Detection and Immunofiuorescence Localization of Antifungal Protein of Gastrodiaelata

Gastrodia data B1. antifungal protein (GAFP) was purified by DEAE-Cellulase column chromatography and Sephadex G-50 column chromatography, and the polyclonal antibody against GAFP (GAFP-PcAbs) was also produced. Using Western blot and Dot blot it was demonstrated that GAFP-PcAbs is specific for GAFP. For further study, immunofluorescence localization of GAFP in secondry corms of G. data has been conducted with fluorescence microscopy. The result showed that GAFP was localized at epidermal and cortical tissues in secondary corm of G. elata,and the content of GAFP was higher in epidermis.     

A novel mannose-binding tuber lectin from Typhonium divaricatum (L.) Decne (family Araceae) with antiviral activity against HSV-II and anti-proliferative effect on human cancer cell lines

A novel mannose-binding tuber lectin with in vitro antiproliferative activity towards human cancer cell lines and antiviral activity against HSV-II was isolated from fresh tubers of a traditional Chinese medicinal herb, Typhonium divaricatum (L.) Decne by a combined procedure involving extraction, ammonium sulfate precipitation, ion exchange chromatography on DEAE-SEPHAROSE, CM-SEPHAROSE and gel-filtration on sephacryl S-200. The apparent molecular mass of the purified Typhonium divaricatum lectin (TDL) was 48 kDa. TDL exhibits hemagglutinating activity toward rabbit erythrocytes at 0.95 microg/ml, and its activity could be strongly inhibited by mannan, ovomucoid, asialofetuin and thyroglobulin. TDL showed antiproliferative activity towards some well established human cancer cell lines, e.g. Pro-01 (56.7 +/- 6.8), Bre-04 (41.5 +/- 4.8), and Lu-04 (11.4 +/- 0.3). The anti-HSV-II activity of TDL was elucidated by testing its HSV-II infection inhibitory activity in Vero cells with TC(50) and EC(50) of 5.176 mg/ml and 3.054 microg/ml respectively. The full-length cDNA sequence of TDL was 1145 bp and contained an 813-bp open reading frame (ORF) encoding a 271 amino acid precursor of 29-kDa. Homology analysis showed that TDL had high homology with many other mannose-binding lectins. Secondary and three-dimensional structures analyses showed that TDL is heterotetramer and similar with lectins from mannose-binding lectin superfamily, especially those from family Araceae.     

Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba

An antifungal peptide from leaves of Ginkgo biloba, designated GAFP, has been isolated. Its molecular mass of 4244.0 Da was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation. GAFP exhibited antifungal activity towards Pellicularia sasakii Ito, Alternaria alternata (Fries) Keissler, Fusarium graminearum Schw. and Fusarium moniliforme. Its activities differed among various fungi. GAFP could also cause increased hyphal membrane permeabilization and a rapid alkalization of the medium when applied at 100 microgram/ml to Pellicularia sasakii Ito hyphae. The amino acid sequence of GAFP shows characteristics of the cysteine/glycine-rich chitin binding domain of many chitin binding proteins. The cysteine residues are well conserved.     

The promoter of an antifungal protein gene from Gastrodia elata confers tissue -specific and fungus-inducible expression patterns and responds to both salicylic acid and jasmonic acid

Gastrodia antifungal proteins (GAFPs) are a group of mannose-binding lectins purified from Gastrodia elata that show strong resistance against a wide spectrum of fungi. The GAFP-2 promoter was analyzed for its ability to control the expression of the reporter gene, -glucuronidase (GUS) in transgenic tobacco plants. The GUS assays revealed that the GAFP-2 promoter is expressed in a tissue-specific manner, which mainly expressed in the vascular cells. The highest GUS activity was observed in roots, followed by stems. GAFP-2-GUS expression was strongly induced by the fungus Trichoderma viride and by the plant stress regulators, salicylic acid and jasmonic acid in the stably transformed tobacco plants. The –537 region of the GAFP-2 promoter was sufficient for its tissue-specific and inducible expression of the promoter.Communicated by H.S. Judelson     

Structure-function relationship of monocot mannose-binding lectins.

The monocot mannose-binding lectins are an extended superfamily of structurally and evolutionarily related proteins, which until now have been isolated from species of the Amaryllidaceae, Alliaceae, Araceae, Orchidaceae, and Liliaceae. To explain the obvious differences in biological activities, the structure-function relationships of the monocot mannose-binding lectins were studied by a combination of glycan-binding studies and molecular modeling using the deduced amino acid sequences of the currently known lectins. Molecular modeling indicated that the number of active mannose-binding sites per monomer varies between three and zero. Since the number of binding sites is fairly well correlated with the binding activity measured by surface plasmon resonance, and is also in good agreement with the results of previous studies of the biological activities of the mannose-binding lectins, molecular modeling is of great value for predicting which lectins are best suited for a particular application.     

