The primary sequence of Ricinus communis agglutinin. Comparison with ricin

A mixture of synthetic oligonucleotides representing all possible sequences of a peptide present in the ricin B chain has been used to screen a cDNA library constructed using ripening castor bean seed poly(A+) RNA. The eight largest recombinant plasmids selected, by hybridization, a single mRNA species whose translational product was identified as preprolectin by immunoprecipitation. Restriction enzyme analysis of these clones demonstrated that two classes were present representing sequences complementary to two distinct but closely related preprolectin mRNA species. The nucleotide sequence of the cloned cDNA from one of these classes encodes preproricin and has been presented elsewhere (Lamb, F. I., Roberts, L. M., and Lord, J. M., (1985) Eur. J. Biochem. 148, 265-270). The nucleotide sequence of the second class is presented here and shown to represent prepro-Ricinus communis agglutinin. The entire coding sequence was deduced from two overlapping cDNA clones having inserts of 1668 and 1151 base pairs. The coding region defines a preproprotein with a 24-amino acid N-terminal signal sequence preceding the A chain (266 amino acids) which is joined to the B chain (262 amino acids) by a 12-amino acid linking peptide. The protein was confirmed as R. communis agglutinin since the deduced B chain N-terminal sequence corresponds exactly with that determined for purified R. communis agglutinin B chain over a region where several residue differences occur in the ricin B chain. The nucleotide and deduced amino acid sequences of the R. communis agglutinin precursor are compared with those of the ricin precursor.     

Lectin affinity high-performance liquid chromatography. Interactions of N-glycanase-released oligosaccharides with Ricinus communis agglutinin I and Ricinus communis agglutinin II

The structural determinants required for interaction of oligosaccharides with Ricinus communis agglutinin I (RCAI) and Ricinus communis agglutinin II (RCAII) have been studied by lectin affinity high-performance liquid chromatography (HPLC). Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with columns of silica-bound RCAI and RCAII. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. RCAI binds oligosaccharides bearing terminal beta 1,4-linked Gal but not those containing terminal beta 1,4-linked GalNAc. In contrast, RCAII binds structures with either terminal beta 1,4-linked Gal or beta 1,4-linked GalNAc. Both lectins display a greater affinity for structures with terminal beta 1,4-rather than beta 1,3-linked Gal, although RCAII interacts more strongly than RCAI with oligosaccharides containing terminal beta 1,3-linked Gal. Whereas terminal alpha 2,6-linked sialic acid partially inhibits oligosaccharide-RCAI interaction, terminal alpha 2,3-linked sialic acid abolishes interaction with the lectin. In contrast, alpha 2,3- and alpha 2,6-linked sialic acid equally inhibit but do not abolish oligosaccharide interaction with RCAII. RCAI and RCAII discriminate between N-acetyllactosamine-type branches arising from different core Man residues of dibranched complex-type oligosaccharides; RCAI has a preference for the branch attached to the alpha 1,3-linked core Man and RCAII has a preference for the branch attached to the alpha 1,6-linked core Man. RCAII but not RCAI interacts with certain di- and tribranched oligosaccharides devoid of either Gal or GalNAc but bearing terminal GlcNAc, indicating an important role for GlcNAc in RCAII interaction. These findings suggest that N-acetyllactosamine is the primary feature required for oligosaccharide recognition by both RCAI and RCAII but that lectin interaction is strongly modulated by other structural features. Thus, the oligosaccharide specificities of RCAI and RCAII are distinct, depending on many different structural features including terminal sugar moieties, peripheral branching pattern, and sugar linkages.     

Characterization of a cDNA encoding ricin E,a hybrid ricin-Ricinus communis agglutinin gene from the castor plant Ricinus communis

Two classes of ricin cDNA clones have been identified and sequenced. The cDNA clone pBL-1 closely matches in nucleotide sequence the ricin genomic clone pAKG previously described by Halling et al., 1985 (Nucl. Acids Res. 13:8019). A second group of cDNA clones, represented by pBL-3, encode a hybrid protein (ricin E), having a ricin-like A chain and N-terminal half of the B chain and an RCA (Ricinus communis agglutinin)-like C-terminal half of the B chain.     

