Received on February 8, 1999; revised on April 6, 1999; accepted on April 9, 1999
Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-[beta]-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)2-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential of dendrimers for applications as lectin-targeting device, as attested by these observations.
Key words: agglutinin/glycodendrimer/immunoglobulin/lactose/lectin/neoglycoprotein/starburst dendrimer
Synthetic carbohydrate and polymer chemistry aims to design artificial glycoconjugates, for which medical applications in, e.g., anti-adhesion therapy can be expected to surface. A milestone in this area is the description of the glycoside cluster effect, describing the nonlinear affinity enhancement of a neoglycoconjugate by stepwise increases in sugar density, and the ensuing adaptation of ligand presentation to fit into the topological presentation of C-type carbohydrate recognition domains (Lee and Lee, 1994, 1997; Bovin and Gabius, 1995; Kiessling and Pohl, 1996; Roy, 1996a-c, 1997a; Bovin, 1998; Mammen et al., 1998; Zanini and Roy, 1998a). Suitable intersugar distances can vary according to the type of presentation of binding sites ranging from a few nanometers to considerably more (Lee and Lee, 1993, 1995). In this way, weak individual protein-carbohydrate interactions are compensated, and the multivalent binding process ensures a biologically relevant affinity which allows to envision applications in drug targeting or anti-adhesion therapy (Gabius, 1988, 1997a; Monsigny et al., 1994; Meijer and Molema, 1995; Rice, 1997; Roy 1997b).
In the quest to achieve a mutual adaptation between the critical cluster design and biochemical and topological features of target lectins new additions to the panel of neoglycoconjugates have recently become available, i.e., starburst glycodendrimers (Roy, 1996a-c, 1997a,b; Jayaraman et al., 1998; Zanini and Roy, 1998a). They represent tree-shaped, monodisperse molecules obtained by iterative assembly cycles with carbohydrate ligands establishing the outer sphere. Such "sugar-ball"-like compounds are capable to expose a high surface density of ligands on their surface. This characteristic renders experiments appealing how binding capacity is correlated with the degree of branching. Initial experiments in cell-free systems with model lectins of different specificity reveal notable variability as to whether increases of affinity can actually be expected (Pagé and Roy, 1997; Zanini and Roy, 1997a,b; Ashton et al., 1998). These studies prompt to systematically evaluate the properties of the new neoglycoconjugates with respect to two pertinent issues: (1) to assay different sugar receptors with identical monosaccharide specificity but varying quaternary structure and binding-site orientation in the same test system, and (2) to compare glycodendrimer performance with that of common neoglycoproteins in order to pinpoint their potential for superior features.
In this line of reasoning, we have selected four galactoside-binding sugar receptors, i.e., the (AB)2-type galactoside-specific lectin from Viscum album L. (VAA) with one galactoside-binding site per B subunit (34 kDa) in the tetramer, the homodimeric galectin-1 (monomer of 14 kDa) with binding sites on opposing sides of the jelly-roll-like motif, the monomeric galectin-3 (MW 35 kDa) harboring a N-terminal accessory collagenase/elastase-sensitive domain besides the C-terminal carbohydrate recognition module and a lactoside-binding immunoglobulin G fraction from human serum (Lee et al., 1992; Dong et al., 1997; Gabius, 1997b). Lactosylated neoglycoproteins with different sugar densities and linkage type served as calibration to judge the relative capacity of ligand binding of the panel of test compounds. In addition to neoglycoproteins asialofetuin (ASF) with its three N-glycans (predominantly triantennary chains with Gal[beta]1-4 (74%) or Gal[beta]1-3 linkage in the [alpha]1-3 arm (9%) and also a biantennary chain (17%)) was used as probe, which has already proven to form lattice-like cross-linked complexes with the three oligomeric sugar receptors (Mandal and Brewer, 1993; Gupta et al., 1996). Since poly(amidoamine) starburst dendrimers (PAMAM) with arylic carbohydrate end group can readily be coated to plastic surfaces (Pagé and Roy, 1997), it is feasible to determine binding affinities in a solid-phase assay. These results will complement the assessment of their inhibitory capacities in tests of lectin binding to asialofetuin or to an immobilized neoglycoprotein with the same type of lactose derivative as end group. Here we describe how the extent of core branching and the binding site presentation in the sugar receptors affect the binding behavior of five generations of starburst glycodendrimers.
