A chitin-binding lectin from stinging nettle rhizomes with antifungal properties.

Rhizomes of stinging nettle contain a small-sized lectin that exhibits binding specificity toward chitin. This lectin inhibits growth of several phytopathogenic and saprophytic chitin-containing fungi in vitro. The antifungal action of the nettle lectin differs from the action of chitinases, which are a ubiquitous class of antifungal plant proteins. Moreover, the nettle lectin acts synergistically with chitinase in inhibiting fungal growth. The nettle lectin may be a promising candidate for possible applications in the genetic engineering of disease-resistant crops.

Antiviral activity of carbohydrate-binding agents against Nidovirales in cell culture.

Coronaviruses are important human and animal pathogens, the relevance of which increased due to the emergence of new human coronaviruses like SARS-CoV, HKU1 and NL63. Together with toroviruses, arteriviruses, and roniviruses the coronaviruses belong to the order Nidovirales. So far antivirals are hardly available to combat infections with viruses of this order. Therefore, various antiviral strategies to counter nidoviral infections are under evaluation. Lectins, which bind to N-linked oligosaccharide elements of enveloped viruses, can be considered as a conceptionally new class of virus inhibitors. These agents were recently evaluated for their antiviral activity towards a variety of enveloped viruses and were shown in most cases to inhibit virus infection at low concentrations. However, limited knowledge is available for their efficacy towards nidoviruses. In this article the application of the plant lectins Hippeastrum hybrid agglutinin (HHA), Galanthus nivalis agglutinin (GNA), Cymbidium sp. agglutinin (CA) and Urtica dioica agglutinin (UDA) as well as non-plant derived pradimicin-A (PRM-A) and cyanovirin-N (CV-N) as potential antiviral agents was evaluated. Three antiviral tests were compared based on different evaluation principles: cell viability (MTT-based colorimetric assay), number of infected cells (immunoperoxidase assay) and amount of viral protein expression (luciferase-based assay). The presence of carbohydrate-binding agents strongly inhibited coronaviruses (transmissible gastroenteritis virus, infectious bronchitis virus, feline coronaviruses serotypes I and II, mouse hepatitis virus), arteriviruses (equine arteritis virus and porcine respiratory and reproductive syndrome virus) and torovirus (equine Berne virus). Remarkably, serotype II feline coronaviruses and arteriviruses were not inhibited by PRM-A, in contrast to the other viruses tested.

Helianthus tuberosus lectin reveals a widespread scaffold for mannose-binding lectins.

BACKGROUND: Heltuba, a tuber lectin from the Jerusalem artichoke Helianthus tuberosus, belongs to the mannose-binding subgroup of the family of jacalin-related plant lectins. Heltuba is highly specific for the disaccharides Man alpha 1-3Man or Man alpha 1-2Man, two carbohydrates that are particularly abundant in the glycoconjugates exposed on the surface of viruses, bacteria and fungi, and on the epithelial cells along the gastrointestinal tract of lower animals. Heltuba is therefore a good candidate as a defense protein against plant pathogens or predators. RESULTS: The 2.0 A resolution structure of Heltuba exhibits a threefold symmetric beta-prism fold made up of three four-stranded beta sheets. The crystal structures of Heltuba in complex with Man alpha 1-3Man and Man alpha 1-2Man, solved at 2.35 A and 2.45 A resolution respectively, reveal the carbohydrate-binding site and the residues required for the specificity towards alpha 1-3 or alpha 1-2 mannose linkages. In addition, the crystal packing reveals a remarkable, donut-shaped, octahedral assembly of subunits with the mannose moieties at the periphery, suggesting possible cross-linking interactions with branched oligomannosides. CONCLUSIONS: The structure of Heltuba, which is the prototype for an extended family of mannose-binding agglutinins, shares the carbohydrate-binding site and beta-prism topology of its galactose-binding counterparts jacalin and Maclura pomifera lectin. However, the beta-prism elements recruited to form the octameric interface of Heltuba, and the strategy used to forge the mannose-binding site, are unique and markedly dissimilar to those described for jacalin. The present structure highlights a hitherto unrecognized adaptability of the beta-prism building block in the evolution of plant proteins.

