Isolation,characterization and molecular cloning of a leaf-specific lectin from ramsons (Allium ursinum L.).

Lectins were isolated from roots and leaves of ramsons and compared to the previously described bulb lectins. Biochemical analyses indicated that the root lectins AUAIr and AUAIIr are identical to the bulb lectins AUAI and AUAII, whereas the leaf lectin AUAL has no counterpart in the bulbs. cDNA cloning confirmed that the leaf lectin differs from the bulb lectins. Northern blot analysis further indicated that the leaf lectin is tissue-specifically expressed. Sequence comparisons revealed that the ramsons leaf lectin differs considerably from the leaf lectins of garlic, leek, onion and shallot.     

Ectopically expressed leaf and bulb lectins from garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.) protect transgenic tobacco plants against cotton leafworm (<Emphasis Type="Italic">Spodoptera littoralis</Emphasis>)

The insecticidal activity of the leaf (ASAL) and bulb (ASAII) agglutinins from Allium sativum L. (garlic) against the cotton leafworm, Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) was studied using transgenic tobacco plants expressing the lectins under the control of the constitutive CaMV35S promoter. PCR analysis confirmed that the garlic lectin genes were integrated into the plant genome. Western blots and semi-quantitative agglutination assays revealed lectin expression at various levels in the transgenic lines. Biochemical analyses indicated that the recombinant ASAL and ASAII are indistinguishable from the native garlic lectins. Insect bioassays using detached leaves from transgenic tobacco plants demonstrated that the ectopically expressed ASAL and ASAII significantly (P < 0.05) reduced the weight gain of 4th instar larvae of S. littoralis. Further on, the lectins retarded the development of the larvae and their metamorphosis, and were detrimental to the pupal stage resulting in weight reduction and lethal abnormalities. Total mortality was scored with ASAL compared to 60% mortality with ASAII. These findings suggest that garlic lectins are suitable candidate insect resistance proteins for the control of S. littoralis through a transgenic approach.     

A new type of cereal lectin from leaves of couch grass (Agropyrum repens)

Extracts from couch grass (Agropyrum repens) leaves contain relatively high lectin concentrations. Preliminary experiments with crude extracts indicated that the leaf lectin differs from the embryo lectin of the same species and other Gramineae embryo lectins with respect to its sugar and blood group specificity, and serological properties. A comparison of the biochemical, physicochemical and biological properties of purified lectins from couch grass leaves and embryos, and wheat germ agglutinin revealed that the leaf lectin has the same molecular structure as the embryo lectins. It is a dimer composed of two identical subunits, which, however, are slightly larger than embryo lectin subunits. Structural differences between both couch grass lectins were further inferred from in vitro subunit exchange experiments and serological analyses. Whereas the embryo lectin readily forms heterodimers with embryo lectins from other cereal species and also is serologically indistinguishable from them, the leaf lectin does not exchange subunits with the same embryo lectins and is serologically different. In addition, couch grass leaf lectin exhibits specificity for N-acetylgalactosamine and agglutinates preferentially blood-group-A erythrocytes whereas the embryo lectin is not inhibited by N-acetylgalactosamine and exhibits no blood-group specificity. It was observed also that the lectin content of couch grass leaves varies enormously during the seasons.     

The closely related homomeric and heterodimeric mannose-binding lectins from garlic are encoded by one-domain and two-domain lectin genes, respectively.

Lectin cDNA clones for two different lectins from garlic (Allium sativum L.) bulbs, ASAI and ASAII (ASA, Allium sativum agglutinin), were isolated and characterized. The first lectin, ASAI, is a heterodimer composed of two different subunits of 11.5 kDa and 12.5 kDa. It is translated from an mRNA of 1400 nucleotides encoding a polypeptide of 306 amino acids with two very similar domains. N-terminal sequencing of the two polypeptides of the mature lectin confirmed that both subunits are derived from the same precursor and that each corresponds to one of the two domains in the sequence. In contrast to ASAI, the second garlic lectin, ASAII, is a homodimer of two identical 12-kDa subunits. It is translated from an mRNA of approximately 800 nucleotides encoding a polypeptide of 154 amino acids. Interestingly, the coding region of the ASAII cDNA clones is almost identical to that of the second domain of the ASAI cDNA clones.     

Comparative study of the post-translational processing of the mannose-binding lectins in the bulbs of garlic (Allium sativum L.) and ramsons (Allium ursinum L.)

