Bark and Leaf Lectins of Sophora japonica Are Sequestered in Protein-Storage Vacuoles

The leguminous tree Sophora japonica contains a family of closely related, but distinct, lectins. Different members of this family are independently expressed in seeds, leaves, and bark (CN Hankins, J Kindinger, LM Shannon 1987 Plant Physiol 83: 825-829; 1988 Plant Physiol 86: 67-10). The inter-, and intracellular distribution of the bark and leaf lectins was studied by indirect postembedding immunogold electron microscopy. Aldehyde fixed bark and leaves postifixed with OsO₄ and embedded in LR White resin permitted sensitive and specific immunogold labeling while maintaining cellular ultrastructure. The leaf and bark tissue cells contain protein-filled storage vacuoles which occupy most the cell's interior volume. The leaf and bark vacuoles closely resemble the protein bodies, or protein storage vacuoles, of seed cotyledons. The leaf and bark lectins were found to be exclusively sequestered in the protein-storage vacuoles of these tissues.     

The Lectins of Sophora japonica: II. Purification, Properties, and N-Terminal Amino Acid Sequences of Five Lectins from Bark

Five N-acetyl-galactosamine-specific lectins were isolated from the bark of the legume tree Sophora japonica. These lectins are immunologically and structurally very similar, but not identical, to the Sophora seed and leaf lectins. The carbohydrate specificities and hemagglutinin activities of these lectins are indistinguishable at pH 8.5 but their activities differ markedly at pH values below 8. All five lectins are tetrameric glycoproteins made up of different combinations of subunits of about 30,000, 30,100, 33,000 M_r containing 3% to 5% covalently attached sugar. These lectins are the overwhelmingly dominant proteins in bark, but they do not appear to be present in other tissues. Amino terminal sequence analysis indicates that at least two distinct lectin genes are expressed in bark.     

Molecular cloning of the bark and seed lectins from the Japanese pagoda tree (Sophora japonica)

cDNA clones encoding the bark and seed lectins from Sophora japonica were isolated and their sequences analyzed. Screening of a cDNA library constructed from polyA RNA isolated from the bark resulted in the isolation of three different lectin cDNA clones. The first clone encodes the GalNAc-specific bark lectin which was originally described by Hankins et al. whereas the other clones encode the two isoforms of the mannose/glucose-specific lectin reported by Ueno et al.. Molecular cloning of the seed lectin genes revealed that Sophora seeds contain only a GalNAc-specific lectin which is highly homologous to though not identical with the GalNAc-specific lectin from the bark. All lectin polypeptides are translated from mRNAs of ca. 1.3 kb encoding a precursor carrying a signal peptide. In the case of the mannose/glucose-specific bark lectins this precursor is post-translationally processed in two smaller peptides. Alignment of the deduced amino acid sequences of the different clones revealed striking sequence similarities between the mannose/glucose-binding and the GalNAc-specific lectins. Furthermore, there was a high degree of sequence homology with other legume lectins which allowed molecular modelling of the Sophora lectins using the coordinates of the Pisum sativum, Lathyrus ochrus and Erythrina corallodendron lectins.     

The Bark Lectin of Robinia pseudoacacia: Purification and Partial Characterization

A lectin was purified from the bark of Robinia pseudoacaciaby sequential ion-exchange chromatography on DEAE-Sepharoseand CM-Toyopearl. The purified lectin was estimated to havea molecular weight of 106 kDa and to be a homotetramer of subunitswith a molecular weight of 29 kDa. Antibodies raised againstthe bark lectin cross-reacted with a 29-kDa polypeptide duringWestern blot analysis, showing that the antibodies are specificfor the bark lectin. The antibodies against the lectin fromRobinia bark cross-reacted with polypeptides in extracts ofthe seeds and bark of Sophora japonica, indicating that thelectin from Robinia bark is immunologically related to the lectinsof Sophora. However, the antibodies did not cross-react withproteins from Robinia seeds and leaves. The first twenty aminoacid residues from the N-terminus of the lectin from Robiniabark were determined and compared with those of the Sophoralectins. (Received July 13, 1991; Accepted December 12, 1991)     