Crystal structure of Urtica dioica agglutinin, a superantigen presented by MHC molecules of class I and class II.

BACKGROUND: Urtica dioica agglutinin (UDA), a monomeric lectin extracted from stinging nettle rhizomes, is specific for saccharides containing N-acetylglucosamine (GlcNAc). The lectin behaves as a superantigen for murine T cells, inducing the exclusive proliferation of Vbeta8.3(+) lymphocytes. UDA is unique among known T cell superantigens because it can be presented by major histocompatibility complex (MHC) molecules of both class I and II. RESULTS: The crystal structure of UDA has been determined in the ligand-free state, and in complex with tri-acetylchitotriose and tetra-acetylchitotetraose at 1.66 A, 1.90 A and 1.40 A resolution, respectively. UDA comprises two hevein-like domains, each with a saccharide-binding site. A serine and three aromatic residues at each site form the principal contacts with the ligand. The N-terminal domain binding site can centre on any residue of a chito-oligosaccharide, whereas that of the C-terminal domain is specific for residues at the nonreducing terminus of the ligand. We have shown previously that oligomers of GlcNAc inhibit the superantigenic activity of UDA and that the lectin binds to glycans on the MHC molecule. We show that UDA also binds to glycans on the T cell receptor (TCR). CONCLUSIONS: The presence of two saccharide-binding sites observed in the structure of UDA suggests that its superantigenic properties arise from the simultaneous fixation of glycans on the TCR and MHC molecules of the T cell and antigen-presenting cell, respectively. The well defined spacing between the two binding sites of UDA is probably a key factor in determining the specificity for Vbeta8.3(+) lymphocytes.

The rye embryo system as an alternative to the wheat system for protein synthesis in vitro.

Isolated rye embryos are a readily available source for the preparation of very active, cell-free, protein-synthesizing systems. Incorporation levels up to 2000 pmol leucine per 50 mul assay are routinely obtained at saturating TMV (Tobacco mosaic virus) RNA concentrations; at limiting messenger RNA concentrations the incorporation exceeds 1000 leucine molecules per TMV RNA molecule. The characteristics of this cell-free system for the translation of TMV RNA are identical with those of a similarly prepared wheat germ system. The major advantage of the rye embryo system is its high reliability as compared to the unpredictable wheat germ system. Sucrose gradient analysis of the reaction mixture during the incubation shows an extensive polysome formation with TMV RNA and demonstrates efficient polypeptide chain release.

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.

The primary structure of stinging nettle (Urtica dioica) agglutinin. A two-domain member of the hevein family.

The primary structure of stinging nettle (Urtica dioica) agglutinin has been determined by sequence analysis of peptides obtained from three overlapping proteolytic digests. The sequence of 80 residues consists of two hevein-like domains with the same spacing of half-cystine residues and several other conserved residues as observed earlier in other proteins with hevein-like domains. The hinge region between the two domains is four residues longer than those between the four domains in cereal lectins like wheat germ agglutinin.

The galactose-binding and mannose-binding jacalin-related lectins are located in different sub-cellular compartments.

A galactose-specific and a mannose-specific lectin of the family of the jacalin-related lectins have been localized by immunofluorescence microscopy. The present localization studies provide for the first time unambiguous evidence for the cytoplasmic location of the mannose-specific jacalin-related lectin from rhizomes of Calystegia sepium, which definitely differs from the vacuolar location of the galactose-specific jacalin from Artocarpus integrifolia. These observations support the hypothesis that the galactose-specific jacalin-related lectins evolved from their mannose-specific homologues through the acquisition of vacuolar targeting sequences.

Isolation and partial characterization of a small chitin-binding lectin from mistletoe (Viscum album).