Point mutations abolishing the mannose-binding capability of boar spermadhesin AQN-1

The mannose-binding capability of recombinant wild-type boar spermadhesin AQN-1 and of its site-directed mutants in the highly-conserved region around of the single glycosylation site (asparagine 50) of some spermadhesins, where the carbohydrate binding site has been proposed to be located, was checked using a solid-phase assay and a biotinylated mannose ligand. Substitution of glycine 54 by amino acids bearing an unipolar side chain did not cause significant decrease in the mannose-binding activity. However, amino acids with uncharged polar side chains or having a charged polar side chain abolished the binding of biotinylated mannose to the corresponding AQN-1 mutants. The results suggest that the higher surface accessibility of amino acids possessing polar side chains compared to those bearing nonpolar groups may sterically interfere with monosaccharide binding. The location of the mannose-binding site in AQN-1 appears to be topologically conserved in other heparin-binding boar spermadhesins, i.e., AQN-3 and AWN, but departs from the location of the mannose-6-phosphate-recognition site of PSP-II. This indicates that different spermadhesin molecules have evolved non-equivalent carbohydrate-binding capabilities, which may underlie their distinct patterns of biological activities.     

Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties

The orchid Gastrodia elata depends on the fungus Armillaria mellea to complete its life cycle. In the interaction, fungal hyphae penetrate older, nutritive corms but not newly formed corms. From these corms, a protein fraction with in vitro activity against plant-pathogenic fungi has previously been purified. Here, the sequence of gastrodianin, the main constituent of the antifungal fraction, is reported. Four isoforms that encoded two different mature proteins were identified at the cDNA level. Another isoform was detected in sequenced peptides. Because the antifungal activity of gastrodianins produced in and purified from Escherichia coli and Nicotiana tabacum was comparable to that of gastrodianin purified from the orchid, gastrodianins are the active component of the antifungal fractions. Gastrodianin accumulation is probably an important part of the mechanism by which the orchid controls Armillaria penetration. Gastrodianin was found to be homologous to monomeric mannose-binding proteins of other orchids, of which at least one (Epipactis helleborine mannose-binding protein) also displayed in vitro antifungal activity. This establishes the gastrodianin-like proteins (GLIPs) as a novel class of antifungal proteins.     

Bioinformatics analyses of the mannose-binding lectins from Polygonatum cyrtonema,Ophiopogon japonicus and Liparis noversa with antiproliferative and apoptosis-inducing activities

In the present study, three typical monocot mannose-binding lectins (e.g., Polygonatum cyrtonema lectin [PCL], Ophiopogon japonicus lectin [OJL] and Liparis noversa lectin [LNL]), were reported to possess a similar tertiary structure with three mannose-binding sites and a close phylogenetic relationship. Subsequently, these lectins were found to bear remarkable inhibitory effects on the growth of MCF-7 cells. Further experiments confirmed that there is a link among the hemagglutinating activity, antiproliferative activity and mannose-binding activity. In addition, these lectins were shown to induce MCF-7 cell apoptosis and caspase was found to be involved in this apoptotic pathway. In conclusion, these findings demonstrate that the different antiproliferative effects may be due to the conserved motifs of mannose-binding sites. Furthermore, our results demonstrate that these lectins induce apoptosis in MCF-7 cells via a caspase-dependent pathway.     

<Emphasis Type="Italic">Narcissus tazetta</Emphasis> lectin shows strong inhibitory effects against respiratory syncytial virus,influenza A (H1N1, H3N2, H5N1) and B viruses