Sugar receptor binding to immobilized glycodendrimers
Covalent incorporation of the p-isothiocyanate derivative of p-aminophenyl-[beta]-D-lactoside (Roy et al., 1992) into ethylenediamine or the different generations of dendritic poly(amidoamine) increased the presentation of lactose units from 2 in the dimer to 128 in the G5 starburst glycodendrimer (Figure 1). To illustrate the constitution of such a molecule, the structure of the G3 molecule (32-mer) is depicted in Figure 2. To start the comparative analysis of the binding properties of the dimer up to the 128-mer for the panel of sugar receptors, labeled proteins retaining their receptor properties were employed as probes for surface-immobilized glycoligands. As exemplarily shown in Figure 3, the binding was saturable and inhibitable by specific inhibitors of the protein-carbohydrate (lactose) interaction. Therefore, affinity constants of the binding between the lactose-exposing matrix and the sugar receptors could be determined. The amount of coated substance was normalized with respect to the total lactose content which is presented with different levels of local density increasing with the degree of branching from G0 to G5.
Fig. 1. Schematic representation of the synthetic design of the lactosylated dimer (D) and starburst poly(amidoamine) glycodendrimers (G0-G5) using p-isothiocyanatophenyl-b-d-lactoside as glycosylation reagent for ethylenediamine and the dendritic poly(amidoamine) core of different generations.
Fig. 2. Structure of the G3 poly(amidoamine) glycodendrimer after covalent incorporation of p-isothiocyanatophenyl-[beta]-d-lactoside into the peripheral sphere of the starburst dendrimer (32-mer).
Fig. 3. Determination of specific (sugar-inhibitable) binding (+) of labeled Viscum album L. agglutinin (VAA) and Scatchard analysis of the binding data (inset) using the lactosylated poly(amidoamine) glycodendrimer G5 (128-mer) as lectin-reactive part of the matrix in the solid-phase assay.
Evidently, the KD values depend on the quaternary structure of the sugar receptor (Table I). The IgG fraction, the tetrameric plant agglutinin and the homodimeric galectin-1 were superior to the monomeric galectin-3 in ligand binding. Increasing local density of the ligand presentation translated nearly invariably into improved affinity. This result intimates actual accessibility of the carbohydrate derivative on the outer sphere. The monomeric nature of galectin-3 can explain why its spatial exclusion was minimal to allow a large number of probe molecules to associate to the matrix. Affinity enhancements are especially pronounced for the plant agglutinin, with a factor of 38.9, while the KD values for galectin-1 and the antibody were only comparatively slightly affected (Table I). The topological presentation of the carbohydrate recognition domains is thus indicated to be a key factor to be reckoned with for the efficiency of the binding process to these new neoglycoconjugates. When considering the potential of the dendrimers to interfere with a lectin's activity, it should be kept in mind that the synthetic inhibitor will be in solution, not exposed on a surface. To address the question on the relative potency of the different synthetic compounds in this respect, their inhibitory capacities were expressed as the concentration which causes a reduction of signal by 50% relative to controls without any interfering substance (IC50 value).
Table I. Determination of the apparent affinity constant (KD) for the interaction of surface-immobilized glycodendrimers with labeled sugar receptors and number of bound probe molecules at saturation (Bmax) in a solid-phase assay
Inhibitory potency of glycodendrimers
Prior to the analysis of the glycodendrimers it was excluded that any noncarbohydrate feature can influence lectin binding in solution. For example, lactosylated G3 was not able to reduce binding of the mannose-specific concanavalin A to immobilized yeast mannan. This reactivity was readily abolished by equivalent mannose concentrations. As already shown in the preceding paragraph, background binding to immobilized glycodendrimers was rather low, underscoring the specific nature of the molecular rendezvous. Nonetheless, total binding was reduced in each case by noninhibitable signal generation, this difference being defined as 100%.