Multiple soluble vertebrate galactoside-binding lectins

All vertebrates synthesize soluble galactoside-binding lectins. Many are expressed at high levels in the embryo and at lower levels in the adult, whereas others show an inverse pattern of expression. Most lectins tend to be concentrated in one or a number of specific cell types. In the past few years, the multiplicity of these lectins has become more apparent. For example, in Xenopus laevis 3 galactoside-binding lectins, 2 with a preference for alpha-galactosides, have been purified and partially characterized. They have subunit molecular weights ranging from 16,000 to 69,000. More detailed studies have been done in mammals. For example, rat lung contains 3 soluble beta-galactoside-binding lectins, RL-14.5, RL-18 and RL-29, with subunit molecular weights, respectively, of 14,500, 18,000 and 29,000. A notable feature of these lectins is that, although they all bind lactose about equally well, their carbohydrate-binding sites are actually quite different, as shown by competitive binding studies with a range of complex mammalian glycoconjugates. Human lung also contains several beta-galactoside-binding lectins, including HL-14, HL-22 and HL-29 with subunit molecular weights, respectively, of 14,000, 22,000 and 29,000. They too show significant differences in their carbohydrate-binding sites when analyzed with naturally occurring mammalian glycoconjugates. Sequencing of purified lectins and cDNA clones indicates that at least 4 distinct genes code for what appears to be a family of HL-14. Heterogeneity is also indicated from isoelectric focusing studies which resolve at least 6 acidic forms of HL-14 and 5 acidic forms of HL-29.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of Dioclea rostrata lectin: insights into understanding the pH-dependent dimer-tetramer equilibrium and the structural basis for carbohydrate recognition in Diocleinae lectins

The legume lectins from the subtribe Diocleinae, often referred to as concanavalin A-like lectins, are a typical example of highly similar proteins that show distinct biological activities. The pH-dependent oligomerization that some of these lectins undergo and the relative position of amino acids within the carbohydrate-binding site are factors that have been reported to contribute to these differences in the activities of Diocleinae lectins. In the present work, we determined the amino acid sequence and the crystal structure of the lectin of Dioclea rostrata seeds (DRL), with the aim of investigating the structural bases of the different behavior displayed by this lectin in comparison to other Diocleinae lectins and determining the reason for the distinct pH-dependent dimer-tetramer equilibrium. In addition, we discovered a novel multimeric arrangement for this lectin.     

Structure of porphobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism

The seeds of jack fruit (Artocarpus integrifolia) contain two tetrameric lectins, jacalin and artocarpin. Jacalin was the first lectin found to exhibit the beta-prism I fold, which is characteristic of the Moraceae plant lectin family. Jacalin contains two polypeptide chains produced by a post-translational proteolysis which has been shown to be crucial for generating its specificity for galactose. Artocarpin is a single chain protein with considerable sequence similarity with jacalin. It, however, exhibits many properties different from those of jacalin. In particular, it is specific to mannose. The structures of two crystal forms, form I and form II, of the native lectin have been determined at 2.4 and 2.5 A resolution, respectively. The structure of the lectin complexed with methyl-alpha-mannose, has also been determined at 2.9 A resolution. The structure is similar to jacalin, although differences exist in details. The crystal structures and detailed modelling studies indicate that the following differences between the carbohydrate binding sites of artocarpin and jacalin are responsible for the difference in the specificities of the two lectins. Firstly, artocarpin does not contain, unlike jacalin, an N terminus generated by post-translational proteolysis. Secondly, there is no aromatic residue in the binding site of artocarpin whereas there are four in that of jacalin. A comparison with similar lectins of known structures or sequences, suggests that, in general, stacking interactions with aromatic residues are important for the binding of galactose while such interactions are usually absent in the carbohydrate binding sites of mannose-specific lectins with the beta-prism I fold.     

Distribution of lectin during the life cycle of Phaseolus vulgaris L.

Phaseolus vulgaris L. cv. ‘garrafal encarnada’ plants have been utilized to study the distribution of lectins accumulated in the seeds and through the life cycle of the plant.The distribution of both, total proteins and lectins was studied in aqueous and saline (1 M NaCl) extracts from different parts and organs in four distinct stages of plant development.Our results showed that lectin concentration decreases sharply during the first weeks of the plant growth, reaching the lowest value in trifoliate leaf stage and increasing during the following phases of plant development. However, the presence of lectins have been detected in all the plant tissues through every phase of the life cycle.The observed differences on lectin levels (RIA) and lectin activity (hemagglutination), suggest the presence of different molecular forms of the lectin in aqueous and saline extracts of plant tissues.These results, as well as the observation about the fixation of lectin on the bacterial surface, support the idea that the function of lectins in the plant may not be limited to storage proteins, but may be involved in specific host-parasite recognition.     