A novel lectin, called VisalbCBA, was isolated from European mistletoe (Viscum album). This lectin differs completely from the classical galactose/N-acetylgalactosamine-binding mistletoe lectins MLI, MLII and MLIII. Biochemical analyses indicated that VisalbCBA is a dimeric protein composed of two identical subunits of approx. 10 kDa. VisalbCBA exhibits specificity towards oligomers of N-acetylglucosamine and shows sequence homology to the previously isolated chitin-binding plant proteins. Although VisalbCBA is less toxic than the other mistletoe lectins, it definitely exhibits cytotoxic properties. The possible involvement of VisalbCBA in the biological and therapeutic effects of mistletoe is discussed.

Ligand-induced perturbations in Urtica dioica agglutinin.

The binding of the trisaccharide, N,N',N"-triacetylchitotriose, to Urtica dioica agglutinin (UDA) was investigated using 1H NMR spectroscopy. UDA is a small antiviral plant lectin containing two homologous 43-amino acid domains. Carbohydrate-induced pertubations occur in one domain of UDA at trisaccharide concentrations below equimolar. Residues in the second domain are shifted at higher carbohydrate concentrations. This data confirms the presence of two binding sites of non-identical affinities per UDA monomer. Qualitative analysis of the 2D NOESY spectra indicates that UDA contains two short stretches of antiparallel beta-sheet. The 1H resonance assignments for both antiparallel beta-sheet sequences have been completed and there is one beta-stretch per domain. A number of these beta-sheet residues are perturbed in the presence of carbohydrate.

Elderberry (Sambucus nigra) contains truncated Neu5Ac(alpha-2,6)Gal/GalNAc-binding type 2 ribosome-inactivating proteins.

Analysis of affinity-purified preparations of the fetuin-binding proteins from elderberry bark and fruits revealed besides the previously reported Neu5Ac(alpha-2,6)Gal/GalNAc-specific type 2 ribosome-inactivating proteins (RIP) the occurrence of single chain proteins of 22 kDa, which according to their N-terminal amino acid sequence correspond to the second part of the B chain of the respective type 2 RIP. Both proteins are very similar except that the polypeptides of the fruit lectin are 10 amino acid residues longer than these from the bark lectin. Our findings not only demonstrate the occurrence of carbohydrate-binding fragments of type 2 RIP but also provide further evidence that type 2 RIP genes give rise to complex mixtures of type 2 RIP/lectins in elderberry.

Molecular cloning of the mitogenic mannose/maltose-specific rhizome lectin from Calystegia sepium.

cDNA clones encoding the mitogenic mannose/maltose-specific lectin from the rhizomes of hedge bindweed (Calystegia sepium) have been isolated and sequenced. Comparison of the deduced amino acid sequence and the molecular weight of the lectin subunit as determined by mass spectrometry indicated that the mature protein comprises the entire open reading frame of the cDNA, which implies that the primary translation product contains no signal peptide and is not proteolytically processed. Searches in the databases revealed sequence homology with the previously described lectins from the taxonomically unrelated Moraceae species Artocarpus integrifolia and Maclura pomifera.

Regulation of gelatinase B (MMP-9) in leukocytes by plant lectins.

The stimulatory or inhibitory effects of plant lectins on the production of gelatinase A (MMP-2) and gelatinase B (MMP-9) by mononuclear white blood cells was investigated by substrate zymography. Leukocyte cultures from 24-h old buffy coats were spontaneously activated and produced high levels of gelatinase B. Using such cultures the suppressing activity of the Datura stramonium, Viscum album, Bauhinia purpurea, Triticum aestivum and Maackia amurensis lectins on gelatinase B induction were demonstrated. When fresh leukocyte preparations from single blood donors were used, low levels of gelatinase B were produced. The induction of gelatinase B was confirmed for concanavalin A and phytohaemagglutinin (PHA-L4). In addition, the Urtica dioica, Calystegia sepium, Convolvulus arvensis and Colchicum autumnale lectins were documented as novel and potent inducers of gelatinase B. Since high circulating gelatinase B levels are associated with specific pathologies, including shock syndromes, the acute toxicity of many lectins might be partially mediated or influenced by gelatinase induction.

Ribosome-inactivating lectins with polynucleotide:adenosine glycosidase activity.