The biosynthesis and processing of the homodimeric and heterodimeric lectins from the bulbs of garlic (Allium sativum) and ramsons (wild garlic;Allium ursinum) were studied using pulse and pulse-chase labelling experiments on developing bulbs. By combining the results of thein vivo biosynthesis studies and the cDNA cloning of the respective lectins, the sequence of events leading from the primary translation products into the mature lectin polypeptides could be reconstructed. From this it is demonstrated that garlic and ramsons use different schemes of post-translational modifications in order to synthesize apparently similar lectins from totally different precursors. Both the homomeric garlic lectin (ASAII) and its homologue in ramsons (AUAII) are synthesized on the endoplasmic reticulum (ER) as nonglycosylated 13.5 kDa precursors, which, after their transport out of the ER are converted into the mature 12.0 kDa lectin polypeptides by the cleavage of a C-terminal peptide. The heterodimeric garlic lectin ASAI is synthesized on the ER as a single glycosylated precursor of 38 kDa, which after its transport out of the ER undergoes a complex processing which gives rise to two mature lectin subunits of 11.5 and 12.5 kDa. In contrast, both subunits of the heterodimeric ramsons lectin AUAI are synthesized separately on the ER as glycosylated precursors, which after their transport out of the ER are deglycosylated and further processed into the mature lectin polypeptides by the cleavage of a C-terminal peptide.     

Purification and Characterization of a Lectin with High Hemagglutination Property Isolated from <Emphasis Type="Italic">Allium altaicum</Emphasis>

A lectin was purified from the leaves of Allium altaicum and corresponding gene was cloned. The lectin namely Allium altaicum agglutinin (AAA) was ~24 kDa homodimeric protein and similar to a typical garlic leaf lectin. It was synthesized as 177 amino acid residues pre-proprotein, which consisted of 28 and 43 amino acid long N and C-terminal signal peptides, respectively. The plant expressed this protein more in scapes and flowers in comparison to the bulbs and leaves. Hemagglutination activity (with rabbit erythrocytes) was 1,428 fold higher as compared to Allium sativum leaf agglutinin (ASAL) although, the insecticidal activity against cotton aphid (Aphis gossypii) was relatively low. Glycan array revealed that AAA had higher affinity towards GlcAb1-3Galb as compared to ASAL. Homology analysis showed 57–94% similarity with other Allium lectins. The mature protein was expressed in E. coli as a fusion with SUMO peptide in soluble and biologically active form. Recombinant protein retained high hemagglutination activity.     

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.     

cDNA cloning, primary structure, and in vitro biosynthesis of the DB58 lectin from Dolichos biflorus

Previous studies have shown that the Dolichos biflorus plant contains a lectin in its stems and leaves, called DB58, that is closely related to the D. biflorus seed lectin. DB58 is a heterodimer composed of two closely related subunits. Immunoprecipitation of total translation products from D. biflorus stem and leaf mRNA suggests a single polypeptide precursor for both of these subunits. Several identical cDNA clones representing the entire coding region of the DB58 mRNA have been isolated from a D. biflorus stem and leaf cDNA library. The DB58 cDNA represents an mRNA encoding a polypeptide of Mr = 29,545. The predicted polypeptide is equal in length to the larger subunit of DB58 with the addition of a 22-amino acid amino-terminal signal sequence. The sequence of the DB58 lectin exhibits 84% homology to the D. biflorus seed lectin at the amino acid level, suggesting that these lectins are encoded by differentially expressed genes and may have evolved to carry out tissue-specific functions. Comparison of the DB58 sequence to other leguminous seed lectins indicates a high degree of structural conservation.     

Purification,characterization, molecular cloning,and expression of novel members of jacalin-related lectins from rhizomes of the true fern Phlebodium aureum (L) J. Smith (Polypodiaceae)

