Site-specific mutagenesis of mistletoe lectin: the role of RIP activity in apoptosis.

Recombinant mistletoe lectin (rML) belongs to the class of type II ribosome-inactivating proteins (RIP) composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding properties. To investigate the contribution of the enzymatic activity of the rML A-chain to the observed cytotoxic and apoptotic effects, an rMLA E166Q R169Q molecule was developed by means of site-specific mutagenesis. Following heterologous expression, the activity of mutant rMLA was measured in a cell-free assay for rRNA-N-glycosidase activity. Moreover, after generation of heterodimer, the activities of mutant rML E166Q R169Q and rML wild type were determined in a cytotoxicity and apoptosis assay. Although the reduction of activity as measured in the cell-free RIP assay was more pronounced (factor 237) than in both cellular assays (factors 20-22), the data clearly indicate a close correlation between cytotoxicity, apoptosis, and the enzymatic activity of the rML A-chain. Thus, RIP activity is an essential feature of rML and therefore a prerequisite for its biological function as an anticancer agent.     

Cytotoxic activity of recombinant bFGF-rViscumin fusion proteins

A fusion protein (bFGF-rMLA), containing the mitogen basic fibroblast growth factor (bFGF) and the cytotoxic component of rViscumin (recombinant mistletoe lectin), the enzymatic A-chain (rMLA), was expressed in Escherichia coli, purified, and functionally characterized. bFGF-rMLA is cytotoxic for mouse B16 melanoma cells expressing the FGF receptor with an IC(50) value of approximately 1 nM. rMLA shows no significant effect on the viability of the B16 cells up to a concentration of 141 nM. Additionally, bFGF-rMLA was associated with the rViscumin B-chain (rMLB) in an in vitro folding procedure. The IC(50) value of bFGF-rMLA/rMLB to B16 cells in the presence of lactose-to block rMLB lectin activity-was 134 pM. Thus, it was possible to enhance the efficacy of a rViscumin A-chain mitotoxin through addition of rMLB. We conclude that rViscumin fusion proteins may be generally applicable for the receptor-specific inactivation of target cells and point out their potential in drug development.     

Molecular characterization of the recombinant A-chain of a type II ribosome-inactivating protein (RIP) from Viscum album coloratum and structural basis on its ribosome-inactivating activity and the sugar-binding properties of the B-chain

Mistletoe (Viscum album) lectins, which are classified as a type II ribosome-inactivating protein (RIP) due to their unique biological function and the potential medical and therapeutic application in cancer cells, receive a rising attention. The heterodimeric glycoproteins contain the Achain with catalytic activity and the B-chain with sugar binding properties. In recent years, studies involving the lectins from the white berry European mistletoe (Viscum album) and the yellow berry Korean mistletoe (Viscum album coloratum) have been described. However, the detailed mechanism in exerting unique cytotoxic effect on cancer cells still remains unclear. Here, we aim to understand and define the molecular basis and biological effects of the type II RIPs, through the studies of the recombinant Korean mistletoe lectin. To this end, we expressed, purified the recombinant Korean mistletoe lectin (rKML), and investigated its molecular characteristics in vitro, its cytotoxicity and ability to induce apoptotic cell death in cancer cells. To gain structural basis for its catalytic activity and sugar binding properties, we performed homology modeling studies based on the high degree of sequence identity and conserved secondary structure prediction between Korean and European, Himalayan mistletoe lectins, and Ricin.     

Coordinated binding of sugar, calcium, and antibody to macrophage C- type lectin

Mouse macrophage galactose/N-acetylgalactosamine-specific C-type lectin (MMGL) is a type II transmembrane glycoprotein belonging to the C-type lectin family. Our development of monoclonal antibodies led us to discover that a calcium-dependent conformational change is detected by an antibody (termed mAb LOM-11) and that the antibody's binding to the respective site locks the lectin in an active conformation. These findings correspond to the divalent cation-mediated regulatory mechanisms in a family of cell adhesion molecule integrins that have gained much attention. We now provide direct evidence that mAb LOM-11 increases the affinity of the lectin for calcium ions as a mechanism for the conformational lock using a soluble recombinant form of MMGL (rML) produced in bacteria. Furthermore, we discovered by using an enzyme-linked immunosorbent assay that specific monosaccharides induced a binding site for mAb LOM-11 on the immobilized rML under low calcium environments. We also demonstrated that cell surface MMGL on a transfectant cell line underwent a conformational change upon addition of calcium or ligands, as detected by the binding of mAb LOM-11. These properties are reminiscent of ligand-induced binding sites defined for integrins. The present results suggest a possibility that the mAb LOM- 11 binding site on the lectin may be a site at which protein-protein interaction helps to fine tune the specificity of the C-type lectins by means of coordinated recognition mechanisms.      

