Glycobiology Advance Access originally published online on June 14, 2006
Glycobiology 2006 16(10):926-937; doi:10.1093/glycob/cwl017
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AB-type lectin (toxin/agglutinin) from mistletoe: differences in affinity of the two galactoside-binding Trp/Tyr-sites and regulation of their functionality by monomer/dimer equilibrium
2 Instituto de Química Física Rocasolano, CSIC, Serrano 119, E-28006 Madrid, Spain; and 3 Institut für Physiologische Chemie, Tierärztliche Fakultät, Ludwig-Maximilians-Universität, Veterinärstrasse 13, D-80539 München, Germany
1 To whom correspondence should be addressed; e-mail: d.solis{at}iqfr.csic.es
Received on February 8, 2006; revised on June 5, 2006; accepted on June 12, 2006
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
|---|
Viscumin of mistletoe (Viscum album L.) has a concentration-dependent activity profile unique to plant AB-toxins. It starts with lectin-dependent mitogenicity and then covers toxicity and cell agglutination, associated with shifts in the monomer/dimer equilibrium. Each lectin subunit harbors two sections for ligand contact. In the dimer, the B-chain sites in subdomain 2
(designated as the Tyr-sites) appear fully accessible, whereas Trp-sites in subdomain 1
are close to the dimer interface. It is unclear whether both types of sites operate similarly in binding glycoligands in solution. By systematically covering a broad range of lactose/lectin ratio in isothermal titration calorimetry, we obtained evidence for two sites showing dissimilar binding affinity. Intriguingly, the site with higher affinity was only partially occupied. To assign the observed properties to the Trp/Tyr-sites, we next performed chemically induced dynamic nuclear polarization measurements of Trp and Tyr accessibility. A Tyr signal, but not distinct Trp peaks, was recorded when testing the dimer. Lactose-quenchable Trp peaks became visible on the destabilization of the dimer by citraconylation, intimating Trp involvement in ligand contact in the monomer. Fittingly, Tyr acetylation but not mild Trp oxidation reduced the dimer hemagglutination activity and the extent of binding to asialofetuin–Sepharose 4B. Altogether, the results attribute lectin activity in the dimer primarily to Tyr-sites. Full access to Trp-sites is gained on dimer dissociation. Thus, the monomer/dimer equilibrium of viscumin regulates the operativity of these sites. Their structural divergence affords the possibility for differences in ligand selection when comparing monomers (Tyr- and Trp-sites) with dimers (primarily Tyr-sites). Key words: agglutinin / glycans / lectin / mistletoe / ribosome-inactivating protein
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
|---|
Cell surfaces are equipped with biochemical signals by the spatially prominent presentation of carbohydrate chains of cellular glycoconjugates. These membrane glycans can interact with endogenous lectins, but they also serve as docking sites for pathogens and bacterial or plant toxins (Solís et al., 2001
; Gabius et al., 2004; Villalobo et al., 2006). An example for the latter case is the family of the AB-type (or type II) ribosome-inactivating proteins (RIPs) (Rüdiger and Gabius, 2001; Hartley and Lord, 2004; Stirpe, 2004). Typically, the A-chain is an rRNA N-glycosidase that starts its deadly action on eukaryotic ribosomes after the toxin contact formation to cells via the lectin activity of the B-subunit and subsequent internalization (Endo, 1989). In monomeric ricin, the best-studied model case, the binding to surface galactosides engages the B-chain subdomains 1 and 2 that share central positioning of an aromatic ring, that is, Trp-37 and Tyr-248 (Robertus and Monzingo, 2004). A mutation in the 2 high-affinity site by introducing a phenylalanine or a histidine instead of the Tyr residue is observed in the nontoxic RIPs ebulin (from Sambucus ebulus L.) and Ricinus communis agglutinin (RCA), respectively, raising the question of whether such a substitution in the lectin domain may be responsible for the loss of toxicity (Hatakeyama et al., 1986; Pascal et al., 2001). In this respect, viscumin, the galactoside-specific AB-type RIP from mistletoe (Viscum album L.), poses a different question.
As shown in Figure 1, its two carbohydrate-binding sites are characterized by Trp-38 at subdomain 1 (hereafter referred to as the Trp-site) and Tyr-249 at subdomain 2 (hereafter referred to as the Tyr-site) (Sweeney et al., 1998; Krauspenhaar et al., 1999; Niwa et al., 2003; Mikeska et al., 2005). An inactive third site at subdomain 1ß lacks the ability of sugar binding because of the replacement of key polar side chains by rather hydrophobic residues (Mikeska et al., 2005). In contrast to ricin, viscumin can form noncovalent [AB]2 homodimers already at submicrogram-per-mL concentrations via contacts at subdomain 1 of adjacent B-chains (Figure 1) (Olsnes et al., 1982; Sweeney et al., 1998; Krauspenhaar et al., 1999; Niwa et al., 2003; Jiménez et al., 2005). Focusing on sugar binding, it is unclear whether and how the two sites may cooperate in binding glycoligands depending on the quaternary structure, although the positioning of the Tyr-sites separated by 87 Å on one face of the dimer and the Trp-sites only 15 Å apart close to the interface (Niwa et al., 2003) might explain its remarkable sensitivity to the topological aspects of ligand display. This property was monitored with glycoclusters and N-glycans substituted by bisecting GlcNAc or core fucosylation (Unverzagt et al., 2002; André et al., 2003, André, Kaltner, et al., 2004, André, Unverzagt, et al., 2004). In line with the mentioned quaternary structure, the biological activity profile, too, shows a unique dependence on concentration: viscumin is mitogenic for immune and tumor cells in vitro and in vivo at low concentrations (ng mL–1; ng [kg body weight]–1), toxic when increasing the concentration and then also a veritable agglutinin and signal elicitor (Olsnes et al., 1982; Hajto et al., 1990; Timoshenko et al., 1999, 2001; Kunze et al., 2000; Gabius et al., 2001; Jiménez et al., 2005). Thus, the occurrence of a dynamic monomer/dimer equilibrium appears to enable viscumin to cover the functional spectrum of the AB-type monomeric/dimeric protein pairs present in other plants, for example, in R. communis, where a strictly dimeric agglutinin (RCA) is synthesized in addition to the monomeric toxin ricin (Jiménez et al., 2005).
