted in 0.1 and 0.2M of salt, respectively, and submitted to reverse-phase chromatography in HPLC. This fraction inhibited the growth, in an in vitro assay, of the phytopathogenic fungi Fusarium oxysporum and colletotrichum lindemuthianum and the yeast Saccharomyces cerevisiae and strongly inhibited glucose-stimulated acidification of the medium by F. oxysporum in a dose-dependent manner. The molecular masses of these proteins, referred to now as Pf1-RP and Pf2-RP, were obtained by MALDI-TOF spectrometry and corresponded to 12,088 Da for Pf1-RP and 11,930 Da for Pf2-RP. These proteins were also subjected to automated N-terminal amino acid sequencing. Sequence comparisons for the heavy subunit of Pf2-RP showed the presence of a protein with a high degree of homology to storage 2S albumins.

Legume seed vicilins (7S storage proteins) interfere with the development of the cowpea weevil (Callosobruchus maculatus (F)).

Vicilins (7S storage proteins) isolated from the seeds of the legumes Vigna unguiculata (cowpea), Vigna angularis (adzuki bean), Canavalia ensiformis (jack bean), Glycine max (soybean), Phaseolus vulgaris (common bean) and Phaseolus lunatus (lima bean) were shown to be immunologically related and to bind to a chitin matrix. The effect of the isolated vicilins on the development of the cowpea weevil Callosobruchus maculatus was examined. Vicilins from all non-host seeds, including those of the C maculatus-resistant cowpea line IT81D-1045, strongly inhibited larval development (ED 50 of 1.07 ± 0.32% to 2.22 ± 0.64%). Vicilins from the C maculatus-susceptible cowpea CE-31 and adzuki bean seeds were the exception with ED 50 of 6.25 ± 0.75% and 5.40 ± 1.54%, respectively. These results correlate well with the host range of C maculatus and are possibly a reflection of the low digestibility of vicilins by insect midgut proteinases in addition to the ability they show in binding to chitin-containing structures like the ones found in the bruchid midguts.

Legume vicilins (7S storage globulins) inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells.

Vicilin (7S storage proteins) isolated from different legume seeds were shown to inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. The degree of growth inhibition varied with the origin of vicilins. It was more than 90% for vicilins from cowpea (Vigna unguiculata, cultivar pitiuba) and equal to 65% for vicilins from Vigna radiata, in the case of Saccharomyces cerevisae. Vicilins from cowpea seeds inhibited the glucose stimulated acidification of the medium by S. cerevisae up to 60%. We have also observed that vicilins bind to yeast cells. We suggest that vicilins bind to chitin-containing structures of yeast cells and that such association could result in inhibition of H+ pumping, cell growth and spore formation. A final consequence of the yeast growth inhibition by vicilins is (probably) the formation of spores.

Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds

The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301-9], with some modifications. A DEAE-Sepharose, equilibrated with 20mM Tris-HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS-Tricine gel electrophoresis with a molecular mass of approximately 6kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.

Purification and physicochemical characterization of a cotyledonary lectin from Luetzelburgia auriculata.

A lectin was purified from the cotyledons of Luetzelburgia auriculata (Fr. All) Ducke by affinity chromatography on agarose-N-acetyl-D-galactosamine. The lectin is a potent agglutinin for rabbit erythrocytes, reacts with human red cells, but is inactive against cow, sheep, and goat erythrocytes. Hemagglutination of rabbit erythrocytes was inhibited by either 0.39 mM N-acetyl-neuraminic acid or N-acetyl-D-galactosamin, 12.5 mM D-lactose or D-melibiose, 50 mM D-galactose or raffinose. Its hemagglutinating activity was lost at 80 degrees C, 5 min, and the activation energy required for denaturation was 104.75 kJ mol(-1). Chromatography on Sephadex G-100, at pH 7.6, showed that at this hydrogenic ionic concentration the native lectin was a homotetramer (123.5 kDa). By denaturing SDS-PAGE, LAA seemed to be composed of a mixture of 29 and 15 kDa polypeptide subunits. At acidic and basic pHs it assumed different conformations, as demonstrated by exclusion chromatography on Superdex 200 HR 10/30. The N-terminal sequence of the 29 kDa band was SEVVSFSFTKFNPNQKDII and the 15 kDa band contained a mixture of SEVVSFSFTKFNPNQKDII and KFNQIVAVEEDTDXESQPQ sequences, indicating that these bands may represent full-length and its endogenous fragments, respectively. The lectin is a glycoprotein having 3.2% neutral carbohydrate, with a pI of 5.8, containing high levels of Asp+Asn and Glu+Gln and hydroxy amino acids, and low amount or absence of sulfur amino acids. Its absorption spectrum showed a maximum at 280 nm and a epsilon (1%) x (1cm) of 5.2. Its CD spectrum was characterized by minima near 228 nm, maxima near 196 nm and a negative to positive crossover at 210 nm. The secondary structure content was 6% alpha-helix, 8% parallel beta-sheet, 38% antiparallel beta-sheet, 17% beta-turn, 31% unordered and others contribution, and 1% RMS (root mean square). In the fluorescence spectroscopy, excitation of the lectin solution at 280 nm gave an emission spectrum in the 285-445 nm range. The wavelength maximum emission was in 334.5 nm, typical for tryptophan residues buried inside the protein.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel α-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1) . Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the proteins biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1) , showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of α-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian α-amylase activity in vitro.