Induction of apoptosis by mistletoe lectin I and its subunits. No evidence for cytotoxic effects caused by isolated A- and B-chains

Type II ribosome inactivating proteins (RIP II) are generally known to induce apoptosis in human cells by the inhibition of protein biosynthesis. Recent data from mistletoe RIP II proteins (eg. mistletoe lectin I; ML1) suggest an additional mode of apoptosis induction through the binding of their lectin part to certain cell surface receptors as is known for some human galectins. In order to clarify this possibility, we used highly sensitive flow cytometric apoptosis assays and mistletoe hololectin subunits of proven purity to show that neither human lymphocytes nor Molt-4 cells undergo apoptosis after treatment with isolated lectin-type B-chains. In contrast to earlier investigations, only the hololectin was able to induce apoptosis in these assays. We conclude that direct apoptosis induction by mistletoe lectins occurs only after uptake of the molecules into the cell due to the action of the ribosome inactivating A-chain.     

Complete structure determination of the A chain of mistletoe lectin III from Viscum album L. ssp. album.

The complete primary structure of the A chain of mistletoe lectin III (ML3A), a type II ribosome-inactivating protein, was determined using proteolytic digests of ML3A, HPLC separation of the peptides, Edman degration and MALDI-MS. Based on our results, ML3A consists of 254 amino acid residues, showing a high homology to the A chain of isolectin ML1 with only 24 amino acid residue exchanges. A striking important structural difference compared with ML1A is the lack of the single N-glycosylation site in ML3A due to an amino acid exchange at position 112 (ML1A: NL112GS ==> ML3A: T112GS). The alignment of ML3A with the A chains of ML1, isoabrins, ricin D, Ricinus communis agglutinin and three lectins, identified from the Korean mistletoe Viscum album ssp. coloratum, demonstrates the rigid conservation of all amino acid residues, responsible for the RNA-N-glycosidase activity as reported for ricin D. In addition, the fully determined primary structure of ML3A will give further information about the biological mechanism of mistletoe lectin therapy.     

Cloning of the mistletoe lectin gene and characterization of the recombinant A-chain.

Mistletoe lectin I (MLI) is the major active constituent of mistletoe extracts, which are widely used for adjuvant tumour therapy. The 66-kDa heterodimeric disulphide-linked glycoprotein is classified as type II ribosome-inactivating protein (RIP) due to the rRNA-cleaving enzyme activity of the A-subunit, also referred to as toxic entity. MLI and the close relative ricin both belong to the family of the two-chain plant type II RIP proteins. Isolation of the glycosylated proteins from plant material yield inhomogeneous material probably due to post-translational modifications. The aim of this study was to prepare pure and homogeneous protein as a prerequisite for structural and mechanistic studies in order to gain insight into the mode of action of this cytotoxic plant protein on tumour and immune cells. Of particular interest was to explain whether the differences in toxicity of ML and ricin are the result of variations of their enzymatic activities. By investigating the sequence homologies between the active sites of different RIPs we were able to deduce a set of primers which were suitable for specific amplification of the mistletoe lectin gene. Applying this PCR strategy the full-length 1923 nucleotide DNA sequence coding for the prepro-protein was obtained showing the existence of a single intron-free gene. In order to elucidate the molecular basis for the observed differences in cytotoxicity within the family of RIP the enzymatic A-subunit was expressed in a heterologous system. Expression of the A-chain in E. coli BL21/pT7 resulted in production of insoluble inclusion bodies constituting 20-30% of total protein. Refolding led to a pure and homogeneous protein species with an apparent molecular mass of 27 kDa and a pI value of 6.4. The ribosome-inactivating activity of the unglycosylated recombinant A-chain (IC50 20.5 pM) protein was in the same range as that of the glycosylated plant-derived ML A-chain (IC50 3.7 pM), which was very similar to that of ricin A-chain (IC50 4.9 pM). Thus, the higher cytotoxicity of ricin cannot be accountable for differences in the enzymatic activities of the type II RIP A-chains.     

