Expression and purification of a novel mannose-binding lectin from Pinellia ternata

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for “native” lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.     

Expression and purification of a novel mannose-binding lectin from Pinellia ternata

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for "native" lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.     

The mistletoe lectin I--phloretamide structure reveals a new function of plant lectins

The X-ray structure at 2.7 Å resolution of the complex between the European mistletoe lectin I (Viscum album, ML-I) and the plant growth hormone, 3-(p-hydroxyphenyl)-propionic acid amide (phloretamide, PA) from xylem sap has revealed the binding of PA at the so far undescribed hydrophobic cavity located between the two subunits of this ribosome-inhibiting protein. No such cavity is observed in related lectins. The binding of PA is achieved through interactions with the non-conserved residues Val228A, Leu230A, Arg388B, and the C-terminal Pro510B. It is conceivable that binding of PA to ML-I is part of a defence mechanism of the parasite against the host, whereby the parasite prevents the growth hormone of the host from interfering with its own regulatory system. The specific binding of PA to ML-I indicates that heterodimeric RIPs are multifunctional proteins whose functions in the cell have not yet been fully recognized and analyzed.     

Two leguminous forest trees new to the flora of French Guiana

Stryphnodendron moricolor (Mimosaceae) is described, illustrated and discussed. The genusMonopteryx (Fabaceae) is reported for the first time outside Amazonian Brazil and adjacent Venezuela. A key distinguishing the three known species ofMonopteryx is presented.     

Screening of a unique lectin from 16 cultivable mushrooms with hybrid glycoprotein and neoproteoglycan probes and purification of a novel N-acetylglucosamine-specific lectin from Oudemansiella platyphylla fruiting body

Hybrid glycoprotein and neoproteoglycan probes were prepared by coupling various glycoproteins or polysaccharides to peroxidase or biotinyl bovine serum albumin, respectively. Lectins recognizable by the neoglycoconjugate probes were extracted from 16 cultivable mushrooms. Dot-blot assay revealed five extracts to be reactive with only hybrid glycoprotein probes, but others also reacted with neoproteoglycan probes. According to the reactivity pattern with probe screening, the one lectin from Oudemansiella platyphylla extract (OPL) bound best with asialotransferrin-- and asialoagalactotransferrin--peroxidase probes and was isolated using an asialotransferrin column, but it did not bind with other hybrid glycoprotein or neoproteoglycan probes. OPL, consisting of two polypeptides with high homology in the N-terminal amino acid sequences, exhibited weak hemagglutinating activity. Purified OPL specifically bound the beta-GlcNAc probe among various biotinylated polymeric sugar probes, while it exhibited essentially the same binding specificity toward neoglycoconjugate probes as that of the crude extract, showing a preference for the asialobiantennary complex type of N-linked glycans. These results indicate that the neoglycoconjugate probes are valuable in lectin screening.     

Interactions between indole-3-acetic acid (IAA) with a lectin from Canavalia maritima seeds reveal a new function for lectins in plant physiology

Indole-3-acetic acid (IAA) bound is considered a storage molecule and is inactive. However, some studies have proposed an additional possible regulatory mechanism based on the ability of lectins to form complexes with IAA. We report the first crystal structure of ConM in complex with IAA at 2.15 Å resolution. Based on a tetrameric model of the complex, we hypothesize how the lectin controls the availability of IAA during the early seedling stages, indicating a possible new physiological role for these proteins. A free indole group is also bound to the protein. The ConM interaction with different forms of IAA is a strategy to render the phytohormone unavailable to the cell. Thus, this new physiological role proposed for legume lectins might be a novel mechanism by which IAA levels are decreased in addition to the destruction and formation of new complexes in the later stages of seed germination.     

A new affinity sorbent for the purification of lectins and its use in the purification of wheat germ agglutinin

A method is developed for obtaining gel from eggwhite and its application as a sorbent for purification of lectins. Eggwhite was treated by 1% glutaraldehyde at pH 5, for 5-6 hours at room temperature, then it was minced and washed by water. The residual aldehyde groups were blocked by glycine treatment. The sorbent obtained possessed high affinity for lectins specific to N-acetylglucosamine and complex oligosaccharides. The galactose- and mannose-specific lectins were adsorbed to a less extent. The purification of the wheat germ agglutinin using the eggwhite gel is described.     