A lectin was purified from rhizomes of the fern Phlebodium aureum by affinity chromatography on mannose-Sepharose. The lectin, designated P. aureum lectin (PAL), is composed of two identical subunits of approximately 15 kDa associated by noncovalent bonds. From a cDNA library and synthetic oligonucleotide probes based on a partial amino acid sequence, 5'- and 3'-rapid amplification of cDNA ends allowed the generation of two similar full-length cDNAs, termed PALa and PALb, each of which had an open reading frame of 438 bp encoding 146 amino acid residues. The two proteins share 88% sequence identity and showed structural similarity to jacalin-related lectins. PALa contained peptide sequences exactly matching those found in the isolated lectin. PALa and PALb were expressed in Escherichia coli using pET-22b(+) vector and purified by one-step affinity chromatography. Native and recombinant forms of PAL agglutinated rabbit erythrocytes and precipitated with yeast mannan, dextran, and the high mannose-containing glycoprotein invertase. The detailed carbohydrate-binding properties of the native and recombinant lectins were elucidated by agglutination inhibition assay, and native lectin was also studied by isothermal titration calorimetry. Based on the results of these assays, we conclude that this primitive vascular plant, like many higher plants, contains significant quantities of a mannose/glucose-binding protein in its storage tissue, whose binding specificity differs in detail from either legume mannose/glucose-binding lectins or monocot mannose-specific lectins. The identification of a jacalin-related lectin in a true fern reveals for the first time the widespread distribution and molecular evolution of this lectin family in the plant kingdom.     

Comparative biochemical analysis of lectin and nuclease from Chelidonium majus L

It has been recently recognized that lectins exhibit other activities besides hemagglutination. Previously we have found that purified lectin from Chelidonium majus showed DNase activity (Fik, Go?dzicka-Józefiak & Kedzia, 1995, Herba Polon. 41, 84-95). Comparison of lectin and DNase from the sap from leaves and roots of Chelidonium majus proved that both these compounds are composed of 24 kDa monomer subunits which have an identical N-terminal sequence but differ in amino-acid composition and degree of glycosylation. Possible interrelationship between lectin and DNase is discussed.     

Developmental Regulation of Lectin and Alliinase Synthesis in Garlic Bulbs and Leaves

Using a combination of northern blot analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a detailed study was made of the temporal and spatial regulation of garlic (Allium sativum L.) lectins and alliinase throughout the life cycle of the plant. The two bulb-specific lectins (ASAI and ASAII), which are the most predominant bulb proteins, accumulate exclusively in the developing garlic cloves and progressively disappear when the old clove is consumed by the plant. On the basis of these observations, ASAI and ASAII can be regarded as typical vegetative storage proteins. The leaf-specific lectin (ASAL), on the contrary, is specifically synthesized in young leaves and remains present until withering. Because ASAL is only a minor protein, it probably fulfills a specific function in the plant. Unlike the lectins, alliinase is present in large quantities in bulbs as well as in leaves. Moreover, intact alliinase mRNAs are present in both tissues as long as they contain living cells. The latter observation is in good agreement with the possible involvement of alliinase in the plant's defense against pathogens and/or predators.     

Isolation, characterization and molecular cloning of the bark lectins from Maackia amurensis

A detailed study was made of the bark lectins of the legume tree Maackia amurensis using a combination of protein purification and cDNA cloning. The lectins, which are the most abundant bark proteins, are a complex mixture of isoforms composed of two types of subunits of 32 and 37 kDa, respectively. Isolation and characterization of the homotetrameric isoforms indicated that the 32 kDa subunit exhibits a 100-fold stronger haemagglutinating activity than the 37 kDa subunit. Molecular cloning confirmed that the two lectin subunits are encoded by different genes. The 32 kDa subunit is apparently encoded by a single gene, whereas two highly homologous genes encode the 37 kDa subunit. A comparison of the deduced amino acid sequences of the bark lectin cDNAs and the previously described cDNA encoding the seed haemagglutinin demonstrated that they are encoded by different genes. Abbreviations: LECMAHb, cDNA clone encoding Maackia amurensis bark haemagglutinin; LECMALb, cDNA clone encoding Maackia amurensis bark leucoagglutinin; MALb, Maackia amurensis bark leucoagglutinin; MAHb, Maackia amurensis bark haemagglutinin This revised version was published online in November 2006 with corrections to the Cover Date.     

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.     

Lectin in Vegetative Tissues of Adult Barley Plants Grown under Field Conditions

A study of the distribution of lectins over different vegetative tissues of barley (Hordeum vulgare L.) plants, which were grown under normal crop conditions, indicated that lectin occurs in roots, leaves, and developing ears. Isolation and characterization of both root and leaf lectins led to the conclusions (a) that they are indistinguishable from the embryo lectin and (b) that the total lectin content of these vegetative organs is many times higher than that of the embryo. Finally, in vivo labeling experiments demonstrated that the lectin is synthesized de novo in roots and leaves.     