Mistletoe lectin dissociates into catalytic and binding subunits before translocation across the membrane to the cytoplasm.

Hybridomas producing monoclonal antibodies (mAbs) against the mistletoe lectin A-chain (MLA) were obtained to investigate the intracellular routing and translocation of ribosome-inactivating proteins. Anti-MLA mAb MNA5 did not bind the holotoxin but interacted with isolated MLA. This epitope was not recognized upon MLA denaturation or conjugation of MLA with the ricin binding subunit (RTB). Furthermore, the mAbs did not appreciably react with a panel of MLA synthetic octapeptides linked to the surface of polyethylene pins. A study of the cytotoxicity of mistletoe lectin, ricin, and chimeric toxin MLA/RTB for the hybridomas revealed that interchain disulfide bond reduction and subunit dissociation are required for cytotoxic activity of mistletoe lectin.     

Production of pea lectin in Escherichia coli

In order to explore the molecular basis for the glycopeptide specificity of legume lectins, we have developed an experimental system in which specific amino acid alterations can be introduced into the carbohydrate binding site of pea lectin. This system is based on the production of pea lectin in Escherichia coli. The plasmid coding for the lectin was constructed from two lectin cDNA sequences isolated from Pisum sativum seeds (Higgins, T. J. V., Chandler, P. M., Zurawski, G., Button, S. C., and Spencer, D. (1983) J. Biol. Chem. 258, 9544-9549) and an expression vector based on the gene for the outer membrane lipoprotein of E. coli (Nakamura, K., and Inouye, M. (1982) EMBO J. 1, 771-775). The lectin is produced as a single polypeptide chain and forms insoluble aggregates in E. coli cells (2-5 mg/liter). Functional lectin is recovered by solubilization of the aggregates in guanidinium hydrochloride, renaturation in the presence of MnCl2 and CaCl2, and affinity purification on Sephadex. This procedure yields a homogeneous 28,000-dalton protein. Comparison of the recombinant lectin with natural pea lectin in an inhibition of hemagglutination assay demonstrated that there is no detectable difference in the carbohydrate binding properties of the two lectins.     

An intestinal galactose-specific lectin mediates the binding of murine IgE to mouse intestinal epithelial cells.

A number of lactose-binding lectins have recently been identified in the rat and mouse intestine, one of which corresponds to the C-terminal domain of IgE-binding proteins, originally identified in rat basophilic leukemia (RBL) cells and mouse 3T3 fibroblasts. In the present report, we describe the affinity purification of a rat intestinal lactose-specific lectin which binds murine IgE antibodies. This binding most likely occurs via the immunoglobulin carbohydrate chains, as it is inhibited by lactose. This intestinal lectin molecule is also immunologically related to the previously described IgE-binding protein (epsilon BP) isolated from RBL cells, since it is recognized by antibodies raised against recombinant epsilon BP. This intestinal form of epsilon BP has a molecular mass of 17.5 kDa, which is much lower than that of its RBL cell analogue (31 kDa). The attachment of IgE to the mouse intestinal epithelium was demonstrated by immunohistochemistry, along with the presence of a corresponding mouse intestinal epsilon BP. The carbohydrate-dependent nature of this attachment was established by demonstrating that IgE binding to mouse epithelium was specifically abolished by lactose (4 mM) and by a blood-group-A-active tetrasaccharide (0.2 mM), but not by mannose (10 mM). Finally, the association of IgE with the mouse intestinal epithelium was prevented by competition with the purified IgE-binding lectin isolated from rat intestine. Although the physiological function of this intestinal protein is still unknown, the finding that IgE binds to a lectin in the intestinal epithelium pinpoints a possible novel mechanism for the regulation of IgE-mediated disorders, such as food allergy.     