|
In this study we have thus first explored the functionality of the carbohydrate-binding sites in dimeric viscumin. The thermodynamic parameters for the binding of lactose to the viscumin dimer have been evaluated calorimetrically covering a wide range of ligand/lectin ratio, hereby moving beyond the scope of a previous study (Bharadwaj et al., 1999). The two sites could be separated not only by the level of occupancy but also by a noticeable difference in binding affinity. To attribute ligand association to either the Trp- or the Tyr-sites, we strategically applied a nuclear magnetic resonance (NMR)-spectroscopic method designed to probe surface accessibility of Trp/Tyr residues in proteins—that is, the laser photo chemically induced dynamic nuclear polarization (CIDNP) approach (Kaptein et al., 1978)—and determined the effect of lactose presence on the Trp and/or Tyr signals. To analyze the monomer functionality, we exploited chemical modification with N-acetylimidazole or citraconic anhydride that led to monomerization as previously reported (Jiménez et al., 2005). Taking advantage of this destabilization of the dimer, the laser photo CIDNP spectra of native and citraconylated viscumin could then be compared to pinpoint a dependence of Trp-site accessibility on the extent of monomerization. In addition, the role of the Trp- and Tyr-sites in binding to glycoproteins and erythrocyte surfaces was probed by target-selective amino acid modification. The results reveal that the functionality of the different sites is regulated by the monomer/dimer equilibrium. Together with the distinct thermodynamics, we therefore delineate a new structural aspect of this astute strategy to combine toxin/agglutinin activities in one AB-type protein and propose its relevance for switching glycan selection.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
|---|
Thermodynamics of lactose binding to viscumin
The structural nonidentity of the two sites per B-chain in the crystal structure of the viscumin–galactose/lactose complexes (Niwa et al., 2003
; Mikeska et al., 2005) engenders interest in determining occupancy with lactose and the characteristics of binding in solution. As a means to evaluate both issues—that is, stoichiometry and thermodynamic properties—we used isothermal titration calorimetry (ITC). Because the two sites per monomer may differ in their affinity for lactose, it was essential to cover a broad ligand/lectin ratio. Toward this aim, a series of measurements were carried out using different viscumin concentrations, from 3 to 30 mg mL–1 (B-chain concentration 50–500 µM), at which the protein definitely occurs as a dimer (Jiménez et al., 2005). Representative titration profiles are shown in Figure 2. None of the results from the individual titrations could be fitted to a one-set-of-site model. The best fit of experimental data was always obtained using a two-site model, consistently providing evidence for the presence of one set of sites for lactose per B-chain with an association constant in the order of (0.8–1.3) x 103 M–1 at 25°C and a second site showing increased affinity for lactose (Ka around [1.1–2.2] x 104 M–1) but only partial occupancy. At this point, it is necessary to take into account that the shape of the binding isotherm is determined by the unitless parameter c, which depends on the total protein concentration in the cell, the site stoichiometry (n), and Ka (c = [viscumin]tot x n x Ka), shifting from rectangular curves for large c values (>5000) to nearly horizontal traces for low c values (
0.1). Only for c values in the range 1 
c 1000, the shape is reasonably sensitive to changes in binding affinity, and the isotherms can be deconvoluted to obtain accurate association constants (Wiseman et al., 1989). Accordingly, for n = 1, protein concentrations above 0.1 and 1 mM should be employed for Ka values of 1 x 104 and 1 x 103 M–1, respectively. Unfortunately, attempts to obtain clear viscumin solutions at concentrations >30 mg mL–1 (B-chain concentration 0.5 mM) were unsuccessful, precluding using more appropriate experimental conditions for a precise determination of the 103 M–1 range constant. However, measurements at viscumin concentrations near the maximum defined by the natural solubility level are suitable for accessing of both the stoichiometry and association constant of the high-affinity site. Accordingly, these parameters could be reasonably estimated by considering those values that simultaneously result in the best fit to datasets from two different titrations at viscumin concentrations of 0.38 and 0.5 mM. Again, the best fit of experimental data was obtained using a two-site model, yielding values for the high-affinity site of 0.43 ± 0.01 for n and (1.6 ± 0.1) x 104 M–1 for Ka. The particular stoichiometry for binding is in principle compatible with an average of one lactose molecule bound to this type of site per viscumin dimer. In addition, data deconvolution came up with a value of (1.10 ± 0.05) x 103 M–1 for the association constant of the low-affinity set of sites, comparable with that found in the previous microcalorimetric analysis (Bharadwaj et al., 1999), and estimated enthalpy values for each set of sites were 50 kJ mol–1. Although these values must be considered approximate, due to the inherent limitations imposed by the conditions used for data acquisition and analysis, it was evident that the binding was enthalpically driven, consistent with van der Waals contacts and hydrogen bonds to establish main forces stabilizing the complexes. Also, it exhibited enthalpy–entropy compensation, as typically observed for lectin–sugar interactions.
|
The previously demonstrated destabilization of the viscumin dimer by citraconylation (Jiménez et al., 2005) could in principle have offered the opportunity to explore the properties of lactose binding in the monomer. Unfortunately, the solubility of the protein decreased noticeably on chemical modification, the highest concentration achieved being just about 70 µM, thus precluding being able to satisfy the concentration requirements for reliable ITC measurements. Having herewith detected differences in the level of occupancy and affinity of the two types of sites per B chain, the ensuing question on the structural assignments for these two sites was addressed spectroscopically. The presence of the aromatic ring of either a Trp or a Tyr residue in the sites as key determinant for stacking with the galactose moiety prompted these experiments. A pilot survey of the effect of lactose binding on the intrinsic fluorescence of viscumin on excitation at 295 nm (Trp fluorescence) yet showed no significant changes on saturation of either the native protein or its citraconylated derivative. This result is not at all surprising considering that the contribution of Trp-38 to the total fluorescence activity of viscumin may be small because of the large number of Trp residues present in the molecule (11 residues). Also the inspection of the circular dichroism behavior showed no changes in the near-UV spectrum of viscumin on saturation with lactose. However, lactose binding to the citraconylated lectin induced a small but significant increase (30 grad·cm2 dmol–1) in negative ellipticity at 290–291 nm (data not shown). This finding was taken as a hint for the involvement of a Trp residue in sugar binding in the viscumin monomer. It warranted further experiments to assign ligand association to the Trp/Tyr-sites, using a more sophisticated method. The laser photo CIDNP technique that determines ligand vicinity to aromatic rings in proteins was therefore recruited toward this end.
Analysis of the involvement of tryptophan and tyrosine side chains in lactose binding by laser photo CIDNP
The laser photo CIDNP approach was used as powerful measure for lactose-dependent changes in tryptophan and/or tyrosine accessibility. At the standard concentration of the laser-reactive dye flavin I mononucleotide (5 mM), and even after stepwise increases of the dye concentration up to 15 mM to avoid missing a signal with the flavin sensor, no distinct Trp peaks could be reliably discerned for dimeric viscumin but a signal attributable to reactive tyrosine side chain(s) was observed (Figure 3A). The formation of protein aggregates had a negative impact on the signal-to-noise ratio that prompted the increase in dye concentration. Should a Tyr residue be involved in ligand binding, the addition of lactose will reduce the signal. The situation, however, is confounded by the presence of five Tyr residues with the areas of surface accessibility between 52 and 126 Å already in the B-chain. We therefore had to adapt the laser power to enable discrimination among the contributing residues. Indeed, under selective conditions, the Tyr-dependent signal was visibly quenched in the presence of lactose (Figure 3B). To explore the response of Tyr and Trp residues to lactose presence in the monomer, we assayed the citraconylated lectin. When examining the monomer at the standard dye concentration (5 mM), the signal for Tyr and, importantly, also the three characteristic Trp peaks (
1, 
3, and 
2) were visible (Figure 3C). Based on surface accessibility data, these signals were assigned to Trp-38, which reaches the largest extent of solvent exposure (72 Å2). The accessible surface area of the other eight Trp residues in the B-chain ranged from 0 to 35 Å, far below a reasonable value for a significant contribution to a CIDNP signal. Trp-38 side chain thus becomes fully reactive to the flavin I mononucleotide dye after the dissociation of the dimer. Evidently, this result implies that its strategic presentation to the solvent in the monomer will also facilitate the binding of sugar ligands. To provide evidence for the involvement of Trp-38 in ligand binding in solution, we carried out a series of experiments in the presence of lactose. Indicative of contact, the sizes of the Trp signals were drastically reduced (Figure 3D). The same happened to the Tyr signal, as likewise has been seen for the dimer. Thus, Trp is definitely shown to be a contact site in the monomer. To add an independent line of evidence for a different operativity of the Trp-sites in dimers and monomers, we pursued the indications that chemical modification of Trp/Tyr affects ligand-binding activity (Gabius et al., 1992) systematically. These experiments used group-specific reagents and were performed with dimers/monomers without/with lactose.