Mannan-Binding Lectin of the Sea Urchin Strongylocentrotus nudus

A novel lectin specific to low-branched mannans (MBL-SN) was isolated from coelomic plasma of the sea urchin Strongylocentrotus nudus by combining anion-exchange liquid chromatography on DEAE Toyopearl 650 M, affinity chromatography on mannan-Sepharose and gel filtration on the Sephacryl S-200. The molecular mass of MBL-SN was estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis under non-reducing conditions to be about 34 kDa. MBL-SN was shown to be a dimer with two identical subunits of about 17 kDa. The native MBL-SN exists as a tetramer. The physico-chemical properties of MBL-SN indicate that it belongs to C-type mannan-binding lectins. The cDNA encoding MBL-SN was cloned from the total cDNA of S. nudus coelomocytes and encodes a 17-kDa protein of 144 amino acid residues that contains a single carbohydrate-recognition domain of C-type lectins. Prediction of the MBL-SN tertiary structure using comparative modelling revealed that MBL-SN is an α/β-protein with eight β-strands and two α-helices. Comparison of the MBL-SN model with available three-dimensional structures of C-type lectins revealed that they share a common fold pattern.     

S-type lectins occur also in invertebrates: High conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium

The marine sponge Geodia cydonium contains several lectins.The main component, called lectin-1, is composed of three tofour identical subunits. The subunits of the lectins were clonedfrom a cDNA library; two clones were obtained. From the deducedaa sequence of one clone, LECT-1, a mol. wt of 15 313 Da iscalculated; this value is in good agreement with mass spectrometricanalysis of 15 453 25 Da. The sequence of another clone, LECT-2,was analysed and the aa sequence was deduced (15 433 Da). Thetwo subunits have a framework sequence of 38 conserved aa whichare characteristic for the carbohydrate-binding site of vertebrateS-type lectins. Clustering of lectin sequences of various speciesfollowing their pairwise comparison establishes a dendrogram,which reveals that the sponge lectin could be considered asthe ancestor for vertebrate S-type lectins. Geodia cydonium lectin sponges S-type lectin     

Molecular cloning of the lectin and a lectin-related protein from common Solomon's seal (Polygonatum multiflorum)

The most prominent protein ofPolygonatum multiflorum (common Solomon's seal) rhizomes has been identified as a mannose-binding lectin. Analysis of the purified lectin demonstrated that it is a tetramer of four identical subunits of 14 kDa. Molecular cloning further revealed that the lectin from this typical Liliaceae species belongs to the superfamily of monocot mannose-binding proteins. Screening of cDNA libraries constructed with RNA isolated from buds, leaves and flowers ofP. multiflorum also yielded cDNA clones encoding a protein, which contains two tandemly arranged domains with an obvious sequence homology to the mannose-binding lectins. Molecular modelling of thePolygonatum lectin and lectin-related protein indicated that the three-dimensional structure of both proteins strongly resembles that of the snowdrop lectin. In addition, this approach suggested that the presumed carbohydrate-binding sites of the lectin can accommodate a mannose residue whereas most of the carbohydratebinding sites of the lectin-related protein cannot.Abbreviations GNA Galanthus nivalis agglutinin - HCA hydrophobic cluster analysis - LECPMA cDNA clone encoding PMA - PM30 30 kDa protein isolated fromPolygonatum multiforum - PMA Polygonatum multiflorum agglutinin - PMLRP Polygonatum multiflorum lectin-related protein     

Evidence for the existence of an intracellular root-lectin in soybeans

Soybean (Glycine max (L.) Merr.) root lectin, identified as extractable agglutination activity, was shown to reappear following 15-h incubations of roots that had previously been stripped of all extractable lectin activity. Additional lectin activity was released following disruption of the root tissues and cellular fractionation. These lectin activities were shown to have binding specificity an antibody cross-ractivity similar to soybean seed and root lectins previously described. Thus, it is possible that this intracellular lectin represents the source of extracellular root lectins.     

Related mannose-specific lectins from different species of the family Amaryllidaceae

Bulbs from three species of the plant family Amaryllidaceae ( Narcissus pseudonurcissus L., Leucojum aestivum L. and Leucojum vernum L.) were found to contain mannose-specific lectins. These lectins were serologically identical to a previously reported Amaryllidaceae lectin from Galanthus nivalis L. bulbs, but had a different molecular structure. The lectins described in this paper are dimeric proteins composed of subunits of 13 kDa, which are not held together by disulphide bridges. In hapten-inhibition assays Amaryllidaceae lectins exhibited exclusive specificity towards mannose. Furthermore, they all had a high specific agglutination activity with trypsin-treated rabbit erythrocytes, whereas human red blood cells were not agglutinated.