Molecular characterization and crystallization of Diocleinae lectins

Molecular characterization of seven Diocleinae lectins was assessed by sequence analysis, determination of molecular masses by mass spectrometry, and analytical ultracentrifugation equilibrium sedimentation. The lectins show distinct pH-dependent dimer-tetramer equilibria, which we hypothesize are due to small primary structure differences at key positions. Lectins from Dioclea guianensis, Dioclea virgata, and Cratylia floribunda seeds have been crystallized and preliminary X-ray diffraction analyses are reported.     

Sambucus nigra agglutinin is located in protein bodies in the phloem parenchyma of the bark

The bark of some young woody stems contains storage proteins which are subject to an annual rhythm: they accumulate in the autumn and are mobilized in the spring. We show here that the bark phoem-parenchyma cells of Sambucus nigra L. contain numerous protein bodies, and that the bark lectin (S. nigra agglutinin) which undergoes an annual rhythm is localized in these protein bodies. The protein bodies in the cotyledons of legume seeds also contain lectin, indicating that lectins may be storage compounds themselves or may have a function in storage and-or mobilization processes.Abbreviations PBS phosphate-buffered saline - IgG immunoglobulin - SNA Sambucus nigra agglutinin     

The Lectins of Sophora japonica: I. Purification Properties and N-Terminal Amino Acid Sequences of Two Lectins from Leaves

Two lectins, Leaf Lectin I and Leaf Lectin II (LLI and LLII) were purified from the leaves of Sophora japonica. Like the Sophora seed lectin, LLI and LLII are tetrameric glycoproteins containing a single subunit with respect to size. The subunits of LLI (32 kilodaltons) and LLII (34 kilodaltons) are slightly larger than those of the seed lectin (29.5 kilodaltons). The three Sophora lectins display indistinguishable specificities, amino acid compositions, specific hemagglutinin activities, and extinction coefficients. Although very closely related to the seed lectin, the leaf and seed lectins are not immunologically identical and they differ in subunit molecular weights, carbohydrate content, and in the pH sensitivity of their hemagglutinin activities. N-terminal amino acid sequence analysis shows that although they are homologous proteins, the three Sophora lectins are products of distinct genes.     

Isolation and characterization of lectins from Vicia villosa. Two distinct carbohydrate binding activities are present in seed extracts

An uncharacterized lectin from Vicia villosa seeds has been reported to bind specifically to mouse cytotoxic T lymphocytes (Kimura, A., Wigzell, H., Holmquist, G., Ersson, B., and Carlsson, P., (1979) J. Exp. Med. 149, 473-484). We have found that V. villosa seeds contain at least three lectins which we have purified by affinity chromatography on a column of immobilized porcine blood group substances eluted with varying concentrations of N-acetylgalactosamine and by anion exchange chromatography. The three lectins are composed of two different subunits with Mr = 35,900 (subunit B) and 33,600 (subunit A), estimated from their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sedimentation equilibrium analysis suggests that the purified lectins are tetramers. They have been designated B4, A4, and A2B2 to indicate their apparent subunit compositions. The purified B4 and A4 lectins contain 6.7-9.8% carbohydrate by weight; in addition, both are rich in the acidic and hydroxylic amino acids and lack cysteine and methionine. The A4 lectin agglutinates A erythrocytes specifically and binds to A1 erythrocytes (273,000 sites/cell) with an association constant of 1.8 X 10(7) M-1. Although a blood group A agglutinating activity was recognized in the original preparation of V. villosa lectins, lectins with this activity were obtained in relatively small amounts from seed extracts. The predominant lectin in V. villosa seeds, B4, does not agglutinate A, B, or O erythrocytes.     