|
Effect of group-specific chemical modification on binding of viscumin to asialofetuin–Sepharose 4B
The impact of lactose presence on the Tyr/Trp-dependent laser photo CIDNP signals and of dimer dissociation on Trp reactivity with dye pointed to an influence of the quaternary structure on the accessibility of the Trp-sites. A binding assay should engage a multivalent ligand to probe for spatial vicinity of operative sites to spot differences. Affinity chromatography on immobilized bovine asialofetuin—a glycoprotein containing both three N- and three O-glycosidically linked carbohydrate chains, which has been amply proven to be an effective ligand for the lectin especially because of its type I-branched triantennary N-glycans (Gupta et al., 1996; Dettmann et al., 2000; André et al., 2006)—was used for this purpose. As expected, the dimeric viscumin was completely retained on an asialofetuin–Sepharose 4B resin in the control experiment and could be specifically eluted by addition of 0.1 M lactose (Figure 4). With this setup, we proceeded to analyze the effect of chemical modification on lectin activity, starting with N-bromosuccinimide treatment. Controlled oxidation of the dimer in the absence of lactose of up to 3.7 tryptophan residues per B-chain had no significant effect on the chromatographic behavior of viscumin, the total amount of modified protein being retained on the column (Table I). To enable to reach a conclusion on the role of Trp-38, it was essential to prove that this residue was subject to modification. The oxidation of Trp-38 under these conditions was confirmed by mass spectrometry of the tryptic digests of viscumin (Figure 5). In detail, the signal intensity of the unmodified peptide B(26–41) (DDDFHDGNQIQLWPSK, monoisotopic mass 1914.86 Da) decreased 40% on oxidation of one Trp residue per monomer and was only 20% compared with the native protein when four Trp residues were modified. Therefore, the integrity of Trp-38 is evidently not essential for binding of the dimer to immobilized asialofetuin. Under these mild oxidation conditions, the presence of lactose resulted in the protection of 0.65 ± 0.15 groups from modification (Table I). To exclude a nonspecific effect, we tested an inert carbohydrate at the same concentration as control. The presence of 0.1 M glucose during the modification yielded no protection, demonstrating that the effect of lactose was specific. The observed partial protection may be accounted for by an average of one Trp residue per viscumin dimer being protected by lactose. The result that one lactose molecule bound to the Trp-sites per viscumin dimer is in full accordance with the ITC-derived stoichiometry for the high-affinity site. Further oxidation of most likely less accessible tryptophan residues at increased N-bromosuccinimide concentrations resulted in a notable decrease in binding activity (Table I), confirming the previously reported data on solid-phase assays of the lectin binding activity (Gabius et al., 1992) and excluding an influence of the different experimental designs on the result. The presence of lactose during the modification did not protect from the loss of activity under these conditions, indicating that a conformational change of the protein rather than the oxidation of the Trp residue directly contacting the ligand is responsible for the observed behavior at this level of oxidation. Having herewith studied the effect of Trp modification under different conditions, we next turned to testing reagents with Tyr specificity.
|
|
|
The iodination of Tyr residues did not reduce binding to asialofetuin–Sepharose 4B. The presence of two di-iodinated Tyr moieties was deduced from measurements of absorbance at 313 nm (see Materials and methods), and peptide mass mapping data confirmed that at least three of them were mono- or di-iodinated. However, peptide B(246–263) was not visible in the mass spectra of the tryptic digest of native viscumin, excluding the possibility of checking the modification of Tyr-249 by analysing the peptide mass fingerprint. We next tested acetylation with N-acetylimidazole, which markedly impaired the lectin activity (Table I). When a 1000-fold molar excess of reagent was used, 50% of the protein was not retained on the column (Figure 4). Under these conditions, 10 of the 17 Tyr residues present per viscumin monomer were modified. Although, as reported above, the modification of Tyr-249 could not be monitored by mass spectrometry, the derivatization of this Tyr residue, one of the five most exposed Tyr residues with a surface accessibility value area of 85 Å2, appears highly likely. The presence of lactose precluding modification only at the contact site should prevent the loss of activity to support this notion. Experimentally, binding activity was preserved when the modification was carried out in the presence of lactose. At this point, it is essential to recall that acetylation with N-acetylimidazole, similar to citraconylation, promotes the dissociation of viscumin dimers into monomers (Jiménez et al., 2005), with the concomitant increase in the accessibility of the Trp-sites as evidenced by the laser photo CIDNP studies. Thus, the involvement of these sites in the binding of viscumin monomers to immobilized asialofetuin may account for the observed partial retention of the Tyr-acetylated protein on the column. To complete systematic monitoring of effects of this chemical modification, we used high reagent concentration. Extensive acetylation using a 10,000-fold molar excess of reagent resulted in a further deterioration of binding activity (Figure 4). It should be noted that, in addition to Tyr residues, primary amino groups could also be acetylated via side reactions, and thus, the acetylation of lysine residues in the binding site could be responsible for the additional loss of binding. Indeed, the side chain of Lys-41 in the Trp-site appears to participate in sugar binding in the crystal (Niwa et al., 2003). Supporting our line of reasoning, the presence of lactose during the modification safeguarded 90% of the binding activity. To be able to separate effects on Tyr residues/amino groups, we consequently proceeded to analyze deliberate modification of amino groups. The selective citraconylation of amino groups resulted in a 50% decrease in binding activity (Table I), and lactose clearly protected one group from modification. The citraconylation reaction also had some deleterious effects on the protein so that the carbohydrate-binding activity was only completely protected by the presence of the ligand when mild modification conditions were used. To complement this interaction study with the glycoprotein as ligand and retention on the resin as parameter, we also studied the potency of chemically modified viscumin as agglutinin.
Effect of group-specific chemical modification on hemagglutination
Hemagglutination assays determine the capacity of the chemically modified lectin as cross-linker for cells. Mild tryptophan oxidation, which did not affect resin binding, had no effect on the agglutination of human erythrocytes (Table I), the modified protein exhibiting the same hemagglutination titer as the native one (12.5 µg mL–1). This behavior points to the Tyr-sites being mainly responsible for the observed activity, and, indeed, extensive acetylation with N-acetylimidazole reduced the activity of viscumin as agglutinin 40-fold (Table I). The presence of lactose during the modification prevented the loss of activity, the concentration of viscumin required to bridge erythrocytes being only 2- to 5-fold increased after derivatization. As noted when monitoring resin binding, citraconylation was also effective to impair binding activity, the titer increasing up to 165 µg mL–1. Again, lactose prevented the loss of activity. Considering that citraconylation and acetylation reduce the integrity of the viscumin dimer (Jiménez et al., 2005), our data on hemagglutination imply cooperation between the two types of carbohydrate-binding site present per B-chain—that is, the Tyr- and the Trp-sites—in erythrocyte cross-linking.
Altogether, the results attribute lectin activity in the dimer primarily to Tyr-sites, whereas the full access of glycoligands to Trp-sites is only gained on dimer dissociation. When considering the evidence from crystallographic studies, this concept is apparently in conflict with the observation that saccharide binding to both the Tyr- and the Trp-sites is observed in the crystal structure of viscumin in complex with lactose. Of note, buffer conditions were conspicuously different in our experiments and in the crystallographic studies. Assuming that this increased accessibility to the Trp-sites in the crystal could result from the conditions used to favor crystal growth, we investigated the stability of the viscumin dimer in that buffer system, namely 0.2 M glycine–HCl, pH 2.5 (Mikeska et al., 2005).
Gel filtration behavior of viscumin at pH 2.5
The issue on a possible impact of the crystallization conditions on the concentration-dependent monomer/dimer equilibrium of viscumin (Jiménez et al., 2005) was addressed by gel filtration on a Superose 12 column equilibrated with the mentioned buffer system (see Materials and methods). The elution time of viscumin at concentrations of 3–10 mg mL–1 was 26.1 ± 0.1 min, somewhat higher than that observed for the dimeric RCA (24.6 ± 0.2 min) (Figure 6). The analysis of increasingly diluted solutions, 1–0.01 mg mL–1, showed a progressive shift of the peak maxima up to 28.5 ± 0.1 min (Figure 6, inset). This position approaches the elution time observed for monomeric ricin (29.5 ± 0.1 min). Thus, the results are in line with the dissociation of viscumin dimers into monomers at the tested protein concentrations. Assuming a typical dilution factor of 10, as estimated from the ratio of width at half-height of the peak to the injection volume, dissociation became detectable at eluted concentrations <100 µg mL–1. This concentration is >30-fold higher than that found at neutral pH (3 µg mL–1) (Jiménez et al., 2005), evidencing a clear shift in the monomer/dimer equilibrium to the monomer and suggesting a weakening of the monomer–monomer interactions in the dimer interface at pH 2.5.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
|---|
Viscumin is special among plant AB-type toxins because of its concentration-dependent quaternary structure and activity. It is mitogenic at very low concentrations on lymphocytes and tumor cells, can act as potent toxin as monomer, and avidly binds to cells as dimer with maintained toxicity (Gabius et al., 2001
; Jiménez et al., 2005). Hereby, it covers the functional spectrum of pairs of separate monomeric/dimeric AB-type proteins found in other plants. The results reported here reveal that the functionalities of the carbohydrate-binding sites of the toxin are also orchestrated by the elegant monomer/dimer equilibrium strategy embodied by viscumin to combine toxin/agglutinin activities in one protein.