A Lectin from Chinese Mistletoe Increases γδ T Cell-mediated Cytotoxicity through Induction of Caspase-dependent Apoptosis

In this study, a mistletoe lectin (ML) was purified from Chinese mistletoe and the effect of this 60 kDa Chinese ML on human γδ T cell cytotoxicity, apoptosis and modulation of the cytokine network was studied. The cytotoxic properties of δ T cells was evaluated by using a ~(51)Cr release test and employed fluorescence-activated cell sorting and enzyme-linked immunosorbent assay analysis to quantify translocation of the cell membrane phospholipid, phosphatidylserine and nuclear DNA fragmentation during apoptosis. It was found that: (ⅰ) ML effectively stimulated γδ T cell proliferation in a dose- and time-dependent manner; (ⅱ) ML increased γδ T cell cytotoxicity; (ⅲ) ML could modulate lipopolysaccharide-induced cytokine release in a pro-inflammatory manner by increasing tumor necrosis factor (TNF)-α release and inhibiting the release of anti-inflammatory interleukin (IL)-10; (ⅳ) ML induced apoptosis in caspase-dependent and CD95-independent manner. The results indicated that ML is a potent immunomodulator to human γδ T cell cytotoxicity, apoptosis and cytokine production.     

Complete structure determination of N‐acetyl‐D‐galactosamine‐binding mistletoe lectin‐3 from Viscum album L. album

The primary structure of the B chain of the N‐acetyl‐D ‐galactosamine‐recognizing mistletoe lectin‐3 (ML‐3B) has been deduced from proteolytic digest peptides of the purified glycoprotein, their HPLC‐separation and Edman degradation and confirmation of the peptide sequences by MALDI‐MS. ML‐3B consists of 262 amino acid residues including 10 cysteine moieties. The structure and linkage of the carbohydrate side chains, connected to two N‐glycosylation sites at positions Asn⁹⁵ and Asn¹³⁵ of the lectin, were determined by a combination of glycosidase treatment and MALDI‐MS of corresponding glycopeptide fragments. The sequence alignment reveals a high homology with other B chains of type‐II RIPs, although there are remarkable differences in the D ‐galactose‐specific mistletoe lectin‐1B chain. The recently published primary structure of the mistletoe lectin‐3A chain 1 and the now available primary sequence of the 3B chain allowed the construction of a preliminary homology model of ML‐3. The model demonstrates, unequivocally, that ML‐3 is a member of the type‐II RIP family with rigid conservation of the enzymatic active site of the A chain and an identical overall protein fold. Specific amino acid residue exchanges and the different glycosylation pattern in comparison with ML‐1 are discussed and related to the properties of the two glycoproteins. The knowledge of the complete primary structure of mistletoe lectin‐3 is a major contribution towards more insight into the mechanism of the biological activity of commercial mistletoe preparations. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

Differences in amino acid sequences of mistletoe lectin I and III B-subunits determining carbohydrate binding specificity

Toxic lectins of European mistletoe Viscum album L.--MLI (viscumin), MLII and MLIII--are present in water extracts of this plant. Earlier we have cloned the full-length gene of MLIII precursor [A.G. Tonevitsky, I.I. Agapov, I.B. Pevzner, N.V. Maluchenko, M.M. Mojsenovich, U. Pfueller, M.P. Kirpichnikov, (2004) Biochemistry (Mosc.), 69 (6), 790-800, in press]. Here for the first time we report the cloning and expression in Escherichia coli cells of MLIII gene fragment encoding the carbohydrate-binding subunit. We have proved with our panel of monoclonal antibodies against ML toxins that the cloned fragment encoded MLIII B-subunit. The immunochemical and sugar-binding activities of renatured recombinant MLIII B-subunit were demonstrated in ELISA and ELLA, respectively. The comparative analysis of amino acid sequences of the cloned rMLIIIB and the B-subunits of other type II RIPs--MLI, ricin, abrin and nigrin b--was performed, revealing the main differences in primary structure of MLI and MLIII B-chains, which could determine their sugar specificity. The antigenicity analysis of MLI and MLIII B-subunits showed one epitope 25RDDDFRDGNQ34 in MLIB that is absent in MLIIIB sequence. The role of the toxic lectins and their subunits in immunological properties of mistletoe extracts is discussed.     

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

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

Induction of apoptosis by the mistletoe lectins: A review on the mechanisms of cytotoxicity mediated by Viscum album L.