A mannose-binding lectin (Narcissus tazetta lectin [NTL]) with potent antiviral activity was isolated and purified from the bulbs of the Chinese daffodil Narcissus tazetta var. chinensis, using ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity chromatography on mannose-agarose and fast protein liquid chromatography (FPLC)-gel filtration on Superose 12. The purified lectin was shown to have an apparent molecular mass of 26 kDa by gel filtration and 13 kDa by SDS-PAGE, indicating that it is probably a dimer with two identical subunits. The cDNA-derived amino acid sequence of NTL as determined by molecular cloning also reveals that NTL protein contains a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension. Three-dimensional modelling study demonstrated that the NTL primary polypeptide contains three subdomains, each with a conserved mannose-binding site. It shows a high homology of about 60%–80% similarity with the existing monocot mannose-binding lectins. NTL could significantly inhibit plaque formation by the human respiratory syncytial virus (RSV) with an IC₅₀ of 2.30 μg/ml and exhibit strong antiviral properties against influenza A (H1N1, H3N2, H5N1) and influenza B viruses with IC₅₀ values ranging from 0.20 μg/ml to 1.33 μg/ml in a dose-dependent manner. It is worth noting that the modes of antiviral action of NTL against RSV and influenza A virus are significantly different. NTL is effective in the inhibition of RSV during the whole viral infection cycle, but the antiviral activity of NTL is mainly expressed at the early stage of the viral cycle of influenza A (H1N1) virus. NTL with a high selective index (SI=CC₅₀/IC₅₀≥141) resulting from its potent antiviral activity and low cytotoxicity demonstrates a potential for biotechnological development as an antiviral agent.     

Mitochondrial processing of bovine adrenal steroidogenic acute regulatory protein

Steroidogenic acute regulatory (StAR) protein is an important regulatory protein in steroidogenesis and rapidly undergoes proteolysis after import into the mitochondria. In this study, we determined the proteolytic cleavage sites and investigated the effects on the stimulation of steroidogenic activity of the blockage of these sites by mutation. The cleaved StAR proteins, which were purified using an anti-StAR immobilized column, reacted with antiserum against the StAR C-terminal oligopeptide. The molecular weights of the purified proteins were determined by MALDI-TOF mass spectrometry, and were found to be identical to those of the 40-285 and 55-285 amino-acid-regions of the StAR protein. To confirm the identification of the cleavage sites, we constructed site-directed mutants of bovine StAR cDNA, which contained the amino acids R37A/R38A/L40A and/or R53A/R54A/R55A. These mutant StAR proteins expressed in COS-1 cells were not cleaved at positions 39-40 and 54-55, and were processed at sites different from those in the wild-type StAR protein. These mutant proteins stimulated pregnenolone formation at almost the same rate as the wild-type StAR protein in COS-1 cells, which suggests that the cholesterol transfer activity was not affected by the mutation.     

A single disulfide bond restores thermodynamic and proteolytic stability to an extensively mutated protein

The potential for engineering stable proteins with multiple amino acid substitutions was explored. Eleven lysine, five methionine, two tryptophan, one glycine, and three threonine substitutions were simultaneously made in barley chymotrypsin inhibitor-2 (CI-2) to substantially improve the essential amino acid content of the protein. These substitutions were chosen based on the three-dimensional structure of CI-2 and an alignment of homologous sequences. The initial engineered protein folded into a wild-type-like structure, but had a free energy of unfolding of only 2.2 kcal/mol, considerably less than the wild-type value of 7.5 kcal/mol. Restoration of the lysine mutation at position 67 to the wild-type arginine increased the free energy of unfolding to 3.1 kcal/mol. Subsequent cysteine substitutions at positions 22 and 82 resulted in disulfide bond formation and a protein with nearly wild-type thermodynamic stability (7.0 kcal/mol). None of the engineered proteins retained inhibitory activity against chymotrypsin or elastase, and all had substantially reduced inhibitory activity against subtilisin. The proteolytic stabilities of the proteins correlated with their thermodynamic stabilities. Reduction of the disulfide bond resulted in substantial loss of both thermodynamic and proteolytic stabilities, confirming that the disulfide bond, and not merely the cysteine substitutions, was responsible for the increased stability. We conclude that it is possible to replace over a third of the residues in CI-2 with minimal disruption of stability and structural integrity.     

Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors.

A cDNA clone, NA-PI-II, encoding a protein with partial identity to proteinase inhibitor (PI) II of potato and tomato has been isolated from a cDNA library constructed from Nicotiana alata stigma and style mRNA. The cDNA encodes a polypeptide of 397 amino acids with a putative signal peptide of 29 amino acids and six repeated domains, each with a potential reactive site. Domains 1 and 2 have chymotrypsin-specific sites and domains 3, 4, 5, and 6 have sites specific for trypsin. In situ hybridization experiments demonstrated that expression of the gene is restricted to the stigma of both immature and mature pistils. Peptides with inhibitory activity toward chymotrypsin and trypsin have been isolated from stigmas of N. alata. The N-terminal amino acid sequence obtained from this protein preparation corresponds to six regions in the cDNA clone NA-PI-II. The purified PI protein preparation is likely to be composed of a mixture of up to five similar peptides of approximately 6 kD, produced in vivo by proteolytic processing of a 42-kD precursor. The PI may function to protect the reproductive tissue against potential pathogens.     