Viscumin poses the intriguing question on how it can act as potent cross-linking protein for eliciting biosignaling and cell aggregation. Structurally, two binding sites per monomer are observed in the crystal structure, which are assumedly operative in solution, whereas a potential third binding site was found to lack the ability of sugar binding because of detrimental amino acid substitutions at key positions (Mikeska et al., 2005). Precipitation analysis and a previous microcalorimetric study had proven at least monovalency for viscumin B-chain (Gupta et al., 1996; Bharadwaj et al., 1999). The cross-linking of cell glycans and the importance of spatial parameters for ligand properties are in line with our results on the dimer affinity level and glycan contact to at least one site per monomer (Gupta et al., 1996; André et al., 1999, 2001; Dettmann et al., 2000; Gabius, 2001). By deliberately covering a broad ligand/lectin ratio, sugar binding to a second set of sites is also detected in solution. Of note, major contact sites engage Trp or Tyr for stacking, and our thermodynamic data thus characterize binding to two sites different from each other in the nature of the aromatic ring. Structurally, the Tyr-sites in the viscumin dimer have no obvious impediments to the binding of lactose, whereas the Trp-sites are located only 15 Å apart from each other and are close to the interface (Figure 1). Inevitably, dimerization may impose steric restrictions on the binding to these sites, accounting for partial lactose occupancy. The weakening of monomer–monomer interactions at pH 2.5, suggested by the marked shift to the monomer in the concentration-dependent monomer/dimer equilibrium of viscumin, should undoubtedly facilitate lactose binding to the Trp-sites, as observed in the X-ray crystal structures. Indeed, laser photo CIDNP measurements on native dimeric and chemically monomerized viscumin demonstrated that the accessibility of the Trp-38 residue in solution, the central residue of this site, to the flavin I mononucleotide dye is significantly improved in the viscumin monomer as compared with the dimer.
To relate these measurements to the physiological situation, it is essential to keep in mind that cellular glycoconjugates are generally extended beyond the disaccharide structure. It thus appears to be reasonable to presume that binding to the Trp-sites of ligands larger than lactose may encounter at least similar or even greater steric constraints as a result of dimerization. Although the accommodation of glycoprotein and/or glycolipid ligands on cell surfaces by these sites cannot be categorically excluded, the results reported here argue in favor of the notion that the carbohydrate-binding profile, commonly determined for the dimer (Lee et al., 1992; Wu et al., 1992; Galanina et al., 1997), can be attributed to the Tyr-sites. Of note, the Trp-sites become fully operative on dissociation into monomers, as evidenced by the lectin hemagglutinating activity after chemical monomerization by either acetylation with N-acetylimidazole or citraconylation under conditions protecting the lectin activity (Jiménez et al., 2005). Because natural dissociation into monomers takes place at viscumin concentrations in the submicrogram-per-mL level (Jiménez et al., 2005), the Trp-site is then expected to be functional in binding to cell surfaces. Its glycan selection in cooperation with the Tyr-site is operative when first mitogenic effects are triggered. Thus, the formation of viscumin dimers could be considered a mechanism to regulate access to the Trp-site and enable separate functions.
Looking at the two sites, viscumin presents an attractive natural model to compare the efficiency of Trp/Tyr as key residue for hydrophobic contact to B-face of galactose. According to the ITC data, the Trp-sites show about 15-fold higher affinity for lactose (Ka = 1.6 x 104 M–1) than the Tyr-sites. This binding affinity is in the range of that calculated from inhibition studies (Rivera-Sagredo et al., 1992; Solís et al., 1993) for the homologous agglutinin from R. communis (Ka = 1.8 x 104 M–1). Here, the Trp-sites are supposed to be the only functional carbohydrate-binding sites because of the mutation of Tyr-248 to His in the relevant region at domain 2 (Roberts et al., 1985; Hatakeyama et al., 1986). Looking at the two sites of viscumin in more detail, the crystal structure of the viscumin–lactose complex provides a possible structural basis for favored binding to the Trp-sites (Mikeska et al., 2005). Protein–lactose interactions common to the Tyr- and Trp-sites involve hydrogen bonds with the hydroxyl groups at positions 3' and 4' of the galactose moiety, as predicted by chemical mapping with ligand derivatives (Rüdiger et al., 2000), and a stacking interaction with the aromatic ring—its size being different—in the binding site. As characteristic of the Trp-site, the 2' hydroxyl group also stabilizes the sugar–protein complex, whereas no interactions of this particular group are visible at the Tyr-site (Mikeska et al., 2005). The glucose moiety is invariably exposed to the solvent at both sites, making no contacts with the protein. As a note of caution, it should be mentioned that the information from this structure obtained at acidic pH may not completely define the protein structure at neutral conditions. In fact, the observed decrease in dimer stability underscores operativity of pH-dependent processes in this special case. Thus, it may also be possible that the enhanced affinity for lactose in the Trp-site could also derive from additional contacts with the glucose unit, plausibly involving Asp-26 as presumed from an inspection of the crystal structure, whereas such contacts are not possible in the Tyr-site. The entropic side of the binding process might also have a distinctive bearing on the binding thermodynamics. The recent observations on ligand binding to hevein and pseudohevein equipped with Trp and Tyr residues, respectively, for stacking with the sugar (Asensio et al., 2000) intimate the idea that restrictions in the mobility of a rather flexible Tyr side chain on ligand stacking may contribute to an increased entropic penalty associated with binding to the Tyr-site compared with that to the Trp-site. Indeed, although the estimation of the enthalpy and entropy contribution to the binding from the present ITC data is admittedly only approximate, the best-fit parameters were consistently in line with less unfavorable entropy changes at the high-affinity site. Moreover, the case study of ebulin 1, with its Tyr/Phe substitution in subdomain 2 and the ensuing impairment of cell binding by a subtle change in the mode the galactose moiety is accommodated, underscores how much placement of the aromatic residue matters for lectin activity (Pascal et al., 2001). This aspect warrants to briefly adding information about bound-state conformation of ligands.
Considering the ligand structure modeling, its conformation within the global minimum is not distorted by binding but the flexibility of the penultimate sugar unit is subject to regulation (Gilleron et al., 1998; Alonso-Plaza et al., 2001; Fernández-Alonso et al., 2004). On this level, the remarkably broad specificity for galactosides irrespective of anomeric linkage and 2,6-sialylation explains the activity of viscumin as a broad-spectrum toxin accommodating ligands with core galactose and tolerating structural extensions (Wu et al., 1992; Lee et al., 1994; Bharadwaj et al., 1999). At this point, it is important to draw attention to the fact that none of the prior specificity studies had considered the existence of two distinct sets for carbohydrate-binding sites at different conditions, a fertile field for future studies using monomers and dimers in direct comparison. Cooperations between Trp-38 and Tyr-249 sites in the monomer and between Tyr-249/Tyr-249 sites in the dimer, the two constellations differing in spatial vicinity of sites and structure, can engender contact to different glycan populations. Eliciting mitogenicity can thus depend on distinct binding (and also cross-linking) to certain target ligands. In this context, the change of quaternary structure can also be considered as an attractive means to modulate activities for human growth-regulatory lectins (Gabius, 1997; André et al., 2005).
