Studies on the molecular recognition of synthetic methyl beta-lactoside analogs by ricin, a cytotoxic plant lectin

The binding of methyl beta-lactoside and of all possible monodeoxy derivatives of methyl beta-lactoside to the galactose-specific highly cytotoxin lectin ricin, has been investigated. The distribution of low-energy conformers of the disaccharide structures has been first determined using molecular-mechanics calculations and high-resolution NMR spectroscopy. The nuclear Overhauser enhancements and specific deshieldings observed are in agreement with a similar distribution of low-energy conformers for all studied compounds which may be described by a major conformer defined by phi (H1'-C1'-O1'-C4) and psi (C1'-O1'-C4-H4) torsion angles of 49 degrees and 5 degrees, respectively, with contribution of conformers with angles phi/psi 24 degrees/-59 degrees, 22 degrees/-32 degrees and 6 degrees/-44 degrees. Assuming that the disaccharides bind to the lectin in these preferred conformations, the apparent dissociation constants for the ricin-disaccharide complexes have been interpreted in terms of specific polar and nonpolar interactions. In agreement with X-ray data, the hydroxyl groups at positions 3, 4 and 6 of the beta-D-galactopyranose moiety appear as key polar groups in the interaction with ricin. These results are in contrast to previous results which have established that position 6 is not involved in lectin binding. An important nonpolar interaction involving position 3 of the beta-D-glucopyranose moiety, seems to be operative. The distribution of low-energy conformers of these disaccharide structures permits this interaction to take place with the hydroxyl group at this position intramolecularly bonded, thus rendering this region of the molecule more lipophylic in character for acceptance into nonpolar regions of the combining site.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Binding and precipitation of lectins from Erythrina indica and Ricinus communis (agglutinin I) with synthetic cluster glycosides

We recently reported that tri- and tetraantennary complex type oligosaccharides with nonreducing terminal galactose residues and the triantennary asialofetuin glycopeptide can bind and precipitate certain galactose specific lectins (L. Bhattacharyya, and C.F. Brewer (1986) Biochem. Biophys. Res. Commun. 141, 963-967; L. Bhattacharyya, M. Haraldsson, and C.F. Brewer (1988) Biochemistry 27, 1034-1041). The present study investigates the binding interactions of two of these lectins, those from Erythrina indica and Ricinus communis (Agglutinin I), with mono-, bi-, and triantennary synthetic cluster glycosides, which have little structural resemblance to complex type oligosaccharides other than they possess nonreducing terminal galactose residues (R.T. Lee, P. Lin, and Y.C. Lee (1984) Biochemistry 23, 4255-4261). The enhanced affinities of the bi- and triantennary glycosides relative to the monoantennary glycoside for the two lectins are consistent with an increase in the probability of binding due to multiple binding residues in the multiantennary glycosides. The triantennary glycoside is capable of precipitating the two lectins, and quantitative precipitation data indicate that it is a trivalent ligand. The results show that the binding and precipitation activities of complex type oligosaccharides with these lectins is due solely to the presence of multiple terminal galactose residues and not to the overall structures of the oligosaccharides.     

Cellular energy dependent agglutination of rat ascites tumor cells mediated by concanavalin A and Ricinus communis agglutinin.

Effect of various metabolic inhibitors on the agglutination of rat ascites tumor cells mediated by concanavalin A and Ricinus communis agglutinin was studied using a quantitative assay method for agglutination in which turbidity of cell suspension is measured. Cell agglutination was inhibited by low temperature, cytochalasin B and inhibitors of energy generating systems without affecting lectin binding, and agglutination was not affected by hydroxyurea, actinomycin D or cycloheximide. The inhibitors of energy generating systems decreased the cellular ATP level and inhibited macromolecular synthesis under the conditions where they inhibited the agglutinations. In contrast, cytochalasin B did not depress the cellular ATP level nor inhibit RNA and protein syntheses. These results suggest that the agglutination is associated with cellular energy dependent processes other than macromolecular synthesis; probably with some cellular surface movements participated by microfilament activity.     

Effect of metabolic inhibitors on the agglutination of tumor cells by concanavalin A and Ricinus communis agglutinin

The agglutinations of rat ascites tumor cells by concanavalin A and by $\text{Ricinus communis}$ agglutinin were inhibited by low temperature, 2,4-dinitrophenol and cytochalasin B but not by cycloheximide. These metabolic inhibitors, however, did not inhibit the binding of the agglutinins to the cells. These results suggest that the agglutination was dependent on an active process; probably on a microfilament system responsible for cell surface movement which requires ATP, but not on protein synthesis.     

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.     

Precursors of ricin and Ricinus communis agglutinin. Glycosylation and processing during synthesis and intracellular transport

During synthesis in vivo the castor bean lectin precursors initially appear in the endoplasmic reticulum as a group of core glycosylated polypeptides of relative molecular mass 64 000-68 000. Pretreatment of intact castor bean endosperm tissue with tunicamycin partially inhibits the cotranslational core glycosylation step and results in the accumulation of a single sized unglycosylated precursor polypeptide of relative molecular mass 59 000. The glycosylated precursors in the endoplasmic reticulum were enzymically converted to the 59 000-Mr form by incubation with endoglucosaminidase H. Intracellular transport of the glycosylated lectin precursors from the endoplasmic reticulum to a denser vesicle fraction was accompanied by modifications to the oligosaccharide moieties which conferred resistance to the action of endoglucosaminidase H. The post-translational addition of fucose to the carbohydrate chain was identified as one of the oligosaccharide modification steps. Fucose addition was catalysed by a glycosyltransferase associated with a smooth-surfaced membrane fraction which was distinct from the endoplasmic reticulum and which was tentatively identified as the Golgi apparatus. Glycosylation was not essential for intracellular transport of the lectin precursors: unglycosylated precursor synthesized in the presence of tunicamycin gave rise to unglycosylated lectin subunits in the protein bodies.     

Fluorescence-polarization studies on binding of 4-methylumbelliferyl beta-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin.

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.     

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.     

Ricin and Ricinus communis agglutinin subunits are all derived from a single-size polypeptide precursor

Antibodies have been raised in rabbits against the individually purified A and B subunits of the toxic castor bean lectin, ricin, and against the A' and B' subunits of Ricinus communis agglutinin type I. Each of the antisera recognised a single polypeptide species of Mr 60 500 when maturing castor bean endosperm mRNA was translated in vitro in a rabbit-reticulocyte-derived system. When dog pancreatic microsomal vesicles were included in the translational system, each subunit antiserum precipitated a group of 66 000-68 000-Mr core-glycosylated polypeptides which had been translocated into the lumen of the vesicles. The 60 500-Mr polypeptide appeared to be a common precursor to all four individual lectin subunits since (a) its glycosylated (66 000-68 000-Mr) forms were readily detected in the endoplasmic reticulum fraction isolated from maturing castor bean endosperm and (b) pulse-chase studies showed that the glycosylated precursors disappeared from the endoplasmic reticulum fraction with the concomittant appearance of authentic lectin subunits in a soluble protein fraction which included protein body matrix components. Antiserum prepared against whole R. communis agglutinin, type I, also precipitated the 65 000-Mr precursor in vitro and in vivo, but in addition precipitated a non-glycosylated 34 000-Mr polypeptide. This smaller protein is not a lectin subunit precursor, contradicting an earlier suggestion. It is most probably a precursor to the 2-S albumin storage proteins found in castor bean endosperm protein bodies.