Genomic cloning and characterization of a ricin gene from Ricinus communis.

A genomic clone that specifies a single polypeptide precursor for ricin, a toxic lectin of Ricinus communis (castor bean), was isolated, sequenced and Sl mapped. The gene encodes a 64 kDa precursor which contains, in the following order: a 24 or 35 amino acid signal peptide, the A chain, a 12 amino acid linker peptide, and the B chain. The 5'-end of the ricin mRNA maps approximately 35 bases upstream from the first methionine codon. Two putative TATA boxes and a possible CAAT box lie in the 5'-flanking region. Two possible polyadenylation signals were found in the 3' flanking region. No introns were found, which is typical of other lectin genes that have been sequenced. Southern blot analysis suggests that the castor bean genome contains approximately six ricin-like genes.     

Ricinus communis agglutinin B chain contains a fucosylated oligosaccharide side chain not present on ricin B chain

Ricinus communis agglutinin (RCA) B chain, in contrast to ricin B chain, contains fucose. Since both RCA and ricin B chain lose two oligosaccharide side chains when treated with β-endo N-acetylglucosaminidase H, it is proposed that fucose is present on a third oligosaccharide. This third oligosaccharide is not present on the ricin B chain and accounts for the larger relative molecular mass of the RCA B chain.Ricinus communis agglutininRicinB chainFucose     

Binding of Ricinus communis agglutinin to the mitochondrial inner membrane as an artifact during preparation.

Endosperm from Ricinus communis was homogenized in the presence of 3H-labelled Ricinus communis agglutinin, with or without addition of lactose. In preparations without the binding-specific sugar the subfraction containing the mitochondrial inner membrane contained sufficient labelled agglutinin to account for the agglutinin reported to be associated with this membrane.     

Resistance of Tetrahymena to ricin, a toxic enzyme from Ricinus communis.

The protozoan $\text{Tetrahymena}\text{pyriformis}$ was found to be resistant to the toxic action of ricin $\text{in}\text{vivo}$. Isolated $\text{Tetrahymena}$ ribosomes were strongly resistant to the A subunit of ricin when tested in a cell free protein synthesis system under different conditions and also lacked the ability to bind A chain stoichometrically. This suggests that $\text{Tetrahymena}$ is resistant $\text{in}\text{vivo}$ because it contains a ribosome which is not susceptible to the toxic action of ricin.     

Specificities of Ricinus communis agglutinin 120 interaction with sulfated galactose

Lectins are used extensively as research tools to detect and target specific oligosaccharide sequences. Ricinus communis agglutinin I (RCA₁₂₀) recognizes non-reducing terminal β-d-galactose (Galβ) and its specificities of interactions with neutral and sialylated oligosaccharides have been well documented. Here we use carbohydrate arrays of sulfated Galβ-containing oligosaccharide probes, prepared from marine-derived galactans, to investigate their interactions with RCA₁₂₀. Our results showed that RCA₁₂₀ binding to Galβ1–4 was enhanced by 2-O- or 6-O-sulfation but abolished by 4-O-sulfation. The results were corroborated with competition experiments. Erythrina cristagalli lectin is also a Galβ-binding protein but it cannot accommodate any sulfation on Galβ.     

Nature of the interaction between Ricinus communis agglutinin and blood cells

Binding of Ricinus communis agglutinin (RCA 120) to carbohydrate receptors of human lymphocytes and erythrocytes is enthalpically driven. As in the case of simple saccharides, the delta S contribution is always unfavorable to the interaction. This result is different from that observed for other lectins and might indicate that hydrophobic interactions do not play a dominant role in binding of RCA 120 to cell surfaces.     