The dynamic monomer/dimer equilibrium for viscumin appears to achieve the same objectives as the development of two separate protein entities in R. communis, a monomeric toxin and cell-cross-linking dimeric agglutinin. The viscumin dimer harbors several contacts between domains 1 of adjacent B-chains, including the hairpin loop of subdomain 1. In ricin and the agglutinin, this loop is stabilized by a disulfide bridge, whereas the substitution of one of these cysteines involved in this bridge, Cys-40, to serine in viscumin endows the loop with increased flexibility. This parameter has been suggested to be important for dimerization, although the relative contributions of the various interactions established at the interface to dimer stability have not yet been rigorously evaluated (Sweeney et al., 1998; Krauspenhaar et al., 1999; Niwa et al., 2003; Mikeska et al., 2005). Evidently, the Ricinus agglutinin would be fatally harmed by such a dimerization, because its Trp-sites would then be positioned at the interface. Alternatively, the dimerization of RCA occurs through the A-chains, and the resulting dimer is covalently stabilized by a disulfide bridge between adjacent A-chains (Sweeney et al., 1997). As a consequence, the distance between the two Trp-sites is about 100 Å, remarkably similar to that between the Tyr-sites in the viscumin dimer. Different patterns are thus adopted by the two proteins to accomplish the ultimate objective of effectively bridging cell ligands. On the contrary, although the agglutinin from Ricinus is weakly cytotoxic despite its rRNA N-glycosidase activity in a cell-free system (Citores et al., 1993), viscumin is a strong cytotoxin, both as monomer like ricin and as dimer (Olsnes et al., 1982; Jiménez et al., 2005). This would imply that mere binding to cell surface oligosaccharides through the Trp-sites is neither sufficient nor even necessary for yielding a toxic effect. Consequently, the occurrence of a concentration-dependent monomer/dimer equilibrium of viscumin is suggested as a means to ensure homing of monomers with two different, fully functional carbohydrate-binding sites to distinct target site of action. Therefore, it is tempting to speculate that the two sites and their positioning exert distinct roles in the biological activity of this and other AB-type RIPs.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
|---|
Isolation of viscumin
The galactoside-specific lectin was purified from the supernatants of the extracts of dried mistletoe leaves by affinity chromatography on lactosylated Sepharose 4B, obtained by divinyl sulfone activation, as crucial step, and analyzed for purity and activity by 1D and 2D gel electrophoresis, an enzyme-linked lectin-binding assay, cell staining, and toxicity as well as hemagglutination (Gabius, 1990
; Gabius et al., 1992, 2001; André et al., 1999; Manning et al., 2004). The protein concentration was determined by the Lowry assay using concanavalin A (Sigma, St. Louis, MO) as standard (Jiménez et al., 2000).
Chemical modifications
The reactions were carried out in the absence or presence of 0.1 M lactose to protect the carbohydrate-binding sites from modification. Tryptophan residues were oxidized by successive addition of 5-µL aliquots of a solution containing 10 mM N-bromosuccinimide to 1 mL solution of viscumin (0.5 mg mL–1) in 0.2 M sodium acetate buffer, pH 4.0 (Sandvig et al., 1978). The decrease in absorbance at 280 nm was monitored after each addition, and the number of modified tryptophan side chains was calculated from this decrease by using an equation given by Spande and Witkop (1967). Tyrosine iodination was carried out in 0.1 M sodium phosphate buffer, pH 7.0, with 0.05 N resublimed iodine in 0.1 M KI, in a proportion of 11 µL per mg of protein (Ishiguro et al., 1977). The amount of di-iodotyrosine was determined by measuring its characteristic absorbance at 313 nm at pH 10, assuming an 313 of 5815 M–1 cm–1 (Ishiguro et al., 1977). The acetylation of tyrosine moieties with N-acetylimidazole was carried out in 5 mM sodium phosphate, pH 7.2, containing 0.2 M NaCl (phosphate-buffered saline [PBS]), using a 2- to 20-fold excess (w/w) of the reagent (Gabius et al., 1992). The number of modified residues was calculated from the decrease in absorbance at 275 nm on tyrosine O-acetylation, considering a 
275 of 1160 M–1 cm–1 (Riordan et al., 1965). Amino groups were modified in 0.1 M Tris–HCl, pH 8.0, using 1.3–8.4 µL of citraconic anhydride per mg of protein (Gabius et al., 1992). Excess reagents were routinely removed by exhaustive dialysis against PBS at 4°C.
Peptide analysis by mass spectrometry
Viscumin samples were resuspended in 50 mM ammonium bicarbonate (99.5% purity; Sigma) and digested with modified porcine trypsin (sequencing grade; Promega, Madison, WI) at a final concentration of 10 ng µL–1 at room temperature for 2 h. The digestion procedure was stopped with the addition of 0.5% trifluoroacetic acid (99.5% purity; Sigma). An aliquot of the digestion solution was mixed with an aliquot of -cyano-4-hydroxycinnamic acid (Bruker-Daltonics, Bremen, Germany) in 33% aqueous acetonitrile and 0.1% trifluoroacetic acid. This mixture was placed onto a 600-µm AnchorChip MALDI probe (Bruker-Daltonics) and allowed to dry at room temperature. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) data were obtained using an Ultraflex time-of-flight mass spectrometer (Bruker-Daltonics) (Suckau et al., 2003). Spectra were recorded in the positive-ion mode at 50 Hz laser frequency, and 200–400 individual spectra were averaged. The detailed analysis of peptide mass mapping data was performed using flexAnalysis software (Bruker-Daltonics). MALDI-MS spectra were calibrated with spectra obtained for the proton adducts of a peptide mixture covering the 800–3200 m/z region.
Isothermal titration calorimetry
The calorimetric titrations were performed at 25°C with an MCS Microcal titration calorimeter (Microcal Inc., Northampton, MA), as previously described (Wiseman et al., 1989). Samples were exhaustively dialyzed against PBS, and solutions with lactose were prepared using the last dialysis buffer. The aliquots of solution containing the ligand at the indicated concentration were added to protein solutions of concentrations 3–30 mg mL–1, kept in a 1.35-mL cell, in a series of injections by means of a rotating stirrer-syringe. The heat developed by ligand dilution was determined separately and subtracted from the total heat produced following each injection. The titration data were analyzed based on a binding model considering either one or two sets of noninteracting binding sites by a nonlinear least-squares algorithm using the MicroCal Origin software. The monomer concentration of the lectin was used as input in the fitting procedures.
Laser photo CIDNP method
Laser photo CIDNP experiments using a continuous-wave argon ion laser (Spectra Physics, Mountain View, CA) as source of light were performed at 33°C with solutions containing 2 mM lactose and 4 mg mL–1 of viscumin, as described in detail in studies on other lectins previously (Siebert, Adar, et al., 1997; Siebert, von der Lieth, et al., 1997). Briefly, the ion laser operated in the multi-line mode with emission wavelengths of 488.0 and 514.5 nm, close to the edge of the 450-nm absorption band of the added dye. The laser light was directed to the sample by an optical fiber and chopped by a mechanical shutter controlled by the spectrometer. This setup of the equipment prevented harmful heating of the protein-containing solution. The laser photo CIDNP radical reaction was initiated by addition of flavin I mononucleotide (N3 of the isoalloxazine ring substituted with CH2COOH and N10 with CH3) as laser-reactive dye, and the irradiation led to the generation of protein–dye radical pairs involving dye-accessible (and therefore surface-exposed) Tyr, Trp, or His residues (Kaptein et al., 1978; Siebert, Adar, et al., 1997; Siebert, von der Lieth, et al., 1997). The resulting light spectrum was subtracted from a dark spectrum, yielding the CIDNP difference spectrum containing only signals of polarized residues. The tyrosine-dependent CIDNP effect corresponds to a spin-density distribution of the intermediate phenoxy radical with strong negative signals of the 1 and 2 protons and less intense positive signals for the 1 and 2 protons. The CIDNP signals of tryptophan are generated by an intermediate radical with strong spin density at the 1, 3, and 2 positions of the indole unit and very small spin density at the 
2 y 3 positions, which all produce positive CIDNP signals. The surface accessibilities of the side chains that can be reactive toward the dye were calculated using the program Surface Racer 3.0 (http://monte.biochem.wisc.edu/~tsodikov/surface.html) (Tsodikov et al., 2002) and the crystal structure of the viscumin dimer (PDB code 1OQL).
