Elderberry (Sambucus nigra L.) seed proteins inhibit protein synthesis and display strong immunoreactivity with rabbit polyclonal antibodies raised against the type 2 ribosome-inactivating protein nigrin b

Affinity chromatography-purifled elderberry (Sambucus nigraL.) seed proteins strongly inhibited protein synthesis and displayedthe 28S rRNA N-glycosidase activity characteristic of all typesof ribosome-inactivating proteins (RIPs). Western blot analysisrevealed several proteins that reacted with antibodies raisedagainst the novel non-toxic type 2 ribosome-inactivating proteinnigrin b isolated from elder bark, thus indicating the presenceof a new type 2 RIP. Key words: Anti-nigrin b antibodies, protein synthesis, seeds, elder seeds, Sambucus nigra     

Presence of polymerized and free forms of the non-toxic type 2 ribosome-inactivating protein ebulin and a structurally related new homodimeric lectin in fruits of Sambucus ebulus L.

Mature leaves of dwarf elder (Sambucus ebulus L.) contain the non-toxic type 2 ribosome-inactivating protein ebulin 1 (Girbés et al., 1993b, J. Biol. Chem. 268: 18195–18199). We have now found that the green fruits of dwarf elder contain both free and polymerized forms of ebulin (ebulin f) and a new homo-dimeric D-galactose-binding lectin (SELfd). Polymerized material containing ebulin and lectin is composed of aggregates of variable relative molecular mass, some of them being close to 250 000. These aggregate forms are maintained in part by reducible disulphide bridges and reconstitute from reductant-free dialyzed material previously reduced with 2-mercaptoethanol. Direct incubation of free ebulin f with the free SELfd did not lead to polymerization, thus indicating that polymerization triggers some kind of substantial and perhaps catalyzed change in the structure of these proteins. Ebulin-containing polymerized material reacts with anti-ebulin f antibodies. Our results indicate that ebulin f is a fruit-form of ebulin 1. In contrast to green fruits, mature fruits lack both polymerized material and ebulin f, thus indicating some kind of reserve role for them in green fruits. Polymerization of ebulin and the dimeric lectin may represent a novel means of storing the non-toxic type 2 ribosome-inactivating proteins and lectins found in highly metabolic tissues, such as green fruits. Received: 4 April 1997 / Accepted: 17 June 1997     

cDNA molecular cloning and seasonal accumulation of an ebulin l-related dimeric lectin of dwarf elder (Sambucus ebulus L) leaves

SELld is a dimeric D-galactose and mucin-binding lectin (apparent Mr 68000) which coexists with the non-toxic type 2 ribosome-inactivating protein (RIP) ebulin l in dwarf elder (Sambucus ebulus L.) leaves. To ascertain a potential structural correlation with ebulin l molecular cloning of a cDNA coding for SELld was performed. SELld shared a 76% of identity with the ebulin l-B chain. Notably, it was found that SELld has Tyr present in the high affinity 2gamma sugar-binding domain of ricin which is absent in ebulin l-B chain and which seems responsible of the low cell and in vivo toxicities of ebulin l. The concentration of ebulin l in leaves decreased along the developmental stage of dwarf elder and almost disappeared in senescence while the content in SELld changed in the opposite way. Our results suggest that SELld and ebulin l play different biological roles in dwarf elder leaves.     

Non-toxic type 2 ribosome-inactivating proteins (RIPs) from Sambucus: occurrence, cellular and molecular activities and potential uses.