Fine specificities of two lectins from Cymbosema roseum seeds: a lectin specific for high-mannose oligosaccharides and a lectin specific for blood group H type II trisaccharide

The legume species of Cymbosema roseum of Diocleinae subtribe produce at least two different seed lectins. The present study demonstrates that C. roseum lectin I (CRL I) binds with high affinity to the "core" trimannoside of N-linked oligosaccharides. Cymbosema roseum lectin II (CRL II), on the other hand, binds with high affinity to the blood group H trisaccharide (Fucα1,2Galα1-4GlcNAc-). Thermodynamic and hemagglutination inhibition studies reveal the fine binding specificities of the two lectins. Data obtained with a complete set of monodeoxy analogs of the core trimannoside indicate that CRL I recognizes the 3-, 4- and 6-hydroxyl groups of the α(1,6) Man residue, the 3- and 4-hydroxyl group of the α(1,3) Man residue and the 2- and 4-hydroxyl groups of the central Man residue of the trimannoside. CRL I possesses enhanced affinities for the Man5 oligomannose glycan and a biantennary complex glycan as well as glycoproteins containing high-mannose glycans. On the other hand, CRL II distinguishes the blood group H type II epitope from the Lewis(x), Lewis(y), Lewis(a) and Lewis(b) epitopes. CRL II also distinguishes between blood group H type II and type I trisaccharides. CRL I and CRL II, respectively, possess differences in fine specificities when compared with other reported mannose and fucose recognizing lectins. This is the first report of a mannose-specific lectin (CRL I) and a blood group H type II-specific lectin (CRL II) from seeds of a member of the Diocleinae subtribe.     

A novel method for the purification of low soluble recombinant C-type lectin proteins

Snake venoms contain a complex mixture of many biological molecules including proteins. The purification of recombinant proteins is a key step in studying their function and structure with affinity chromatography as the common method used in their purification. In bacterial expression systems, hydrophobic recombinant proteins are usually precipitated into inclusion bodies, and contaminants are typically associated with tagged proteins after purification. The purpose of this study was to develop a procedure to purify hydrophobic recombinant proteins without an affinity tag. Snake venom mature C-type lectin-like proteins (CLPs) with a tag were cloned, expressed, and purified by repeated sonication and wash steps. The effects of the signal peptide on the expression and solubility of the recombinant protein were investigated. The CLPs in washed inclusion bodies were solubilized and refolded by dialysis. The CLPs without a tag were successfully purified with a yield 38 times higher than the traditional method, and inhibited blood platelet aggregation with an IC(50) of 100.57μM in whole blood. This novel procedure is a rapid, and inexpensive method to purify functional recombinant hydrophobic CLPs from snake venoms useful in the development of drug therapies.     

Studies on lectins. XXXVIII. Isolation and characterization of the lectin from black locust bark (Robinia pseudacacia L.)

The lectin of black locust (Robinia pseudacacia) bark was isolated by specific adsorption on formaldehyde-fixed human erythrocytes and elution with a borate solution. The lectin is homogeneous on disc electrophoresis and ultracentrifugation (s20,w = 5.8 S) but yields three bands on isoelectric focusing. It has a molecular weight of approximately 110,000 and consists of two types of subunit (mol. wt 29,000 and 31,500). Its pI is approximately 5.9; it contains high amounts of aspartic acid, threonine and serine, no cysteine and very little methionine. Also 7.2% of covalently bound neutral sugar and 0.47% of glucosamine are present. The lectin is nonspecific in agglutination of human erythrocytes, it is inhibited by high concentrations of N-acetyl-D-galactosamine and is mitogenic in rabbit lymph node lymphocytes.     

我国豆科树种结瘤调查(续)

据国内资料报道,我国豆科树种约94属763种,已调查过结瘤状况的有含羞草亚科的123种(包括近几年来从国外引种的54种)、蝶形花亚科的211种和苏木亚科的70种,其中可以结瘤的有含羞草亚科的116种、蝶形花亚科的207种和苏木亚科的18种。含羞草亚科中的2种、蝶形花亚科中的3种均有结瘤和不结瘤的报道。     

High-resolution structure of a new Tn antigen-binding lectin from Vatairea macrocarpa and a comparative analysis of Tn-binding legume lectins

Plant lectins have been studied as histological markers and promising antineoplastic molecules for a long time, and structural characterization of different lectins bound to specific cancer epitopes has been carried out successfully. The crystal structures of Vatairea macrocarpa (VML) seed lectin in complex with GalNAc-α-O-Ser (Tn antigen) and GalNAc have been determined at the resolution of 1.4 Å and 1.7 Å, respectively. Molecular docking analysis of this new structure and other Tn-binding legume lectins to O-mucin fragments differently decorated with this antigen provides a comparative binding profile among these proteins, stressing that subtle alterations that may not influence monosaccharide binding can, nonetheless, directly impact the ability of these lectins to recognize naturally occurring antigens. In addition to the specific biological effects of VML, the structural and binding similarities between it and other lectins commonly used as histological markers (e.g., VVLB4 and SBA) strongly suggest VML as a candidate tool for cancer research.     