Affinity chromatography on asialofetuin–Sepharose 4B
Asialofetuin (Sigma) was immobilized on cyanogen-bromide-activated Sepharose 4B (Amersham Biosciences, Uppsala, Sweden) following a described protocol (Tercero and Díaz-Mauriño, 1988). Routinely, 100 µg of lectin in PBS was applied to a 2.5-mL column, containing 4.5 mg asialofetuin per mL of resin, equilibrated with the same buffer. The flow rate was 15 mL h–1 and elution monitored at 280 nm using an LKB Uvicord S2 UV detector (LKB Biotechnology, Uppsala, Sweden) and a Shimadzu C-R8A chromatographic integrator (Shimadzu Corporation, Tokyo, Japan). The bound protein was eluted by addition of 0.1 M lactose to the buffer.
Hemagglutination assays
For the evaluation of the hemagglutinating activity, samples of Rh-positive human blood of A or B status, kindly provided by the Haematology Service at the Hospital La Princesa (Madrid, Spain), were used for erythrocyte preparation and testing yielding reproducible results. Blood was diluted 1:6 (v/v) with 5 mM sodium phosphate buffer, pH 7.6, containing 0.15 M NaCl and 10 mM sodium citrate. After centrifugation at 2800 g for 1 min, the sedimented erythrocytes were washed twice with this buffer and another two times with the same buffer without citrate. Finally, the erythrocytes were resuspended in buffer without citrate at a ratio of 1:10 (v/v). Hemagglutination assays were carried out in flat-bottomed polystyrene 96-well microtiter plates (Costar, Cambridge, MA) by incubating 25 µL of the cell suspension with 25 µL of 2-fold serial dilutions of the lectin in PBS for 2 h at 20°C. The hemagglutination titer was defined as the highest dilution still showing visual agglutination of erythrocytes.
Gel filtration
Gel filtration was carried out with a Superose 12 HR 10/30 column (Pharmacia Biotech, Uppsala, Sweden; void volume: 8.3 mL) equilibrated with 0.2 M glycine–HCl buffer, pH 2.5, with 0.02% NaN3, containing 0.1 M lactose to prevent interactions of the lectin with the agarose-based matrix. The flow rate was 0.5 mL min–1, and the elution was monitored at 280 nm. Ricin and the agglutinin RCA, obtained from R. communis seeds (Jardín Botánico, C.S.I.C., Madrid, Spain) (Nicolson et al., 1974), were fractionated as controls under similar conditions.
|
Conflict of interest statement |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
|---|
None declared.
|
Acknowledgments |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
|---|
We thank Dr. Margarita Menéndez for invaluable help with acquisition and critical evaluation of ITC data and L. Emilio Camafeita from the Proteomics Unit of Centro Nacional de Investigaciones Cardiovasculares (Madrid, Spain) for peptide mass analyses. This work was supported by DGICYT (BQU2000-1501-C02-02 and BQU2003-03550-C03-03), an EC Marie Curie Research Training Network grant (contract number MRTN-CT-2005-019561), the Mizutani Foundation for Glycoscience (Tokyo, Japan), and the Verein zur Förderung des biologisch-technologischen Fortschritts in der Medizin. V. (Heidelberg, Germany).
|
Abbreviations |
|---|
CIDNP, chemically induced dynamic nuclear polarization; ITC, isothermal titration calorimetry; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; PBS, phosphate-buffered saline; RCA, Ricinus communis agglutinin; RIP(s), ribosome-inactivating protein(s)
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
|---|
Alonso-Plaza, J.M., Canales, M.A., Jiménez, M., Roldán, J.L., García-Herrero, A., Iturrino, L., Asensio, J.L., Cañada, F.J., Romero, A., Siebert, H.-C., and others (2001) NMR investigations of protein–carbohydrate interactions: insights into the topology of the bound conformation of a lactose isomer and ß-galactosyl xyloses to mistletoe lectin and galectin-1. Biochim. Biophys. Acta, 1568, 225–236.[Medline]
André, S., Cejas Ortega, P.J., Perez, M.A., Roy, R., and Gabius, H.-J. (1999) Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties. Glycobiology, 9, 1253–1261.
André, S., Kaltner, H., Furuike, T., Nishimura, S.-I., and Gabius, H.-J. (2004) Persubstituted cyclodextrin-based glycoclusters as inhibitors of protein–carbohydrate recognition using purified plant and mammalian lectins and wild-type and lectin-gene-transfected tumor cells as targets. Bioconjug. Chem., 15, 87–98.[CrossRef][Web of Science][Medline]
André, S., Kaltner, H., Lensch, M., Russwurm, R., Siebert, H.-C., Fallsehr, C., Tajkhorshid, E., Heck, A.J.R., von Knebel Doeberitz, M., Gabius, H.-J., and Kopitz, J. (2005) Determination of structural and functional overlap/divergence of five proto-type galectins by analysis of the growth-regulatory interaction with ganglioside GM1 in silico and in vitro on human neuroblastoma cells. Int. J. Cancer, 114, 46–57.[CrossRef][Web of Science][Medline]
André, S., Kojima, S., Gundel, G., Russwurm, R., Schratt, X., Unverzagt, C., and Gabius, H.-J. (2006) Branching mode in complex-type triantennary N-glycans as regulatory element of their ligand properties. Biochim. Biophys. Acta, 1760, 768–782.
André, S., Liu, B., Gabius, H.-J., and Roy, R. (2003) First demonstration of differential inhibition of lectin binding by synthetic tri- and tetravalent glycoclusters from cross-coupling of rigidified, 2-propynyl lactoside. Org. Biomol. Chem., 1, 3909–3916.[CrossRef][Web of Science][Medline]
André, S., Pieters, R.J., Vrasidas, I., Kaltner, H., Kuwabara, I., Liu, F.-T., Liskamp, R.M.J., and Gabius, H.-J. (2001) Wedgelike glycodendrimers as inhibitors of binding of mammalian galectins to glycoproteins, lactose maxiclusters, and cell surface glycoconjugates. Chembiochem, 2, 822–830.[CrossRef][Web of Science][Medline]
André, S., Unverzagt, C., Kojima, S., Frank, M., Seifert, J., Fink, C., Kayser, K., von der Lieth, C.-W., and Gabius, H.-J. (2004) Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico, in vitro and in vivo. Eur. J. Biochem., 271, 118–134.[CrossRef]
Asensio, J.L., Siebert, H.-C., von der Lieth, C.-W., Laynez, J., Bruix, M., Soedjanaatmadja, U.M.S., Beintema, J.J., Cañada, F.J., Gabius, H.-J., and Jiménez-Barbero, J. (2000) NMR investigations of protein–carbohydrate interactions: studies on the relevance of Trp/Tyr variations in lectin binding sites as deduced from titration microcalorimetry and NMR studies on hevein domains. Determination of the NMR structure of the complex between pseudohevein and N,N',N''-triacetylchitotriose. Proteins, 40, 218–223.[CrossRef][Web of Science][Medline]
Bharadwaj, S., Kaltner, H., Korchagina, E.Y., Bovin, N.V., Gabius, H.-J., and Surolia, A. (1999) Microcalorimetric indications for ligand binding as a function of the protein for galactoside-specific plant and avian lectins. Biochim. Biophys. Acta, 1472, 191–196.