Homology between ricin and Ricinus communie agglutinin: Amino terminal sequence analysis and protein synthesis inhibition studies

The existence of three forms of ricin and two forms of the Ricinus communis agglutinin (RCA) was established using cation exchange chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis. The preparation of the RCA we obtained was 60–75 times more potent than ricin in the agglutination of erythrocytes, but was about 4% as effective as an inhibitor of cell-free protein synthesis. When reduced with 2-mercaptoethanol, the RCA was activated 3000-fold as an inhibitor of cell-free protein synthesis, whereas ricin was activated about 600-fold by the same treatment. A mixture of the RCA A chains was about one-fifth as effective as the ricin A chain in the inhibition of cell-free protein synthesis. The purified polypeptide subunits of the castor bean lectins were subjected to automated Edman degradation. The sequence for 17 of the first 19 residues of the agglutinin A chain was determined. The first seven residues of the ricin A chain were determined and they are identical with those of the RCA A chain. Nineteen turns of Edman degradation on the RCA B chain resulted in the identification of 18 amino acids. The sequence determined for the first 17 residues of the ricin B chain was identical with that of the RCA B chain. It is likely that the identity of the ricin/RCA A and B chain sequences extends further along the polypeptide chains than the sequences we have determined. The similar structural and catalytic potentials of the RCA and ricin suggest that they bear a precursor-product relationship.     

Preparation and evaluation of Ricinus communis agglutinin affinity adsorbents using polymeric supports

A practicable and efficient procedure for preparation of Ricinus communis agglutinin (RCA) affinity adsorbents has been developed. For immobilization of RCA two different polymer-based supports, Toyopearl and TSKgel (TosoHaas), were used. RCA has been successfully immobilized onto these supports with amounts of coupled ligand between 15 and 23 mg/g dry support and corresponding coupling yields of 69-93% (w/w). The prepared affinity adsorbents were characterized concerning their binding capacity for the glycoprotein asialofetuin (ASF) and accessibility of the ligand binding sites. The high accessibility of 80% showed that steric hindrance was negligible at the present ligand density. RCA-Toyopearl was successfully applied in affinity chromatography of glycoproteins indicating its high specificity. A long-term stability test proved no change in capacity for a period of at least 12 months. High-performance affinity chromatography (HPLAC) was carried out using RCA-TSKgel. Experimental results showed that the prepared adsorbents are suitable for selective separation of glycoproteins and oligosaccharides and therefore can be used for investigations of adsorption characteristics of glycoconjugates and for laboratory-scale preparations.     

Variants of hamster fibroblasts resistant to Ricinus communis toxin (ricin).

1. Variant baby-hamster kidney (BHK) cell lines were isolated that grow in the presence of high concentrations of ricin, the toxic lectin of castor beans (Ricinus communis). The variant lines were independently derived from several cultures of normal BHK cells which had been exposed to the mutagen, methyl-N-nitro-N-nitrosoguanidine, before selection by ricin. 2. The cell lines maintain a high degree of resistance to ricin after growth in lectin-free medium for prolonged periods and therefore exhibit stable phenotypes that are different from normal BHK cells. 3. A preliminary classification of the phenotypes was made. Several cell lines bind normal amounts of 125I-labelled ricin, whereas other bind the lectin poorly. 4. A loss of surface receptors for two other lectins, R. communis RCA and Axinella polyploides, which have specificities similar to ricin, was also found in some but not all of the cell lines showing decreased surface concentrations of ricin receptors. 5. The binding to the ricin-resistant cells of lectins of different sugar specificity, namely Lens culinaris lectin and concanavalin A, was similar to, or higher than, to normal BHK cells. 6. Several of the ricin-resistant cell lines were shown to be cross-resistant to the weak cytotoxicity of Phaseolus vulgaris lectin. By contrast, some cell lines were more sensitive to concanavalin A than were normal BHK cells.     