This review focuses on the cytotoxic properties of Viscum album L. (VAL). Apart from well-established results of protein synthesis inhibition by the mistletoe lectins (MLs), namely their catalytic A chain, there is now convincing evidence that the VAL-mediated cytotoxicity is mainly due to an induction of apoptosis. Among the more than 1,000 proteins detected in VAL, the MLs and the viscotoxins (VTs) are the predominant toxic proteins. Using purified components, such as the D-galactose-specific ML I, the N-acetyl-D-galactosamine-specific ML II and ML III, crude VTs and oligosaccharides, only the MLs induced apoptosis. The in vitro studies suggest that interaction of lectin B chains with appropriate receptors on the cell surface activates distinct signalling pathways that ultimately leads to apoptosis in a large fraction of cells, while others survive, however, with a conservation of their DNA. Inhibition of protein synthesis by the A chain of the hololectin probably accelerates the B chain-induced course of events.     

Cloning and expression of mistletoe lectin III B-subunit

Aqueous extracts of mistletoe (Viscum album L.) contain toxic proteins (lectins) MLI (viscumin), MLII, and MLIII. We previously cloned the gene encoding MLIII precursor. In the present study, a gene fragment encoding the carbohydrate-binding subunit of mistletoe toxic lectin MLIII was cloned and expressed in Escherichia coli cells. The structure and immunochemical properties of recombinant MLIII B-subunit were investigated using a panel of monoclonal antibodies against ML-toxins. Sugar-binding activity of recombinant MLIII B-subunit was determined by ELISA. Amino acid sequence analysis of the cloned MLIII compared with known mistletoe toxins and other ribosome inactivating type II proteins (ricin, abrin a, and nigrin b B-subunits) revealed essential features of the recombinant MLIIIB primary structure that could determine sugar specificity of the lectin as well as immunomodulating and anti-tumor properties of mistletoe extracts.Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 378–389.Original Russian Text Copyright © 2005 by Pevzner, Agapov, Pfueller, Pfueller, Maluchenko, Moisenovich, Tonevitsky, Kirpichnikov.     

Further refinement of the description of the ligand-binding characteristics for the galactoside-binding mistletoe lectin,a plant agglutinin with immunomodulatory potency

The galactoside-binding lectin from mistletoe (Viscum album L.) is a biological response modifier, eliciting e.g. enhanced secretion of cytokines. This immunological activity warrants the further analysis of its ligand-binding properties with special attention paid to blood group epitopes. To avoid the microheterogeneity and complexity of naturally occurring glycoproteins, chemically strictly defined neoglycoconjugates and a panel of synthetic oligosaccharides were employed in solid-phase assays for direct binding and assessment of the relative inhibitory capacity. Since label incorporation into the lectin, although performed under protective conditions, or surface immobilization by adsorption to plastic may affect its affinity characteristics, the extent of neoglycoconjugate binding in the absence of any interfering substance and in the presence of oligosaccharides was determined comparatively with labeled and with immobilized lectin. In principle, these two factors could be excluded to markedly alter binding features. In addition to lactose, the blood group determinants H and B were strongly reactive. A fucose residue can thus especially be accommodated to the binding site when linked to the non-reducing unit. N-Acetyllactosamine was nearly as potent as an inhibitor as lactose. Le^c and the A determinant were notably inferior to the other ABH blood group epitopes. Le^a and Le^x and their sialylated derivatives displayed only very weak binding capacity. Among the two natural isomers of sialyllactose, the α2,6-form displayed a higher level of inhibitory capacity than the α2,3-derivative. Isomeric variants of the Thomsen-Friedenreich antigen, too, reduced lectin binding to the lactose-carrying polymer. Their capacities were surpassed by those of the H and the B determinants and a related form of the latter, the P₁ epitope. An overlap of specificity with the immunomodulatory human galectin-3 is thus measurable for H/B-like structures. The documented differential reactivity of the mistletoe lectin to blood group oligosaccharides may have a bearing on the responsiveness of blood group-positive cell populations. © 1997 John Wiley & Sons, Ltd.     