Ribosome-inactivating proteins (RIPs) are a family of enzymes that trigger the catalytic inactivation of ribosomes. The most known member of the family is the highly poisonous two-chain ricin isolated from Ricinus communis L. Sambucus species contain a number of two-chain RIPs structurally and enzymatically related to ricin which have the noteworthy feature that, having an enzymatic activity on ribosomes, leading to the inhibition of protein synthesis, higher than ricin, they are lacking of the tremendous unspecific toxicity of ricin. Therefore, they have been called non-toxic type 2 RIPs. The most representative and studied members are nigrin b present in the bark of the common (black) elder Sambucus nigra L. and ebulin 1 present in the leaves of the dwarf elder Sambucus ebulus L. The molecular basis for the low unspecific activities of nigrin b and ebulin 1 as compared with ricin seems to be related with single changes of amino acids in the high affinity sugar binding sites of the B chains. These changes determine the intracellular traffic of these proteins and thus the cellular toxicity. Conjugation ofnigrin b or ebulin 1 to either transferrin or monoclonal antibodies provided highly active conjugates targeting cancer. Thus these non-toxic type 2 RIPs are promising tools for cancer therapy.     

Isolation and characterization of a new non-toxic two-chain ribosome-inactivating protein from fruits of elder (Sambucus nigra L.)

Sambucus (Caprifoliaceae) species contain nigrin b and ebulinI, which are two-chain ribosomeinactivating proteins (RIPs)belonging to a new type of RIPS which are non-toxic to miceand cultured human cells. In this work the presence in fruitsof elder (S. nigra L.) of a new non-toxic type 2 RIP (nigrinf) that co-exists with a lectin known as SNA IV is described.Nigrin f strongly inhibited protein synthesis in mammalian,but not in plant, ribosomes, promoting the depurination of sensitiveribosomes and thus allowing the release of the RIP diagnosticRNA fragment. Nigrin f is composed of two dissimilar subunitslinked by disulphide bridges with apparent M_r values of 31 600and 26 300. The N-terminal amino acid sequence revealed closehomology of the catalytic A chain with type 1 RIPs, especiallythose from Cucurbitaceae, and the B chain with several lectinspreviously isolated from Sambucus species. Nigrin f was nottoxic to mice when injected intraperitoneally up to 2 mg kg^–1.In addition, NHC human cells were also insensitive to nigrinf up to 60 µg ml^–1. Anti-nigrin b rabbit polyclonalantibodies reacted with nigrin f, indicating that nigrin b andnigrin f are proteins with similar structures. Key words: Sambucus nigra, elder fruits, nigrin f, ribosomeinactivating protein, characterization     

Transient occurrence of an ebulin-related D-galactose-lectin in shoots of Sambucus ebulus L

Young shoots of Sambucus ebulus L. contain a monomeric d-galactose binding lectin (SELlm), which disappears upon shoot development, and was previously undetected since it co-purifies with the non-toxic type 2 ribosome-inactivating protein ebulin l and the dimeric lectin SELld. Molecular cloning of cDNA coding for SELlm and mass spectrometry analysis revealed a protein with a molecular mass of 34,239 Da, which displays 80%, 77% and 45% of amino acid sequence identity with the ebulin l-B chain, SELld and ricin-B chain, respectively. Furthermore, the cloned precursor, with respect to the ebulin l precursor is truncated and contains the signal peptide, a piece of the A chain, a piece of the connecting peptide and the B chain. Further processing yields the lectin protein, which contains only the B chain. Despite the fact that SELlm displays the same d-galactose-binding sites than ricin, it was found that the lectin has different binding properties to D-galactose-containing matrix than ricin. Notably, and unlike ricin, the binding of SELlm and other Sambucus lectins to such matrix was maximum in range of 0-10 degrees C and abolished at 20 degrees C.     

The major elderberry (Sambucus nigra) fruit protein is a lectin derived from a truncated type 2 ribosome-inactivating protein

The major protein of elderberry ( Sambucus nigra L.) fruits is a lectin, called Sambucus nigra agglutinin IVf or SNAIVf. This lectin is composed of subunits that strongly resemble the B chain of the type 2 ribosome-inactivating protein (RIP), called SNAVf, present in the same tissue. To corroborate the possible relationship between both proteins their corresponding cDNAs were cloned and compared. Alignment of the deduced amino acid sequences revealed that the cDNA encoding SNAIVf is almost identical to that of SNAVf except that its A chain is truncated. Northern blot analysis confirmed that the mRNA encoding SNAIVf is about 500 nucleotides shorter than the SNAVf mRNA. In addition, the occurrence of a truncated type 2 RIP gene was unambiguously demonstrated by the analysis of PCR amplified genomic sequences. These results not only demonstrate for the first time that a plant lectin is encoded by a truncated type 2 RIP gene but also address important questions with respect to the molecular evolution of RIP and lectins.     