To provide a measure of relative affinity even for weakly interacting structures, the actual percentage of inhibition (<50%) at a certain concentration will be given. The shape of the resulting inhibition curves is exemplarily illustrated in Figure 4. As matrix, a neoglycoprotein (lactosylated bovine serum albumin) and the natural glycoprotein asialofetuin with its three triantennary N-glycans as ligand part were employed to assess the relevance of disparate ligand presentation (clustered lactosides vs. three tri-antennary N-glycans). A further factor as potential source of error warrants a comment. In the tested concentration range no precipitation of sugar receptor-dendrimer complexes was observed. Such a reaction would reduce the effective concentrations in solution and lead to erroneous interpretations. This reaction was visible for VAA using 50 µg protein in a final volume of 100 µl with 10 nmol of lactose ligands. As indicated in Table II and Table III, only 1.5 µg VAA was used in standard assays for IC50-determinations. The G2-G4 molecules were very effective inducers of precipitate formation, G1/G5 had a moderate activity, whereas G0 and the dimer failed to elicit a detectable level of precipitation. Galectin-1 also exhibited a respective activity, albeit weakly, with G3 as test substance.
Fig. 4. Inhibition curves of binding of biotinylated Viscum album L. agglutinin (VAA) to surface-immobilized asialofetuin using lactosylated D (solid diamonds), G0 (open diamonds), G1 (open circles), G2 (open triangles), G3 (×), G4 (+) and G5 (bullets); for structures of these substances, please see Figure 1) as competitive inhibitor to interfere with lectin binding.
Table II. Determination of the IC50-values and the relative inhibitory potency of multivalent (neo)glycoproteins and glycodendrimers in a solid-phase assay with surface-immobilized lactosylated bovine serum albumin (Lac-BSA(thio.)) and four different labeled sugar receptors in solution Table III. Determination of the IC50-values and the relative inhibitory potency of multivalent (neo)glycoproteins and glycodendrimers in a solid-phase assay with surface-immobilized asialofetuin (ASF) and four different labeled sugar receptors in solution To judge the inhibitory properties of the glycodendrimers, galactose, lactose, and (neo)glycoproteins were first subjected to processing by this analytical method. The relative difference in inhibitory potency between galactose and lactose in this assay for the plant versus the mammalian sugar receptors is in line with the preferential recognition of the terminal sugar by the mistletoe lectin (Lee et al., 1992; Galanina et al., 1997). Since reductive amination leads to a ring opening and thus a loss of the hexopyranose integrity for the reducing glucose unit of lactose, this factor together with the sugar density can be of importance to explain the disparate behavior towards the different sugar receptors (Tables II, III). It appears that the presence of an aromatic linker can compensate higher density of ligand presentation, as corroboratively apparent in glycohistochemical studies (Gabius et al., 1990). Thus, already on the level of neoglycoproteins differences in the efficiency of blocking access of a sugar receptor were detectable. Notably, the natural asialoglycoprotein with its restricted set of interglycosidic distances is invariably less favorable as inhibitor than the neoglycoprotein with the highest tested sugar density. Its ligands had deliberately been conjugated to the carrier as p-isothiocyanatophenyl derivatives as in the glycodendrimers to keep this chemical factor constant. Having assured that saccharides and (neo)glycoproteins can readily be analyzed in this system and that the carbohydrate part of the glycodendrimer panel will be crucial for the response in this test system, the questions were addressed, if and how the shape and valency of glycodendrimers up to the 128-mer will modulate the reactivity of the inhibitor to different sugar receptors. Two types of matrix, i.e., neoglycoprotein with clustered ligand and asialofetuin, were employed to simulate different kinds of ligand presentation. As exemplarily shown in Figure 4, the inhibitory potency varied conspicuously over a log-scale, unveiling the necessity for custom-made design to reach an optimal level. It is remarkable that the inhibitory capacities for the tetrameric plant lectin with two binding sites passed through an optimum at G4. The 64-mer was even markedly more potent for this lectin than the most efficient neoglycoprotein (Table II, III). To indicate the relative potency of the individual glycan unit, the different content of