Purification and characterization of an acidic trypsin/subtilisin inhibitor from tortoise egg white

Egg whites of three species of tortoise and turtle have been compared by gel chromatography for inhibitory activity against proteases. The egg white of Geomda trijuga trijuga Schariggar contains trypsin/subtilisin inhibitor while the egg white of Caretta caretta Linn. contains both trypsin and chymotrypsin inhibitors. No protease inhibitory activity has been detected in the egg white of Trionyx gangeticus Cuvier. An acidic trypsin/subtilisin inhibitor has been purified to homogeneity from the egg white of tortoise (G. trijuga trijuga). It is a single polypeptide chain of 100 amino acid residues, having a molecular weight of 11 700. It contains six disulphide bonds and is devoid of methionine and carbohydrate moiety. Its isoelectric point is at pH 5.95 and is stable at 100°C for 4 h at neutral pH. The inhibitor inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio close to unity. Their dissociation contants are 7.2·10^?9 M for bovine trypsin adn 5.5·10^?7 M for subtilisin. Chemical modification of amino groups with trinitrobenzene sulfonate has reduced its inhibitory activities against both trypsin and subtilisin, but the loss of its trypsin inhibitory activity is faster than of its subtilisin inhibitory activity. It has independent binding sites for inhibition of trypsin and subtilisin.     

Carbohydrate-binding site of a novel mannose-specific lectin from fugu (Takifugu rubripes) skin mucus

Pufflectin-s, identified in the skin mucus of the fugu Takifugu rubripes, is a novel mannose-specific lectin with similar structure to monocotyledonous plant lectins. In the present study, mutational analysis was used to reveal the mannose-binding sites of pufflectin-s. Putative binding sites were mutated as follows: binding site 1; rPL-D32E (Asp³² → Glu³²), rPL-N34S (Asn³⁴ → Ser³⁴) and rPL-V36A (Val³⁶ → Ala³⁶) whereas binding site 2; rPL-D61E (Asp⁶¹ → Glu⁶¹), rPL-N63S (Asn⁶³ → Ser⁶³) and rPL-V65A (Val⁶⁵ → Ala⁶⁵). All recombinant proteins were expressed in Escherichia coli, purified with two chromatographic steps, and then subjected to mannose-binding assay by affinity chromatography. Recombinant wild-type pufflectin-s (rPL-wt) as well as three mutants with changes in binding site 2 could bind to mannose, in contrast to the three mutants with changes in binding site 1 in which mannose-binding activity was completely lost. These results clearly demonstrate that, at the least, binding site 1 is critical to mannose-binding activity in pufflectin-s.     

Mannose-binding proteins in human serum: identification of mannose-specific immunoglobulins and a calcium-dependent lectin, of broader carbohydrate specificity, secreted by hepatocytes

Human serum contains lectins which inhibit the uptake of mannose- and N-acetylglucosamine-terminated glycoproteins by isolated rat hepatic sinusoidal cells. In these experiments, calcium-dependent and calcium-independent human serum mannose-binding proteins have been isolated by affinity chromatography using mannan linked to four different supports. In electroblots both calcium-dependent and -independent serum mannose-binding proteins bound radioiodinated mannan and invertase in the presence of calcium ions, but the binding of calcium-dependent serum mannose-binding proteins was abolished by EDTA. Chicken antibodies were raised against serum mannose-binding proteins and an ELISA was developed. The principal calcium-independent serum mannose-binding protein is mannose-specific IgG as judged by immunodiffusion and electroblotting with anti-human IgG antibodies. The calcium-dependent serum mannose-binding protein is probably the secreted form of an intracellular hepatocyte mannose-binding protein since: antibodies raised against the 30 kDa subunit of the calcium-dependent serum mannose-binding protein also bound 30 kDa subunits of whole liver homogenate and purified human liver mannose-binding protein; antibodies to the human liver mannose-binding protein bound to the 30 kDa subunit of the calcium-dependent serum mannose-binding protein; and the binding specificities of the calcium-dependent serum mannose-binding protein for N-acetylglucosamine and fucose as well as mannose, and its recognition of the core region of an oligosaccharide rather than only the peripheral sugars, were identical to those reported for the hepatocyte mannose-binding protein. The physiological ligands of these serum mannose-binding proteins are unknown but they could bind noxious glycoproteins which enter the circulation prior to their removal by the sinusoidal mannose receptor.     

Expression and purification of the cytoplasmic tail of an endocytic receptor by fusion to a carbohydrate-recognition domain.