Seasonal Fluctuations of Lectins in Barks of Elderberry (Sambucus nigra) and Black Locust (Robinia pseudoacacia)

Elderberry (Sambucus nigra) and black locust (Robinia pseudoacacia) agglutinins, which are abundantly present in the bark of both species, display seasonal fluctuations with regard to their content in this tissue. These seasonal changes result apparently from a circa-annual rhythm of lectin accumulation and depletion during autumn and spring, respectively. Because the bark of trees can be considered as a type of vegetative storage tissue, the results suggest that bark lectins behave as typical storage proteins.     

Ebulitins: A new family of type 1 ribosome-inactivating proteins (rRNA N-glycosidases) from leaves of Sambucus ebulus L. that coexist with the type 2 ribosome-inactivating protein ebulin 1

A new family of single chain (type 1) ribosome-inactivating proteins (RIPs), that we have named ebulitins, have been found in mature leaves of Sambucus ebulus L., a caprifoliaceae plant also known to contain a non-toxic two chain (type 2) RIP named ebulin 1 in its leaves. Ebulitins are basic proteins of M_r 32,000, 29,000 and 29,000 for ebulitins , β and γ, respectively. The simultaneous presence of different basic type 1 and acidic type 2 RIPs in the same plant and in the same tissue is described here for the first time and opens a new door in research into RIPs.     

Isolation and partial characterization of nigrin b,a non-toxic novel type 2 ribosome-inactivating protein from the bark ofSambucus nigra L.

The bark ofSambucus nigra L. contains a non-toxic novel type 2 ribosome-inactivating protein that we named nigrin b.In vitro, nigrin b strongly inhibited mammalian protein synthesis but did not affect plant nor bacterial protein synthesis. The protein (M _r 58 000) contains two subunits, A (M _r 26 000) and B (M _r 32 000); linked by disulphide bridge(s). Nigrin b was found to be an rRNA N-glycosidase of the rRNA of intact mammalian ribosomes and shares a very good N-terminal amino-acid sequence homology with the anti-HIV-1 proteins TAP 29 and trichosanthin.     

Sialic acid-binding dwarf elder four-chain lectin displays nucleic acid N-glycosidase activity

Sialic acid-binding dwarf elder agglutinin (SEA) present only in rhizomes of the medicinal plant Sambucus ebulus L., was found to be a tetrameric glycoprotein consisting of two covalently-associated dimers of an enzymic A chain with rRNA N-glycosidase activity (EC 3.2.2.22) linked to a B chain with agglutinin properties. The lectin inhibited protein synthesis by a cell-free system and depurinated ribosomes. Cloning of the corresponding gene and molecular modeling of the deduced amino acid sequence demonstrated that SEA has a three-dimensional structure which resembles that reported for other two tetrameric type 2 RIPs from Sambucus (SNAI and SSA). The lectin agglutinated red blood cells and displayed sugar affinity for sialic acid residues apart from d-galactose, binding to the mucin-producing gut goblet cells. Since sialic acid is present in animal cells, especially in epithelial lining gut cells, but not in plants, SEA could play a role in the defense against insect attack.     