lactose residues in each dendrimer type was taken into account. When the inhibitory capacity was arithmetically normalized to individual lactose units, this result still held true (please see calculated numbers in brackets in Tables II and III). Inhibition was especially effective in assays with the natural glycoprotein. Concerning galectins the performance of the glycodendrimers to interfere with binding to ligands in immobilized (neo)glycoproteins was reduced relative to the plant lectin. This result could be inferred from the already reported comparatively small affinity. Competition with matrix ligands was more pronounced with the homodimeric galectin-1 than with galectin-3. For galectin-1 the lactosylated dendrimers with 64 or 128 ligands were rather potent. In contrast to the experiments with VAA, lactosylated albumin surpassed dendrimers in inhibitory capacity for galectin-3 and equaled the properties of the 128-mer for galectin-1 on asialofetuin (Tables II, III). Antibody binding was by far most sensitive to this synthetic product. Although the relative potency continuously increased from G0-G4 with G4 or G3 reaching optimal values on the basis of the individual sugar unit, spatial presentation on the dendrimers appeared to be least favorable for this sugar receptor. On the contrary, the panel of interglycosidic distances on the neoglycoprotein appeared to furnish an excellent cluster inhibitor for this representative of a class of common carbohydrate-binding proteins. Spectroscopic analysis and small-angle x-ray scattering point to a similar overall density of poly(amidoamine) dendrimers as a function of the generation of core assembly and to secondary interactions between end groups on the surface, arguing in favor of a sugar-ball-like morphology (Prosa et al., 1997; Bosman et al., 1998). The feasibility to coat glycodendrimers with arylic parts in the end groups onto a plastic surface has enabled binding studies with the four sugar receptors. Dimeric sugar receptors facing the sugar surface with their binding sites profit in terms of their KD-values from the increased valency considerably, while galectin-1 with binding sites on opposite sides of the protein experienced a merely 3-fold affinity improvement. Only a slight affinity enhancement was measurable for the monomeric galectin-3. Fittingly, the possibility for an establishment of a nearly linear array of monomers on the surface of the dendrimers can translate into high densities of probe binding. Surface aggregation of galectin-3 is supported by its potential for homotypic protein-protein interactions via the N-terminal domain (Hsu et al., 1992; Massa et al., 1993; Ochieng et al., 1993; Mehul et al., 1994). Since the sugar content on the coating solution has been normalized, the Bmax-values can readily be reconciled with ligand exposure and no major extent of backfolding. Concerning binding-site orientation and its consequences for the affinity reflected in kinetic parameters it is informative to refer to a dimeric form of an anti-carbohydrate antibody. It exhibited an off-rate 20-fold slower and an on-rate 5-fold increased relative to the monomeric form (MacKenzie et al., 1996). For a related AB-toxin-type lectin of VAA, namely RCA120, association and dissociation rate constants decreased by a factor of 2-3 in the course of elevating the number of galactose-terminated branches in bi- to tetra-antennary N-glycans (Shinohara et al., 1995). Evidently, kinetic rate constants appeared to be favorably altered for glycodendrimers, as reflected in the affinity increases. These results start to shed light on the correlation of dendrimer features and binding properties. That no simple a priori predictions are valid is underscored by a recent comparative analysis of neoglycoconjugate binding to E.histolytica and rat liver membranes (Yi et al., 1998). Similarly instructive in terms of characteristics of different lectins it is remarkable that relative to an animal C-type lectin with galactose specificity, rate constants for association to glycopeptides from asialofetuin differ considerably by a factor of 20 for kon and a factor of 8 for koff for the VAA-homologue RCA120 (Shinohara et al., 1994; Yamamoto et al., 1995). Together with the differential presentation of binding sites this result primes the expectation that glycodendrimer effects can be nonuniform for different sugar receptors. To examine the influence of matrix composition, both a neoglycoprotein and a natural glycoprotein served as sensors in attempts to mimic a cell surface with spatial restrictions on ligand display. It is notable that the neoglycoprotein furnishes a heterogeneous