Citores, L., Ferreras, J.M., Iglesias, R., Carbajales, M.L., Arias, F.J., Jiménez, P., Rojo, M.A., and Girbés, T. (1993) Molecular mechanism of inhibition of mammalian protein synthesis by some four-chain agglutinins. Proposal of an extended classification of plant ribosome-inactivating proteins (rRNA N-glycosidases). FEBS Lett., 329, 59–62.[CrossRef][Web of Science][Medline]
Dettmann, W., Grandbois, M., André, S., Benoit, M., Wehle, A.K., Kaltner, H., Gabius, H.-J., and Gaub, H.E. (2000) Differences in zero-force and force-driven kinetics of ligand dissociation from ß-galactoside-specific proteins (plant and animal lectins, immunoglobulin G) monitored by plasmon resonance and dynamic single molecule force microscopy. Arch. Biochem. Biophys., 383, 157–170.[CrossRef][Web of Science][Medline]
Endo, Y. (1989) Mechanism of action of ricin and related toxic lectins on the inactivation of eukaryotic ribosomes. Adv. Lectin Res., 2, 60–73.
Fernández-Alonso, M.C., Cañada, F.J., Solís, D., Cheng, X., Kumaran, G., André, S., Siebert, H.-C., Mootoo, D.R., Gabius, H.-J., and Jiménez-Barbero, J. (2004) Computational and NMR experimental definition of differences in the conformational behavior of free and lectin-bound glycomimetic aza/carba-lactosides. Eur. J. Org. Chem., 7, 1604–1613.[CrossRef]
Gabius, H.-J. (1990) Influence of type of linkage and spacer on the interaction with ß-galactoside-binding proteins with immobilized affinity ligands. Anal. Biochem., 189, 91–94.[CrossRef][Web of Science][Medline]
Gabius, H.-J. (1997) Animal lectins. Eur. J. Biochem., 243, 543–576.[Web of Science][Medline]
Gabius, H.-J. (2001) Probing the cons and pros of lectin-induced immunomodulation: case studies for the mistletoe lectin and galectin-1. Biochimie, 83, 659–666.[Medline]
Gabius, H.-J., Darro, F., Remmelink, M., André, S., Kopitz, J., Danguy, A., Gabius, S., Salmon, I., and Kiss, R. (2001) Evidence for stimulation of tumor proliferation in cell lines and histotypic cultures by clinically relevant low doses of the galactoside-binding mistletoe lectin, a component of proprietary extracts. Cancer Invest., 19, 114–126.[CrossRef][Web of Science][Medline]
Gabius, H.-J., Siebert, H.-C., André, S., Jiménez-Barbero, J., and Rüdiger, H. (2004) Chemical biology of the sugar code. Chembiochem, 5, 740–764.[CrossRef][Medline]
Gabius, H.-J., Walzel, H., Joshi, S.S., Kruip, J., Kojima, S., Gerke, V., Kratzin, H., and Gabius, S. (1992) The immunomodulatory galactoside-specific lectin from mistletoe: partial sequence analysis, cell and tissue binding, and impact on intracellular biosignalling of monocytic leukemia cells. Anticancer Res., 12, 669–676.[Web of Science][Medline]
Galanina, O.E., Kaltner, H., Khraltsova, L.S., Bovin, N.V., and Gabius, H.-J. (1997) Further refinement of the description of the ligand-binding characteristics for the galactoside-binding mistletoe lectin, a plant agglutinin with immunomodulatory potency. J. Mol. Recognit., 10, 139–147.[CrossRef][Web of Science][Medline]
Gilleron, M., Siebert, H.-C., Kaltner, H., von der Lieth, C.-W., Kozár, T., Halkes, K.M., Korchagina, E.Y., Bovin, N.V., Gabius, H.-J., and Vliegenthart, J.F.G. (1998) Conformer selection and differential restriction of ligand mobility by a plant lectin. Conformational behaviour of Galß1-3GlcNAcß1-R, Galß1-3GalNAcß1-R and Galß1-2Galß1-R' in the free state and complexed with galactoside-specific mistletoe lectin as revealed by random walk and conformational clustering molecular-mechanics calculations, molecular dynamics simulations and nuclear Overhauser experiments. Eur. J. Biochem., 252, 416–427.[Web of Science][Medline]
Gupta, D., Kaltner, H., Dong, X., Gabius, H.-J., and Brewer, C.F. (1996) Comparative cross-linking activities of lactose-specific plant and animal lectins and a natural lactose-binding immunoglobulin G fraction from human serum with asialofetuin. Glycobiology, 6, 843–849.
Hajto, T., Hostanska, K., Frei, K., Rordorf, C., and Gabius, H.-J. (1990) Increased secretion of tumor necrosis factor-, interleukin-1, and interleukin-6 by human mononuclear cells exposed to galactoside-specific lectin from clinically applied mistletoe extract. Cancer Res., 50, 3322–3326.
Hartley, M.R. and Lord, J.M. (2004) Cytotoxic ribosome-inactivating proteins from plants. Biochim. Biophys. Acta, 1701, 1–14.[Medline]
Hatakeyama, T., Yamasaki, N., and Funatsu, G. (1986) Identification of the tryptophan residue located at the low-affinity saccharide binding site of ricin D. J. Biochem., 100, 781–788.
Ishiguro, M., Taira, E., Mashima, K., Funatsu, G., Funatsu, M., and Kikutani, M. (1977) Iodination of ricin. Agric. Biol. Chem., 41, 299–307.
Jiménez, M., André, S., Gabius, H.-J., and Solís, D. (2000) Calibration of colorimetric protein assays for quantitation of plant AB toxins. Anal. Biochem., 284, 418–420.[CrossRef][Web of Science][Medline]
Jiménez, M., Sáiz, J.L., André, S., Gabius, H.-J., and Solís, D. (2005) Monomer/dimer equilibrium of the AB-type lectin from mistletoe enables combination of toxin/agglutinin activities in one protein: analysis of native and citraconylated proteins by ultracentrifugation/gel filtration and cell biological consequences of dimer destabilization. Glycobiology, 15, 1386–1395.
Kaptein, R., Dijkstra, K., and Nicolay, K. (1978) Laser photo-CIDNP as a surface probe for proteins in solution. Nature, 274, 293–294.[CrossRef][Medline]
Krauspenhaar, R., Eschenburg, S., Perbandt, M., Kornilov, V., Konareva, N., Mikailova, I., Stoeva, S., Wacker, R., Maier, T., Singh, T., and others (1999) Crystal structure of mistletoe lectin I from Viscum album. Biochem. Biophys. Res. Commun., 257, 418–424.[CrossRef][Web of Science][Medline]
Kunze, E., Schulz, H., Adamek, M., and Gabius, H.-J. (2000) Long-term administration of galactoside-specific mistletoe lectin in an animal model: no protection against N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced urinary bladder carcinogenesis in rats and no induction of a relevant local cellular immune response. J. Cancer Res. Clin. Oncol., 126, 125–138.[CrossRef][Web of Science][Medline]
Lee, R.T., Gabius, H.-J., and Lee, Y.C. (1992) Ligand binding characteristics of the major mistletoe lectin. J. Biol. Chem., 267, 23722–23727.
Lee, R.T., Gabius, H.-J., and Lee, Y.C. (1994) The sugar-combining area of galactose-specific toxic lectin of mistletoe extends beyond the terminal sugar residue: comparison with a homologous toxic lectin, ricin. Carbohydr. Res., 254, 269–276.[CrossRef][Web of Science][Medline]
Manning, J.C., Seyrek, K., Kaltner, H., André, S., Sinowatz, F., and Gabius, H.-J. (2004) Glycomic profiling of developmental changes in bovine testis by lectin histochemistry and further analysis of the most prominent alteration on the level of the glycoproteome by lectin blotting and lectin affinity chromatography. Histol. Histopathol., 19, 1043–1060.[Web of Science][Medline]
Mikeska, R., Wacker, R., Arni, R., Singh, T.P., Mikhailov, A., Gabdoulkhakov, A., Voelter, W., and Betzel, C. (2005) Mistletoe lectin I in complex with galactose and lactose reveals distinct sugar-binding properties. Acta Crystallogr., F61, 17–25.[Medline]
Nicolson, G.L., Blaustein, J., and Etzler, M.E. (1974) Characterization of two plant lectins from Ricinus communis and their quantitative interaction with a murine lymphoma. Biochemistry, 13, 196–204.[CrossRef][Medline]
Niwa, H., Tonevitsky, A.G., Agapov, I.I., Saward, S., Pfüller, U., and Palmer, R.A. (2003) Crystal structure at 3 Å of mistletoe lectin I, a dimeric type-II ribosome-inactivating protein, complexed with galactose. Eur. J. Biochem., 270, 2739–2749.[Web of Science][Medline]
Olsnes, S., Stirpe, F., Sandvig, K., and Pihl, A. (1982) Isolation and characterization of viscumin, a toxic lectin from Viscum album L. (mistletoe). J. Biol. Chem., 257, 13263–13270.