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     

Carbohydrate-binding activity of the type-2 ribosome-inactivating protein SNA-I from elderberry (Sambucus nigra) is a determining factor for its insecticidal activity

In recent years, different classes of proteins have been reported to promote toxic effects when ingested. Type-2 ribosome-inactivating proteins (RIPs) are a group of chimeric proteins built up of an A-chain with RNA N-glycosidase activity and a B-chain with lectin activity. These proteins are thought to play a role in plant protection. Sambucus nigra agglutinin I (SNA-I) is a type-2 RIP, isolated from the bark of elderberry (S. nigra L.). This study demonstrated the insecticidal potency of SNA-I on two Hemipteran insect species using two different methods. An artificial diet supplemented with different concentrations of the purified RIP reduced survival and fecundity of pea aphids Acyrthosiphon pisum. In addition, feeding of tobacco aphids, Myzus nicotianae, on leaves from transfected plants constitutively expressing SNA-I, resulted in a delayed development and reduced adult survival and also the fertility parameters of the surviving aphids were reduced, suggesting that a population of aphids would build up significantly slower on plants expressing SNA-I. Finally, a series of experiments with transgenic lines in which a mutant RIP was expressed, revealed that the carbohydrate-binding activity of SNA-I is necessary for its insecticidal activity. In a first set of mutants, the B-chain was mutated at one position (Asp231ΔGlu), and in the second set both carbohydrate-binding sites were mutated (Asn48ΔSer and Asp231ΔGlu). Mutation of one carbohydrate-binding site strongly reduced the insecticidal activity of SNA-I, whereas mutation of both lectin sites (almost) completely abolished the SNA-I effect on tobacco aphids.     

2.8-A crystal structure of a nontoxic type-II ribosome-inactivating protein, ebulin l

Ebulin l is a type-II ribosome-inactivating protein (RIP) isolated from the leaves of Sambucus ebulus L. As with other type-II RIP, ebulin is a disulfide-linked heterodimer composed of a toxic A chain and a galactoside-specific lectin B chain. A normal level of ribosome-inactivating N-glycosidase activity, characteristic of the A chain of type-II RIP, has been demonstrated for ebulin l. However, ebulin is considered a nontoxic type-II RIP due to a reduced cytotoxicity on whole cells and animals as compared with other toxic type-II RIP like ricin. The molecular cloning, amino acid sequence, and the crystal structure of ebulin l are presented and compared with ricin. Ebulin l is shown to bind an A-chain substrate analogue, pteroic acid, in the same manner as ricin. The galactoside-binding ability of ebulin l is demonstrated crystallographically with a complex of the B chain with galactose and with lactose. The negligible cytotoxicity of ebulin l is apparently due to a reduced affinity for galactosides. An altered mode of galactoside binding in the 2gamma subdomain of the lectin B chain primarily causes the reduced affinity.     

Purification and Partial Characterization of a Lectin from the Bark of Japanese Elderberry (Sambucus sieboldiana)

A lectin [Sambucus sieboldiana agglutinin (SSA)] was purifiedfrom the twigs of Sambucus sieboldiana by repeated affinitychromatography on fetuin-Sepharose. SSA had a molecular weight(Mr) of approximately 160 K on gel filtration and consistedof two types of subunit of which the molecular weights rangedfrom 31 to 37 K. SSA agglutinated human erythrocytes irrespectiveof their blood type and the hemagglutination was inhibited bya very low concentration of Neu5Ac(2-6)lactose, suggesting thatSSA has a carbohydrate-binding specificity similar to that ofthe lectin previously isolated from the bark of S. nigra (SNA).However, the Ouchterlony double-diffusion analysis of theselectins with an antibody raised against SSA showed that SSAwas immunologjcally related but not identical to SNA. (Received January 17, 1989; Accepted June 19, 1989)     

Influence of Sambucus nigra bark lectin on cell DNA under different in vitro conditions

The biological activity of Sambucus nigra bark lectin on Chinese hamster cells in vitro was investigated by comet-assay and cytotoxicity testing. Mitogenic properties at the concentrations 0.063-0.25 microg/ml (but not higher) were found, and the induction of DNA breaks at concentrations 0.5 microg/ml and higher is demonstrated. S. nigra bark lectin at mitogenic concentrations decreased the level of nickel-induced DNA damage. The character and mechanism of this lectin protective activity was probably related to the induction of DNA reparation in the cells, decreasing nickel uptake in cells, and non-specific binding of nickel ions by protein molecules.     