Ultraviolet difference spectroscopic analysis of the saccharide-binding properties of Ricinus communis agglutinin

The nature of the binding of saccharides to Ricinus communis agglutinin was studied by ultraviolet difference spectroscopy. Upon binding of galactose and galactose-containing saccharides, R. communis agglutinin displayed difference spectra with an extreme maximum at 291-293 nm and a smaller maximum at 284-285 nm. Such difference spectra suggest that the environment of a tryptophan residue located at or near the saccharide-binding site of R. communis agglutinin is being changed by an interaction between a tryptophan residue and the bound saccharides. The value of the difference spectra (delta epsilon) increased upon progressive addition of saccharide until the saccharide binding site was saturated with ligand. From the increase in delta epsilon at 291-293 nm, the association constants were obtained for the R. communis agglutinin-saccharide interaction over the temperature range 5-35 degrees C and various pH values. The results clearly demonstrate that the association constants are nearly equal in the range of pH 5-8, but decrease beyond the above pH range and with elevation of temperature. From the thermodynamic parameters for the binding of various saccharides to R. communis agglutinin, we suggest that there exists a subsite structure in the saccharide-binding site of the R. communis agglutinin molecule.     

Interaction of human erythrocyte Band 3 with Ricinus communis agglutinin and other lectins

Agglutination and competition studies suggest that human erythrocyte Band 3 can interact with both mannose/glucose- and galactose-specific lectins. Purified Band 3 reconstituted into lipid vesicles binds concanavalin A, but the nonspecific binding component, measured in the presence of alpha-methylmannoside, is very high. This glycoprotein also carries binding sites for the galactose-specific lectin Ricinus communis agglutinin. Binding was inhibited poorly by lactose, but much more effectively by desialylated fetuin glycopeptides, suggesting that the lectin recognizes a complex oligosaccharide sequence on Band 3. The glycoprotein bears two separate classes of binding sites for R. communis agglutinin. High-affinity binding sites exist which show strong positive cooperativity and correspond in number to the outward-facing Band 3 molecules. A low-affinity binding mode is abolished by 40% ethyleneglycol, suggesting the involvement of hydrophobic lectin-glycoprotein interactions. Studies on binding of R. communis agglutinin to human erythrocytes indicate positively cooperative binding to 7 X 10(5) very-high-affinity sites per cell, and lectin binding is completely inhibitable by lactose. Based on its binding characteristics in vesicles, it seems likely that Band 3 forms the major receptor for this lectin in human erythrocytes. Properties such as positive cooperativity thus appear to be a common feature of the interaction of Band 3 with a variety of lectins of different specificity, both in erythrocytes and lipid bilayers.     

Phloem transport of abscisic acid in Ricinus communis L. seedlings

Sieve tube sap exuded from the cut hypocotyl of castor bean seedlings (Ricinus communis L.) was found to contain 0.2–0.5 mmol m^?3abscisic acid (ABA). The ABA concentration in the sieve tube sap always exceeded that in root pressure exudate under a wide range of water supply. Exudation of sieve tube sap from the cut hypocotyls caused water loss, and this induced ‘water shortage’ in the cotyledons which resulted in the ABA concentration in the cotyledons increasing by 3-fold and that in the sieve tube sap increasing by up to 50-fold within 7h. The wounded surface of the cut hypocotyl was not responsible for the ABA increase. Incubation of the cotyledons of endosperm-free seedlings in various ABA concentrations (up to 100 mmol m^?3) increased the ABA concentration in sieve tube sap. The concomitant increase in ABA, both in cotyledons and in sieve tube sap, had no effect on the phloem loading of sucrose, K⁺ and Mg²⁺ within the experimental period, i.e. up to 10h. It can be concluded that (i) the phloem is an important transport path for ABA, (ii) water stress at the phloem loading sites elevates phloem-mobile ABA, which may then serve as a water stress signal for sinks, for example stem and roots (not only for stomata), and (iii) the ABA concentration of cells next to or in the phloem is more important than the average ABA content in the whole cotyledon for determining the ABA concentration in sieve tube sap.