Lectins from Aegopodium podagraria (APA), Bryonia dioica (BDA), Galanthus nivalis (GNA), Iris hybrid (IRA) and Sambucus nigra (SNAI), and a new lectin-related protein from Sambucus nigra (SNLRP) were studied to ascertain whether they had the properties of ribosome-inactivating proteins (RIP). IRA and SNLRP inhibited protein synthesis by a cell-free system and, at much higher concentrations, by cells and had polynucleotide:adenosine glycosidase activity, thus behaving like non-toxic type 2 (two chain) RIP. APA and SNAI had much less activity, and BDA and GNA did not inhibit protein synthesis.

Structural characterisation of the native fetuin-binding protein Scilla campanulata agglutinin: a novel two-domain lectin.

The three-dimensional structure of a 244-residue, multivalent, fetuin-binding lectin, SCAfet, isolated from bluebell (Scilla campanulata) bulbs, has been solved at 3.3 A resolution by molecular replacement using the coordinates of the 119-residue, mannose-binding lectin, SCAman, also from bluebell bulbs. Unlike most monocot mannose-binding lectins, such as Galanthus nivalis agglutinin from snowdrop bulbs, which fold into a single domain, SCAfet contains two domains with approximately 55% sequence identity, joined by a linker peptide. Both domains are made up of a 12-stranded beta-prism II fold, with three putative carbohydrate-binding sites, one on each subdomain. SCAfet binds to the complex saccharides of various animal glycoproteins but not to simple sugars.

Mannose-binding plant lectins: different structural scaffolds for a common sugar-recognition process.

Mannose-specific lectins are widely distributed in higher plants and are believed to play a role in recognition of high-mannose type glycans of foreign micro-organisms or plant predators. Structural studies have demonstrated that the mannose-binding specificity of lectins is mediated by distinct structural scaffolds. The mannose/glucose-specific legume (e.g., Con A, pea lectin) exhibit the canonical twelve-stranded beta-sandwich structure. In contrast to legume lectins that interact with both mannose and glucose, the monocot mannose-binding lectins (e.g., the Galanthus nivalis agglutinin or GNA from bulbs) react exclusively with mannose and mannose-containing N-glycans. These lectins possess a beta-prism structure. More recently, an increasing number of mannose-specific lectins structurally related to jacalin (e.g., the lectins from the Jerusalem artichoke, banana or rice), which also exhibit a beta-prism organization, were characterized. Jacalin itself was re-defined as a polyspecific lectin which, in addition to galactose, also interacts with mannose and mannose-containing glycans. Finally the B-chain of the type II RIP of iris, which has the same beta-prism structure as all other members of the ricin-B family, interacts specifically with mannose and galactose. This structural diversity associated with the specific recognition of high-mannose type glycans highlights the importance of mannose-specific lectins as recognition molecules in higher plants.

Lectin histochemistry of the spleen: a new lectin visualizes the stromal architecture of white pulp and the sinuses of red pulp.

The subcompartmentalization of the white pulp in the spleen is the result of interactions of specific resident stromal cells and migrating subtypes of lymphocytes. Because carbohydrate residues of cell membranes and extracellular matrices are involved in cell-cell and cell-matrix interactions, they were investigated in rat spleen by a broad panel of lectins. Splenic macrophages, which were also demonstrated by Perls' Prussian blue reaction, were labeled selectively by most mannose-specific lectins and gave the characteristic distribution patterns in all splenic (sub)compartments. One recently isolated lectin, Chelidonium majus agglutinin (CMA), visualized predominantly central arterioles, the reticular meshwork (RM) in the periarteriolar lymphatic sheaths (PALS), the circumferential reticulum cells limiting PALS and follicles, and some follicular dendritic cells (FDCs) in white pulp. The endothelial cells of venous sinuses in red pulp were also labeled by CMA and, if frozen sections were used, CMA also labeled the macrophages of the red pulp. Compared to CMA, the monoclonal antibody CD11, which can be used only in frozen sections, stained almost solely the fibrous (extracellular) component of the RM. Because CMA stains the reticulum cells in particular, it is better suited to visualize the stromal architecture of splenic white pulp than the monoclonal antibody. Because CMA can be applied to paraffin-embedded material, it is a particularly useful tool to study the splenic stromal architecture in archival material.