Gene fusion has been used to produce the cytoplasmic domain of an endocytic receptor. DNA sequences coding for the 52 COOH-terminal amino acids of the mannose receptor from human macrophages, including the 41-amino acid cytoplasmic tail, were fused to the codons specifying the carbohydrate-recognition domain (CRD) of rat mannose-binding protein. The fusion protein was expressed in Escherichia coli and purified in one step on mannose-Sepharose, making use of the carbohydrate-binding activity of the CRD. The tail peptide was released from the fusion protein using endoproteinase Arg-C. This method provides an alternative to chemical synthesis for the production of midlength peptides.     

Molecular Cloning of the cDNA Encoding an Antifungal Protein in Gastrodia elata Bl.

Antifungal protein is the main inhibitor of fungal infection in the secondary corm of Gastrodia elata B1. was isolated and purified antifungal protein (GAFP) from the plant. Its molecular weight was about 14 kD. Polyclonal antibody against GAFP was produced. In vitro test, this antifungal protein inhibited the growth of some fungi in some crop including Gibberella zeae. cDNA was synthesized from poly (A) mRNA purified from G. elata. The cDNA was ligated into phage vector λgtll DNA and packaged in vitro and the phages were propagated on E. coli Y1090 and a λgtll expression library was constructed. A cDNA clone encoding for antifungal protein was screened out by immunoscreening of the library using the protein as a probe. The λDNA containing insert was digested by Eco RI after isolated and purified recombinants λDNA, the insert was obtained. The cDNA was 300 bp in length. The authors had isolated the cDNA clone encoding antifungal protein from G. elata.     

Cloning and characterization of a monocot mannose-binding lectin from Crocus vernus (family Iridaceae).

The molecular structure and carbohydrate-binding activity of the lectin from bulbs of spring crocus (Crocus vernus) has been determined unambiguously using a combination of protein analysis and cDNA cloning. Molecular cloning revealed that the lectin called C. vernus agglutinin (CVA) is encoded by a precursor consisting of two tandemly arrayed lectin domains with a reasonable sequence similarity to the monocot mannose-binding lectins. Post-translational cleavage of the precursor yields two equally sized polypeptides. Mature CVA consists of two pairs of polypeptides and hence is a heterotetrameric protein. Surface plasmon resonance studies of the interaction of the crocus lectin with high mannose-type glycans showed that the lectin interacts specifically with exposed alpha-1,3-dimannosyl motifs. Molecular modelling studies confirmed further the close relationships in overall fold and three-dimensional structure of the mannose-binding sites of the crocus lectin and other monocot mannose-binding lectins. However, docking experiments indicate that only one of the six putative mannose-binding sites of the CVA protomer is active. These results can explain the weak carbohydrate-binding activity and low specific agglutination activity of the lectin. As the cloning and characterization of the spring crocus lectin demonstrate that the monocot mannose-binding lectins occur also within the family Iridaceae a refined model of the molecular evolution of this lectin family is proposed.     

Molecular characterization of the mouse mannose-binding proteins. The mannose-binding protein A but not C is an acute phase reactant.

Mannose-binding proteins play a role in first line host defense against a variety of pathogens. We report the molecular cloning of two mouse mannose-binding proteins designated A and C based on their close identity with their rat homologues. The deduced amino acid sequence of the mouse mannose-binding proteins, as with rat and the human forms, have an NH2 terminus that is rich in cysteine that stabilizes a collagen alpha helix followed by a carboxyl- terminal carbohydrate binding domain. We further show that the mouse mannose-binding protein A mRNA, as with the human, is induced like the acute phase reactant serum amyloid P protein, yet the expression of mouse mannose-binding protein C mRNA is not regulated above its low baseline level. The expression of both mannose-binding proteins A and C mRNA is restricted to the liver under basal and stress conditions.     

Purification, characterization, and molecular cloning of lectin from winter buds of Lysichiton camtschatcensis (L.) Schott

A novel lectin was purified to homogeneity from winter buds of Lysichiton camtschatcensis (L.) Schott of the Araceae family. It was a tetramer composed of two non-covalently associated polypeptides with small subunits (11 kDa) and large subunits (12 kDa). Sequencing of both subunits yielded unique N-terminal sequences. A cDNA encoding the lectin was cloned. The isolated cDNA contained an open reading frame that encoded 267 amino acids. It encoded both subunits, indicating that the lectin is synthesized as a single precursor protein that is post-translationally processed into two different subunits with 45% sequence identity. Each subunit contained a mannose-binding motif known to be conserved in monocot mannose-binding lectins, but its activity was not inhibited by monosaccharides, including methyl α-mannoside. Asialofetuin and yeast invertase were potent inhibitors. Lectin activity was detected in the buds formed during the winter season but not in the expanded leaves.