Lectin receptors in the salivary glands of the rat during postnatal ontogenesis

Distribution of lectin-binding sites in rat submandibular and sublingual salivary glands during postnatal development has been investigated. Lectin preparations include con A, lentin lectin, castor beans agglutinin, peanut, soybean and Sophora japonica agglutinins, wheat germ agglutinin and lectin from the bark of Laburnum anagyroides. The direct and indirect peroxidase techniques are used. According to the similarities of histochemical patterns, all lectins are divided into four groups. Besides the general patterns of lectin binding sites, some details are noted. Lectins of peanut and Sophora japonica possess an extremely high affinity to mast cells, con A, lens lectin, castor beans and wheat germ agglutinins--to serous demilunes cells. Laburnum lectin--to salivary ducts epithelia in adult rat salivary glands. Lentin lectin, con A and Laburnum lectin preferentially stain cells with specific granularity in granular ducts at early stages of postnatal development. Considering the character of staining, we propose for further histochemical investigations of the salivary glands lentin lectin, peanut agglutinin, wheat germ agglutinin and Laburum anagyroides lectin.     

Two crystal forms of the lentil lectin diffract to high resolution.

The legume lectins are an important class of polysaccharide-binding proteins with a wide range of biochemical and immunological applications. Two high-resolution crystal forms are obtained for the lentil (Lens culinaris) lectin: a monoclinic P21 and an orthorhombic P212121. The unit cell dimensions for the monoclinic form are a = 58.0 A, b = 56.0 A, c = 82.1 A, beta = 104.4 degrees, while for the orthorhombic form a = 56.4 A, b = 74.6 A, c = 124.9 A. The asymmetric unit contains one dimer in both cases. The crystals diffract to 1.7 A resolution using synchrotron radiation. Preliminary data have been collected to 2.3 A on both crystal forms using a conventional X-ray source.     

Crystal structure of native and Cd/Cd-substituted Dioclea guianensis seed lectin. A novel manganese-binding site and structural basis of dimer-tetramer association.

Diocleinae legume lectins are a group of oligomeric proteins whose subunits display a high degree of primary structure and tertiary fold conservation but exhibit considerable diversity in their oligomerisation modes. To elucidate the structural determinants underlaying Diocleinae lectin oligomerisation, we have determined the crystal structures of native and cadmium-substituted Dioclea guianensis (Dguia) seed lectin. These structures have been solved by molecular replacement using concanavalin (ConA) coordinates as the starting model, and refined against data to 2.0 A resolution. In the native (Mn/Ca-Dguia) crystal form (P4(3)2(1)2), the asymmetric unit contains two monomers arranged into a canonical legume lectin dimer, and the tetramer is formed with a symmetry-related dimer. In the Cd/Cd-substituted form (I4(1)22), the asymmetric unit is occupied by a monomer. In both crystal forms, the tetrameric association is achieved by the corresponding symmetry operators. Like other legume lectins, native D. guianensis lectin contains manganese and calcium ions bound in the vicinity of the saccharide-combining site. The architecture of these metal-binding sites (S1 and S2) changed only slightly in the cadmium/cadmium-substituted form. A highly ordered calcium (native lectin) or cadmium (Cd/Cd-substituted lectin) ion is coordinated at the interface between dimers that are not tetrameric partners in a similar manner as the previously identified Cd(2+) in site S3 of a Cd/Ca-ConA. An additional Mn(2+) coordination site (called S5), whose presence has not been reported in crystal structures of any other homologous lectin, is present in both, the Mn/Ca and the Cd/Cd-substituted D. guianensis lectin forms. On the other hand, comparison of the primary and quaternary crystal structures of seed lectins from D. guianensis and Dioclea grandiflora (1DGL) indicates that the loop comprising residues 117-123 is ordered to make interdimer contacts in the D. grandiflora lectin structure, while this loop is disordered in the D. guianensis lectin structure. A single amino acid difference at position 131 (histidine in D. grandiflora and asparagine in D. guianensis) drastically reduces interdimer contacts, accounting for the disordered loop. Further, this amino acid change yields a conformation that may explain why a pH-dependent dimer-tetramer equilibrium exists for the D. guianensis lectin but not for the D. grandiflora lectin.     

Multiple molecular forms of glutamine synthetase in pea seeds

Summary Multiple molecular forms of glutamine synthetase (GS, EC 6.3.1.2) have been studied in pea seeds of different varieties. The number of GS molecular forms in the seeds proved to be related to their colour. Two GS forms in the green seeds have been found and only one of them in the yellow seeds. Green seeds had chlorophyll content amounted to 0.4% of the total pigment content in the leaves. Chloroplasts, somewhat smaller than those in pea leaves of the same variety, have been isolated from green seeds. The presence of the second GS form in the pea green seeds we relate to the chloroplasts. By electrophoretic mobility both forms of GS from the green seeds are not identical to the chloroplast GS and the cytosol GS of leaves. Thus, we believe pea plant to contain, at least, four GS forms.     