Lectin and proteoglycan histochemistry of feline pacinian corpuscles.

We studied carbohydrate residues of glycoproteins and proteoglycans (PGs) in peritoneal Pacinian corpuscles of five adult cats. Terminal monosaccharides of glycoproteins and related polysaccharides were identified by lectin histochemistry and the PGs and glycosaminoglycans (GAGs) by specific antibodies. The most intensive lectin staining reactions indicated an abundance of glycoconjugates with terminal mannose (Man) or sialic acid residues, but no complex-type oligosaccharides were detected within the corpuscles. Terminal fucose (Fuc) and galactose (Gal) residues typical for O-linked mucin-type glycoproteins generally associated with high water binding capacity were also absent. Antibodies against unsulfated chondroitin (C-0-S), chondroitin-4-sulfate (C-4-S), and decorin showed positive reactions in the interfibrillar spaces between the lamellae, around collagen fibers, and around the lamellae of the perineural capsule, especially in the outer parts known to contain Type II collagen. Biglycan showed a preference for the innermost part of the perineural capsule (intermediate layer), known to contain Type V collagen. Collagen V and biglycan are both linked to growth processes. Hyaluronic acid (HA), chondroitin-6-sulfate (C-6-S) chains, and a chondroitin sulfate proteoglycan (CSPG) were co-localized in the terminal glia. The study of carbohydrates with high water binding capacity may contribute to our understanding of the high viscoelasticity of Pacinian corpuscles.

A comparative study of bark lectins from three elderberry (Sambucus) species.

Three elderberry lectins isolated from the bark of three different species of the genus Sambucus which are native to Europe (S. nigra), North America (S. canadensis), and Japan (S. sieboldiana) were studied comparatively with regard to their carbohydrate binding properties and some structural features. All three lectins contained two identical carbohydrate binding sites per molecule and showed a very high specificity for the Neu5Ac(alpha 2-6)-Gal/GalNAc sequence. However, relative affinities for various oligosaccharides were significantly different among them, suggesting differences in the detailed structure of the carbohydrate binding sites of these lectins. The three lectins were immunologically related, but not identical, and all were composed of hydrophobic and hydrophilic subunit regions, although the molecular sizes of these subunits were slightly different among the three lectins. N-terminal sequence analysis of the subunits of these lectins suggested that they have a very similar structure in this region but also indicated the occurrence of N-terminal processing such as the deletion of several amino acid residues at the N-termini for both hydrophobic and hydrophilic subunits of all three lectins. Tryptic peptide mapping of the three lectins showed a similar pattern for all of them but also showed the presence of some unique peptides for each lectin.

The agglutination of beta-haemolytic streptococci by lectins.

The ability of 25 lectins, isolated from different plants and fungi, to agglutinate 95 clinical isolates of beta-haemolytic streptococci was examined. Cell suspensions were untreated, trypsin-treated or boiled at pH 2.0. None of the 95 untreated cell suspensions gave a visible reaction with any of the lectins. When the cells were trypsinised, 42 strains were agglutinated with one or more lectin and after boiling at pH 2, all the strains were agglutinated. After treatment with trypsin, 20 different agglutination patterns were observed, and after boiling, 19 patterns, four of which were similar. A correlation was found between Lancefield group C and some of these patterns. Some lectins reacted specifically with group C streptococci; DBA and WFA, both specific for D-GalNAc, DSA, a GlcNAc-specific lectin, and RPA, which showed a complex specificity, reacted only with group C strains. Furthermore, the lectin of Maackia amurensis reacted with 50% of group B streptococci only. Agglutination assays with lectins were reproducible, easy to perform, relatively inexpensive and, therefore, applicable to studies of cell-wall structure and epidemiology of beta-haemolytic streptococci.