FGO: a novel ontology for identification of ligand functional group

Small molecules play crucial role in the modulation of biological functions by interacting with specific macromolecules. Hence small molecule interactions are captured by a variety of experimental methods to estimate and propose correlations between molecular structures to their biological activities. The tremendous expanse in publicly available small molecules is also driving new efforts to better understand interactions involving small molecules particularly in area of drug docking and pharmacogenomics. We have studied and designed a functional group identification system with the associated ontology for it. The functional group identification system can detect the functional group components from given ligand structure with specific coordinate information. Functional group ontology (FGO) proposed by us is a structured classification of chemical functional group which acts as an important source of prior knowledge that may be automatically integrated to support identification, categorization and predictive data analysis tasks. We have used a new annotation method which can be used to construct the original structure from given ontological expression using exact coordinate information. Here, we also discuss about ontology-driven similarity measure of functional groups and uses of such novel ontology for pharmacophore searching and de-novo ligand designing.     

A novel method for the purification of recombinant subunit I of theDolichos biflorus seed lectin

Lectins are carbohydrate-binding proteins that are ubiquitous in nature. Their ability to specifically bind carbohydrates has been used as a means of purification mainly through affinity chromatography techniques. Plant lectins are one of the most thoroughly studied class of lectins, however, details of theirin situ function remains elusive. Recent advances in recombinant DNA techniques have been used in several laboratories to study the function of these lectins by heterologous over-expression. The larger subunit of theDolichos biflorus seed lectin was described by Chao et al. in 1994 and purification through affinity chromatography techniques was described. Here we report on a new method for the purification of this recombinant protein with techniques that are not dependent on the ability of the lectin to bind sugars. This method may have uses in the purification of mutant proteins that may not bind carbohydrates. Characterization of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization (MALDI) mass spectroscopy shows that the lectin is over 99% pure with a molecular weight of 27,090±16.17 Da, and hemagglutination assays confirm that the lectin retains its biological activity.     

我国豆科树种结瘤调查

据国内资料报道,我国豆科树种约94属763种,已调查过结瘤状况的有含羞草亚科的123种(包括近几年来从国外引种的54种)、蝶形花亚科的211种和苏木亚科的70种,其中可以结瘤的有含羞草亚科的116种、蝶形花亚科的207种和苏木亚科的18种。含羞草亚科中的2种、蝶形花亚科中的3种均有结瘤和不结瘤的报道。     

Uses and potential uses of leguminous trees for minimal energy input agriculture

Leguminous tree orchards are proposed as an agricultural system requiring minimal input of fossil fuel, machinery, and capital. Legume orchards fix nitrogen, require neither tillage nor irrigation, and, potentially, provide large yields of nutritious pods. Further, our analytical data show seed protein contents ranging from 16 to 69 percent, with one species having an amino acid profile superior to that of casein.     

Cloning of a lectin cDNA and seasonal changes in levels of the lectin and its mRNA in the inner bark ofRobinia pseudoacacia

A cDNA clone encoding a lectin was isolated by immunological screening of an expression library prepared from poly(A)⁺ RNA from the inner bark ofRobinia pseudoacacia. The cDNA clone (RBL104) had an open reading frame of 858 bp that encoded a polypeptide with a predicted molecular weight of 31210. This molecular weight corresponded closely to that of a polypeptide immunoprecipitated from products of translationin vitro of the poly(A)⁺ RNA. Thus, RBL104 appeared to be a full-length cDNA. The N-terminal amino acid sequence of the purified lectin protein matched a portion of the predicted amino acid sequence. It appeared that the lectin was synthesized as a precursor that consisted of a putative signal peptide of 31 amino acids and a mature polypeptide of 255 amino acids. Southern blot analysis of the genomic DNA revealed that the lectin was encoded by a small multigene family. The lectin was mostly localized in the axial and ray parenchymal cells of the inner bark. A small amount of lectin was also found in the axial and ray parenchymal cells of the xylem. The lectin accumulated in the inner bark in September, remained at high levels during the winter and disappeared in May. The mRNA for the lectin was detected from August to the following March. The appearance and disappearance of the mRNA were observed prior to those of the lectin protein.     

Two new Marasmiellus species found on the bark of living coniferous and broad-leaved trees in Japan

Two new species of Marasmiellus (Tricholomataceae, Agaricales) are described and illustrated from Japan: (1) Marasmiellus crassitunicatus sp. nov. (subsection Inodermini of section Marasmiellus) forms dark reddish-brown, orbicular to reniform basidiomata constantly accompanied by tufts of short filiform rhizomorphs, growing on the bark of living coniferous or broad-leaved trees; and (2) Marasmiellus vernalis sp. nov. (subsection Quercini of section Dealbati) forms small brown basidiomata with nondiverticulate pileipellis elements, growing on the bark of living Pinus thunbergii in spring (April to June).     

Screening for novel enzymes for biocatalytic processes: accessing the metagenome as a resource of novel functional sequence space

Historically, biotechnology has missed up to 99% of existing microbial resources by using traditional screening techniques. Strategies of directly cloning 'environmental DNA' comprising the genetic blueprints of entire microbial consortia (the so-called 'metagenome') provide molecular sequence space that along with ingenious in vitro evolution technologies will act synergistically to bring a maximum of available sequence-space into biocatalytic application.