Pascal, J.M., Day, P.J., Monzingo, A.F., Ernst, S.R., Robertus, J.D., Iglesias, R., Pérez, Y., Férreras, J.M., Citores, L., and Girbés, T. (2001) 2.8 Å crystal structure of a non-toxic type-II ribosome-inactivating protein, ebulin 1. Proteins, 43, 319–326.[CrossRef][Web of Science][Medline]
Riordan, J.F., Wacker, W.E.C., and Vallee, B.L. (1965) N-Acetylimidazole: a reagent for determination of "free" tyrosyl residues of proteins. Biochemistry, 4, 1758–1765.[CrossRef]
Rivera-Sagredo, A., Jiménez-Barbero, J., Martín-Lomas, M., Solís, D., and Díaz-Mauriño, T. (1992) Studies on the molecular recognition of synthetic methyl ß-lactoside analogues by Ricinus communis agglutinin. Carbohydr. Res., 232, 207–226.
Roberts, L.M., Lamb, F.I., Pappin, D.J., and Lord, J.M. (1985) The primary sequence of Ricinus communis agglutinin. Comparison with ricin. J. Biol. Chem., 260, 15682–15686.
Robertus, J.D. and Monzingo, A.F. (2004) The structure of ribosome inactivating proteins. Mini Rev. Med. Chem., 4, 477–486.[Web of Science][Medline]
Rüdiger, H. and Gabius, H.-J. (2001) Plant lectins. Glycoconj. J., 18, 589–613.[CrossRef][Web of Science][Medline]
Rüdiger, H., Siebert, H.-C., Solís, D., Jiménez-Barbero, J., Romero, A., von der Lieth, C.-W., Díaz-Mauriño, T., and Gabius, H.-J. (2000) Medicinal chemistry based on the sugar code: fundamentals of lectinology and experimental strategies with lectins as targets. Curr. Med. Chem., 7, 389–416.[Web of Science][Medline]
Sandvig, K., Olsnes, S., and Pihl, A. (1978) Chemical modifications of the toxic lectins abrin and ricin. Eur. J. Biochem., 84, 323–331.[Web of Science][Medline]
Siebert, H.-C., Adar, R., Arango, R., Burchert, M., Kaltner, H., Kayser, G., Tajkhorshid, E., von der Lieth, C.-W., Kaptein, R., Sharon, N., and others (1997) Involvement of laser photo-CIDNP (chemically induced dynamic nuclear polarization)-reactive amino acid side chains in ligand binding by galactoside-specific lectins in solution. Eur. J. Biochem., 249, 27–38.[Web of Science][Medline]
Siebert, H.-C., von der Lieth, C.-W., Kaptein, R., Beintema, J.J., Dijkstra, K., van Nuland, N., Soedjanaatmadja, U.M.S., Rice, A., Vliegenthart, J.F.G., Wright, C.S., and Gabius, H.-J. (1997) Role of aromatic amino acids in carbohydrate binding of plant lectins: laser photo chemically induced dynamic nuclear polarization study of hevein domain-containing lectins. Proteins, 28, 268–284.[CrossRef][Web of Science][Medline]
Solís, D., Fernández, P., Díaz-Mauriño, T., Jiménez-Barbero, J., and Martín-Lomas, M. (1993) Hydrogen-bonding pattern of methyl ß-lactoside binding to the Ricinus communis lectins. Eur. J. Biochem., 214, 677–683.[Web of Science][Medline]
Solís, D., Jiménez-Barbero, J., Kaltner, H., Romero, A., Siebert, H.-C., von der Lieth, C.-W., and Gabius, H.-J. (2001) Towards defining the role of glycans as hardware in information storage and transfer: basic principles, experimental approaches and recent progress. Cells Tissues Organs, 168, 5–23.[CrossRef][Web of Science][Medline]
Spande, T.F. and Witkop, B. (1967) Determination of the tryptophan content of proteins with N-bromosuccinimide. Methods Enzymol., 11, 498–506.
Stirpe, F. (2004) Ribosome-inactivating proteins. Toxicon, 44, 371–382.[Medline]
Suckau, D., Resemann, A., Schuerenberg, M., Hufnagel, P., Franzen, J., and Holle, A. (2003) A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Anal. Bioanal. Chem., 376, 952–965.[CrossRef][Web of Science][Medline]
Sweeney, E.C., Tonevitsky, A.G., Palmer, R.A., Niwa, H., Pfüller, U., Eck, J., Lentzen, H., Agapov, I.I., and Kirpichnikov, M.P. (1998) Mistletoe lectin I forms a double trefoil structure. FEBS Lett., 431, 367–370.[CrossRef][Web of Science][Medline]
Sweeney, E.C., Tonevitsky, A.G., Temiakov, D.E., Agapov, I.I., Saward, S., and Palmer, R.A. (1997) Preliminary crystallographic characterization of ricin agglutinin. Proteins, 28, 586–589.[CrossRef][Web of Science][Medline]
Tercero, J.C. and Díaz-Mauriño, T. (1988) Affinity chromatography of fibrinogen on Lens culinaris agglutinin immobilized on CNBr-activated Sepharose: study of the active groups involved in nonspecific adsorption. Anal. Biochem., 174, 128–136.[CrossRef][Web of Science][Medline]
Timoshenko, A.V., Gorudko, I.V., Kaltner, H., and Gabius, H.-J. (1999) Dissection of the impact of various intracellular signaling pathways on stable cell aggregate formation of rat thymocytes after initial lectin-dependent cell association using a plant lectin as model and target-selective inhibitors. Mol. Cell Biochem., 197, 137–145.[CrossRef][Web of Science][Medline]
Timoshenko, A.V., Lan, Y., Gabius, H.-J., and Lala, P.K. (2001) Immunotherapy of C3H/HeJ mammary adenocarcinoma with interleukin-2, mistletoe lectin or their combination: effects on tumor growth, capillary leakage and nitric oxide (NO) production. Eur. J. Cancer, 37, 1910–1920.[CrossRef][Web of Science][Medline]
Tsodikov, O.V., Record, M.T., Jr., and Sergeev, Y.V. (2002) A novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature. J. Comput. Chem., 23, 600–609.[CrossRef][Web of Science][Medline]
Unverzagt, C., André, S., Seifert, J., Kojima, S., Fink, C., Srikrishna, G., Freeze, H., Kayser, K., and Gabius, H.-J. (2002) Structure–activity profiles of complex biantennary glycans with core fucosylation and with/without additional 2,3/2,6-sialylation: synthesis of neoglycoproteins and their properties in lectin assays, cell binding, and organ uptake. J. Med. Chem., 45, 478–491.[CrossRef][Web of Science][Medline]
Villalobo, A., Nogales-González, A., and Gabius, H.-J. (2006) A guide to signaling pathways connecting protein–carbohydrate interaction with the emerging versatile effector functionality of mammalian lectins. Trends Glycosci. Glycotechnol., 18, 1–37.
Wiseman, T., Williston, S., Brandts, J.F., and Lin, L.N. (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem., 179, 131–137.[CrossRef][Web of Science][Medline]
Wu, A.M., Chin, L.K., Franz, H., Pfüller, U., and Herp, A. (1992) Carbohydrate specificity of the receptor sites of mistletoe toxic lectin-I. Biochim. Biophys. Acta, 1117, 232–234.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||