NMR investigations of protein–carbohydrate interactions: insights into the topology of the bound conformation of a lactose isomer and β-galactosyl xyloses to mistletoe lectin and galectin-1

A hallmark of oligosaccharides is their often limited spatial flexibility, allowing them to access a distinct set of conformers in solution. Viewing each individual or even the complete ensemble of conformations as potential binding partner(s) for lectins in protein–carbohydrate interactions, it is pertinent to address the question on the characteristics of bound state conformation(s) in solution. Also, it is possible that entering the lectin’s binding site distorts the low-energy topology of a glycosidic linkage. As a step to delineate the strategy of ligand selection for galactosides, a common physiological docking point, we have performed a NMR study on two non-homologous lectins showing identical monosaccharide specificity. Thus, the conformation of lactose analogues bound to bovine heart galectin-1 and to mistletoe lectin in solution has been determined by transferred nuclear Overhauser effect measurements. It is demonstrated that the lectins select the syn conformation of lactose and various structural analogues (Galβ(1→4)Xyl, Galβ(1→3)Xyl, Galβ(1→2)Xyl, and Galβ(1→3)Glc) from the ensemble of presented conformations. No evidence for conformational distortion was obtained. Docking of the analogues to the modeled binding sites furnishes explanations, in structural terms, for exclusive recognition of the syn conformer despite the non-homologous design of the binding sites.     

Preferential binding of the anticancer drug rViscumin (recombinant mistletoe lectin) to terminally alpha2-6-sialylated neolacto-series gangliosides

Production of biochemically defined recombinant mistletoe lectin was achieved by cloning and separate expression of the single catalytically active A-chain and the B-chain with carbohydrate binding properties in Escherichia coli, yielding an active heterodimeric protein named rViscumin (Eck et al. [1999] Eur. J. Biochem., 265, 788-797). Employing solid phase binding assays, rViscumin was shown to preferentially bind to terminally alpha2-6-sialylated neolacto-series gangliosides IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer isolated from human granulocytes. Only marginal binding of rViscumin to galactose-terminated neutral GSLs was determined, whereas reinvestigation of ricin specificity demonstrated this lectin as a galactose-binding protein. Human promyelotic HL-60 cells exhibited an IC(50) value (half maximum cytotoxicity) of 1.16 pM and human bladder carcinoma 5637 cells of 12.1 pM rViscumin; CHO-K1 cells were resistant to rViscumin treatment up to a concentration of 5.26 nM tested. Quantification of the predominant receptor ganglioside IV(6)Neu5Ac-nLc4Cer by means of a specific anti-Neu5Acalpha2-6Galbeta1-4GlcNAc-R antibody revealed 3.68 x 10(6) and 1.54 x 10(6) receptor molecules per HL-60 and 5637 cell, respectively; CHO-K1 cells were negative, lacking alpha2-6-sialylated gangliosides. The data imply a direct correlation of rViscumin cytotoxicity and the expression of receptor ganglioside. Moreover, CHO-K1 cells were rendered susceptible toward rViscumin cytotoxicity after exogenous application of human granulocyte gangliosides. Thus, (1) rViscumin has to be considered as a sialic acid-specific rather than a galactose-specific type II ribosome-inactivating protein, and (2) neolacto-series gangliosides with Neu5Acalpha2-6Galbeta1-4GlcNAc-terminus are true functional and physiologically relevant rViscumin receptors.     

Influence of type of linkage and spacer on the interaction of beta-galactoside-binding proteins with immobilized affinity ligands

Affinity chromatography provides a powerful tool for isolation of carbohydrate-binding proteins. However, the choice of the ligand and spacer has an important impact on effectiveness. The influence of several different ligands on qualitative and quantitative aspects of the purification of two beta-galactoside-specific lectins has been evaluated. Sepharose was modified by coupling four types of neoglycoproteins (galactosylated or lactosylated bovine serum albumin with increasing sugar content) and two naturally occurring asialoglycoproteins at similar densities. Carbohydrate ligands at essentially equal density were made accessible to the lectins by seven commonly used methods. The yield of mistletoe lectin was high when lactosylated neoglycoproteins were used for separation. For these resins the sugar incorporation exceeded 10 sugar groups per protein carrier molecule. The yield was similarly high with the asialoglycoproteins and with lactose; the sugar was coupled to the resin as a p-aminophenyl derivative or by means of divinyl sulfone activation. An epoxy group in linkages of galactose or lactose decreased the binding capacity. A quantitatively similar degree of protein yields was obtained for the beta-galactoside-binding protein of bovine heart, although different proteins were obtained when neoglycoproteins were used as ligand. The nature of the affinity ligand in lectin purification can increase the yield and may also influence the profile of the carbohydrate-binding proteins.