population of interglycoside distances, whereas asialofetuin with its N-glycans represents a less variable kind of clustering. Indeed, nonuniform capacities to reduce receptor binding to matrices were measured, with most favorable potency of the 32- and 64-mers observed for the plant agglutinin. Evidently, even on the per-lactose level, the compounds from the third and fourth generation of core extension excelled the inhibitory capacity of the other dendrimers and of the most efficient neoglycoprotein. In view of previous studies with concanavalin A delineating high efficiency of 9- and 18-mers (Pagé and Roy, 1997; Ashton et al., 1998), this result emphasizes an obvious activity beyond this limit. In contrast to the plant agglutinin, immunoglobulin G definitely prefers the sugar presentation of a neoglycoprotein, when impairment of matrix binding with protein and dendrimers in solution was measured. On this basis, it is conceivable that the tested sugar balls cannot offer suitably spaced lactose units to this receptor site orientation, possibly indicative of restricted mobility of end groups. This factor and also an occurrence of spatial restrictions on carbohydrate conformer flexibility, which can strongly influence the capability of an oligosaccharide to fit snugly into a binding site in cases of conformer selection (Gabius, 1998; von der Lieth et al., 1998), will have to be taken into account in further attempts to improve the performance of glycodendrimers. By the way, a deliberate freezing of unfavorable dynamics of the interglycosidic torsion angles of oligosaccharides could engender a desirable affinity increase by reducing the entropic penalty during receptor binding. Monomeric galectin-3 similarly failed to be object of a glycoside cluster effect, while tetramers and 32-mer were operative inhibitors for galectin-1. Overall, these differential effects underscore a potential for marked selectivity of certain starburst glycodendrimers towards distinct classes of sugar receptors. If, e.g., prevention of AB-toxin-mediated cell death is desirable, the 32- or 64-mers are clearly preferable to a neoglycoprotein. Further studies thus appear to be warranted to exploit the inhibitory potential of glycodendrimers in lectin-mediated contacts as well as to rigorously assess their efficiency to serve as drug-targeting or radioimaging devices as conceivably promising competitors to neoglycoproteins, polyacrylamide-based neoglycoconjugates or carbohydrate-bearing liposomes (Gabius et al., 1996; Kojima et al., 1997). Moreover, systematic extension of the experimental basis along the given route will enable definition of rules to predict the relative affinities with the eventual aim of a custom-made design. As recently shown for flu virus inhibition of adhesion (Reuter et al., 1999), carbohydrate clusters scaffolded onto random coil polymers may offer the advantages intrinsically present on both polymers and dendrimers. Synthesis of glycodendrimers and neoglycoproteins The synthesis of lactosylated starburst poly(amidoamine) dendrimers up to generation five with an unprotected lactoside derivative followed the strategy described for mannosylated and sialylated dendrimers (Pagé and Roy, 1997; Zanini and Roy, 1998b). Briefly, per-O-acetylated p-nitrophenyl-[beta]-D-lactoside, obtained by a novel phase transfer catalysis method (Roy et al., 1992), was de-O-acetylated under standard conditions (NaOMe, MeOH) to give product 1 in Figure 1 quantitatively (mp 249-250°C, [[alpha]]D -39.1° (c = 1.25, DMSO)). Catalytic transfer hydrogenation of 1 using ammonium formiate and 10% Pd-C in methanol according to Roy et al. (1992) yielded the p-aminophenyl derivative 2 also quantitatively (mp 237-238.5°C from ethanol, [[alpha]]D -24.2° (c = 1.25, DMSO)). Treatment of amine 2 with excess thiophosgene in 80% aqueous ethanol according to McBroom et al. (1972) resulted in its conversion to the isothiocyanate 3 in 69% yield after crystallization from water. Its treatment with ethylenediamine or dendritic poly(amidoamine) (PAMAM), kindly provided by Dr. R.Spindler (Dendritech Inc., Midland, MI), at room temperature overnight in a mixture of dimethylformamide-H2O (1:1, v/v) containing diisopropylethylamine provided access to the dimer and G0-G5 glycodendrimers, shown in Figure 1, in yields of ~80%. The dimer was purified by size exclusion chromatography on a Biogel P2 column after lyophilization of the reaction mixture, while the glycodendrimers were purified by removal of residual reactants by dialysis against water. The products gave consistent high-field 1H- and 13C-NMR as well as MALDI-TOF spectra. Molecular weights are as follows: Lac-dimer: 1011.0; G0 PAMAM-[Lac]4: 2418.9; G1 PAMAM-[Lac]8: 5233.8; G2 PAMAM-[Lac]16: 10,863.8; G3 PAMAM-[Lac]32: 22,124; G4 PAMAM-[Lac]64: 44,645; G5 PAMAM-[Lac]128: 89,684. The neoglycoproteins were prepared by covalent conjugation of the diazonium (diaz.) and p-isothiocyanatophenyl (thio) derivatives of p-aminophenyl-[beta]-D-lactoside and of lactose in the presence of sodium cyanoborohydride by reductive amination (red. amin.) to carbohydrate-free bovine serum albumin as carrier (McBroom et al., 1972; Gray, 1974). Isolation and labeling of the sugar receptors The galactoside-specific lectins from mistletoe (Viscum album L. agglutinin, VAA) and bovine heart (galectin-1) as well as from expression vector-carrying E.coli JA221 cells (murine galectin-3; Agrwal et al., 1993) were purified by affinity chromatography on lactosylated Sepharose 4B, obtained by divinyl sulfone activation, as described previously (Gabius, 1990). The [beta]-galactoside-binding immunoglobulin G (IgG) fraction from human serum was obtained by similar affinity chromatography including a negative refinement step on melibiosylated Sepharose 4B to exclude any subfractions with preferential affinity towards [alpha]-galactosides, thus yielding IgG ([alpha]-[beta]+) (Dong et al., 1997). Concanavalin A was prepared as described previously (Gabius et al., 1989). Biotinylation was performed under activity-preserving conditions with either the N-hydroxysuccinimide ester derivative for the plant agglutinin and the two mammalian galectins or the amidocaproyl hydrazide derivative for the human IgG fraction, as described previously (Gabius et al., 1991; Dong et al., 1995). Solid-phase assays Thermodynamic binding parameters of the interaction between the glycodendrimers and the sugar receptors were determined by solid-phase assays in microtiter plate wells (Greiner, Nürtingen, Germany), as described previously (Zeng and Gabius, 1993; André et al., 1997). Signal generation to quantitate binding of labeled sugar receptors to surface-immobilized glycodendrimers was achieved by streptavidin-peroxidase (Sigma, Munich, Germany; used at a concentration of 0.5 µg/ml) and o-phenylenediamine (1 mg/ml)/H2O2 (1 µl/ml). The extent of sugar-inhibitable binding was assessed by controls, where incubation of wells proceeded in the presence of the inhibitor mixture (20 mM phosphate-buffered saline, pH 7.2, containing 0.5 mg asialofetuin/ml and 75 mM lactose). The individual experimental series with at least duplicates were carried out independently at least four times up to the level of saturation of binding of the labeled protein in solution. The amount of glycodendrimer used for coating was normalized to the exposed lactose density (i.e., from 36.4 ng dimer to 50.4 ng G5). The data sets were algebraically transformed to calculate the KD value and the number of bound probe molecules at saturation. To assess the relative inhibitory potency of glycodendrimers and neoglycoproteins, expressed as the concentration of carbohydrate or conjugate (in µM) required for 50% inhibition of the OD490-value which is measured in the absence of any added test substance (IC50 value), constant amounts of a (neo)glycoconjugate were coated to the surface of microtiter plate wells for assays with each sugar receptor, and the test substance was coincubated with the labeled sugar receptor (Galanina et al., 1997). Blank values were determined in the presence of 0.5 mg asialofetuin/ml and 75 mM lactose to exclude to incorporate any sugar-independent probe binding into the calculations. At least four independent experimental series were performed with duplicates. Induction of precipitate formation by cross-linking glycodendrimers and a sugar receptor was measured in a final volume of 100 µl with 50 µg VAA and a dendrimer amount equivalent to 10 nmol lactose ligand quantity, as described previously (Pagé and Roy, 1997). We thank Dr. J.L.Wang for kindly donating the expression vector for murine galectin-3, Dr.-M.-Scheel-Stiftung für Krebsforschung for generous financial support, and B.Hofer and L.Mantel for expert technical assistance. ASF, asialofetuin; IgG, immunoglobulin G; Lac-BSA, lactosylated bovine serum albumin derived from covalent conjugation of the diazonium (diaz.) or p-isothiocyanato (thio) derivatives of p-aminophenyl lactoside or by reductive amination (red. amin.); PAMAM, poly(amidoamine); VAA, Viscum album L. agglutinin.
1To whom correspondence should be addressed
DISCUSSION
MATERIALS AND METHODS
ACKNOWLEDGMENTS
ABBREVIATIONS
REFERENCES
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: jnl.info{at}oup.co.uk
Last modification:
Copyright© Oxford University Press, 1999.