Structural analysis of ConBr reveals molecular correlation between the carbohydrate recognition domain and endothelial NO synthase activation

Diocleinae lectins are highly homologous in their primary structure which features metal binding sites and a carbohydrate recognition domain (CRD). Differences in the biological activity of legume lectins have been widely investigated using hemagglutination inhibition assays, isothermal titration microcalorimetry and co-crystallization with mono- and oligosaccharides. Here we report a new lectin crystal structure (ConBr) extracted from seeds of Canavalia brasiliensis, predict dimannoside binding by docking, identify the α-aminobutyric acid (Abu) binding pocket and compare the CRD of ConBr to that of homologous lectins. Based on the hypothesis that the carbohydrate affinity of lectins depends on CRD configuration, the relationship between tridimensional structure and endothelial NO synthase activation was used to clarify differences in biological activity. Our study established a correlation between the position of CRD amino acid side chains and the stimulation of NO release from endothelium.     

灰毛豆甲醇提取物的杀虫活性

研究灰毛豆(Tephrosia purpurea)各部位提取物的杀虫活性及其作用方式。结果表明,灰毛豆对白纹伊蚊Aedes albopictus Skuse幼虫、菜青虫Pieris rapae(L.)、斜纹夜蛾Prodenialitura(Fabricius)幼虫和黄曲条跳甲Phyllotreta striolata(Fabricius)成虫都有杀虫活性。灰毛豆种子、树皮、根皮、豆荚、枝条和树叶的甲醇提取物对白纹伊蚊4龄幼虫24h的LC50值分别是22.1,97.7,36.6,142.6,165.6和618.3mg.L-1,树干木质部没有杀虫活性;灰毛豆种子、树皮和根皮甲醇提取物对3龄菜青虫24h的触杀毒力LC50值分别是232.1,206.3和236.7mg.L-1;种子、树皮、根皮、枝条、树叶和豆荚对3龄菜青虫的24h胃毒毒力LC50值分别是192.6,168.4,249.7,524.5,1001.0,和510.7mg.L-1。在取食或接触有提取物的叶碟后,5龄菜青虫会出现取食量减少和生长发育变慢的亚致死现象。这些研究结果表明,灰毛豆除茎干木质部以外的其它各部位均含有杀虫活性成分,其作用方式为胃毒和触杀。     

Isolation and characterization of a lectin gene from seeds of chickpea (Cicer arietinum L.).

A cDNA library was constructed in lambda TriplEx2 vector using poly (A(+)) RNA from immature seeds of Cicer arietinum. The lectin gene was isolated from seeds of chickpea through library screening and RACE-PCR. The full-length cDNA of Chichpea seed lectin(CpGL)is 972 bp and contains a 807 bp open reading frame encoding a 268 amino acid protein. Analysis shows that CpSL gene has strong homology with other legume lectin genes. Phylogenetic analysis showed the existence of two main clusters and clearly indicated that CpSL belonged to mannose-specific family of lectins. RT-PCR revealed that CAA gene expressed constitutively in various plant tissues including flower, leaf, root and stem. When chickpea lectin mRNA level was checked in developing seeds, it was higher in 10 DAF seeds and decreased throughout seed development.     

Interactions between lectins and other components of leguminous protein bodies

Previous work from this laboratory had shown that Leguminosa seed extracts contain lectin-bound proteins. In the present paper, the isolation of protein bodies from the seeds of 7 Leguminosa species (Canavalia ensiformis, Lens culinaris, Pisum sativum, Glycine max, Sophora japonica, Wisteria floribunda and Phaseolus vulgaris) is described. Protein bodies were characterized microscopically and by their constituents, storage proteins, lectins and some glycosidases. From the protein bodies, lectin-bound proteins were isolated and were shown to be identical with those from whole seed extracts. This indicates a common localization of lectin-bound proteins and of lectins. Lectin-bound proteins belong to the storage proteins and to the proteins with glycosidase activity. The common localization of these proteins and interactions between them suggest a biological role of seed lectins: during maturation they may act as a packaging aid for storage proteins and enzymes into developing protein bodies. Lectins thus may contribute to an ordered construction and degradation of protein bodies.