A method of selective histochemical analysis of sialoglycoproteins using lectins from elder (Sambucus nigra L.)]

Good potentialities in application of elderberry (Sambucus nigra L.) bark lectin for selective histochemical identification of sialylated glycoconjugates has been demonstrated using lectin-peroxidase technique. In order to omit this lectin binding to D-galactose and N-acetyl-D-galactosamine residues, preincubation of tissue sections with non-marked PNA and SBA (or other lectins with similar carbohydrate specificity) is proposed. By means of neuraminidase digestion it has been ascertained, that oligosaccharide chains of secretory glycopolymers, synthesised in ovine submandibular gland mucocytes, contain DGal and DGalNAc residues penultimate to terminal sialic acids.     

Sambucus ebulus L. lectin: detection of H character of newborn red cells

The O(H) foetal red cells are not always constantly agglutinated by the eel test serum, but the Sambucus ebulus L. fruit lectin provides a regular agglutination. Different experiments with treated red cells suggest that the lectin acts upon the membrane's inner sites. Besides the H sites, it is possible that other receptors might be bonded with the lectin.     

Analysis of the in planta antiviral activity of elderberry ribosome-inactivating proteins.

Although the type-2 ribosome-inactivating proteins (SNA-I, SNA-V, SNLRP) from elderberry (Sambucus nigra L.) are all devoid of rRNA N-glycosylase activity towards plant ribosomes, some of them clearly show polynucleotide-adenosine glycosylase activity towards tobacco mosaic virus RNA. This particular substrate specificity was exploited to further unravel the mechanism underlying the in planta antiviral activity of ribosome-inactivating proteins. Transgenic tobacco (Nicotiana tabacum L. cv Samsun NN) plants expressing the elderberry ribosome-inactivating proteins were generated and challenged with tobacco mosaic virus in order to analyze their antiviral properties. Although some transgenic plants clearly showed antiviral activity, no clear correlation was observed between in planta antiviral activity of transgenic tobacco lines expressing the different ribosome-inactivating proteins and the in vitro polynucleotide-adenosine glycosylase activity of the respective proteins towards tobacco mosaic virus genomic RNA. However, our results suggest that the in planta antiviral activity of some ribosome-inactivating proteins may rely on a direct mechanism on the virus. In addition, it is evident that the working mechanism proposed for pokeweed antiviral protein cannot be extrapolated to elderberry ribosome-inactivating proteins because the expression of SNA-V is not accompanied by induction of pathogenesis-related proteins.     

Comparative analysis of carbohydrate binding properties of Sambucus nigra lectins and ribosome-inactivating proteins

In the past three decades a lot of research has been done on the extended family of carbohydrate-binding proteins from Sambucus nigra, including several so-called type 2 RIPs as well as hololectins. Although all these proteins have been studied for their carbohydrate-binding properties using hapten inhibition assays, detailed carbohydrate specificity studies have only been performed for a few Sambucus proteins. In particular SNA-I, has been studied extensively. Because of its unique binding characteristics this lectin was developed as an important tool in glycoconjugate research to detect sialic acid containing glycoconjugates. At present much less information is available with respect to the detailed carbohydrate binding specificity of other S. nigra lectins and RIPs, and as a consequence their applications remain limited. In this paper we report a comparative analysis of several lectins from S. nigra using the glycan microarray technology. Ultimately a better understanding of the ligands for each lectin can contribute to new/more applications for these lectins in glycoconjugate research. Furthermore, the data from glycan microarray analyses combined with the previously obtained sequence information can help to explain how evolution within a single lectin family eventually yielded a set of carbohydrate-binding proteins with a very broad specificity range.