Combination of immunological and lectin reactions in affinity histochemistry: Proposition of the term affinitin

Summary Using the series system cell receptormistletoe lectin antiferritin-antibodyferritin, the possibilities for combination of lectin and immunological reactions for histochemistry are discussed.The system cell antigen antibody labelled mistletoe (or other) lectin is recommended for visualization of cell antigens (mistletoe lectin as common immunoglobulin reagent). It is pointed out that lectin reactions do not belong to immunhistochemistry but to affinity histochemistry. For all receptor specific proteins (antibodies, lectins, enzymes, haptoglobin and other) the term affinitin is proposed. In consideration of this new definition a common scheme is formulated: Affinitin reacts with affinitin receptor forming affinity product.     

Mistletoe lectin has a shiga toxin-like structure and should be combined with other Toll-like receptor ligands in cancer therapy

Mistletoe extract (ME) is applied as an adjuvant treatment in cancer therapy in thousands of patients each year in Europe. The main immunostimulating component of mistletoe extract, mistletoe lectin, recently has been shown to be a pattern recognition receptor ligand and hence is binding to an important class of pathogen-sensing receptors. Pattern recognition receptor ligands are potent activators of dendritic cells. This activation is a prerequisite for a full-blown T-cell response against cancer cells. Pattern recognition receptor ligands are increasingly recognized as important players in cancer immunotherapy. We collect evidence from case studies on spontaneous regression, from epidemiology, from experiments in a mouse cancer model, and from protein structure comparisons to argue that a combination of mistletoe therapy with other pattern recognition receptor ligand substances leads to an increased immune stimulatory effect. We show that mistletoe lectin is a plant protein of bacterial origin with a 3D structure very similar to shiga toxin from Shigella dysenteriae, which explains the remarkable immunogenicity of mistletoe lectin. Secondly, we show that a combination of pattern recognition receptor ligands applied metronomically in a cancer mouse model leads to complete remission, while single pattern recognition receptor ligands slowed tumor growth. Taken together, we propose to combine mistletoe drugs with other pattern recognition receptor ligand drugs to increase its efficacy in adjuvant or even primary cancer therapy.     

Carbohydrate specificity of the receptor sites of mistletoe toxic lectin-I.

The carbohydrate specificity of mistletoe toxic lectin-I (ML-I) was studied by haemagglutination-inhibition assay. The results indicated that ML-I has a broad range of affinity for Gal alpha,beta linked sequences. The galabiose (E, Gal alpha 1----4Gal) sequence, a receptor of the uropathogenic E. coli ligand, was one of the best disaccharide inhibitors tested. The lectin also exhibits affinity for Lac(Gal beta 1----4Glc), T(Gal beta 1----3GalNAc), I/II(Gal beta 1----3/4GlcNAc) and B(Gal alpha 1----3Gal) sequences. Gal alpha 1----4Gal and Gal beta 1----4Glc are frequently occurring sequences of many glycosphingolipids located at the mammalian cell membranes, such as intestinal and red blood cell membranes, for ligand binding and toxin attachment. This finding provides important information concerning the possible mechanism of intoxication of cells by the mistletoe preparation.     

Isolation and properties of three lectins from mistletoe (Viscum album L.).

Three lectins have been isolated from an extract of mistletoe (Viscum album) by affinity chromatography on partially hydrolysed Sepharose and human immunoglobulin- Sepharose. The lectins differ in molecular weight and sugar specificity (lectin I, mol.wt. 11500, D-galactose-specific; lectin II, mol.wt. 60000, both D-galactose- and N-acetyl-D-galactosamine-specific; lectin III, mol. wt. 50000, N-acetyl-D-galactosamine-specific). All three lectins react with human erythrocytes without specificity for the A, B, and O blood groups. In contrast with abrin and ricin the mistletoe lectins cannot be divided into "toxins" and "haemagglutinins".     

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.     

Cell-Type Dependence of Stability Modulation of Lectin-initiated Contacts by Impairment of Multivalent Carbohydrate Binding and Intracellular Signaling

Initial ligand selection and the intermolecular spatial arrangement ofglycan-lectin complexes are assumed to be essential to induce formationof stable cell aggregates by a lectin. To distinguish effects of thesetwo processes, the tetrameric mistletoe lectin and its isolated B-chainwere used. A reduced impact of multivalency for Ehrlich ascites tumorcells in contrast to rat thymocytes was revealed. Signaling is thusinitiated in a cell-type-dependent manner. Using selective metabolicinhibitors to reduce signal transfer for aggregate stability, decreasein cellular SH-group level was shown to be a common effect accompanyingsuppression of lectin-dependent aggregate stability. The resultsunderscore an intrinsic variability in the relative importance oflectin-dependent glycan aggregation on the cell surface for triggeringpost-binding lectin effects.     

Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.     

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.     

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

The emerging concept of the sugar code attributes functional significance to oligosaccharides of cellular glycoconjugates by protein (lectin)-carbohydrate interactions. Hence it follows that monitoring of glycan expression (glycomic profiling) is not only valuable to delineate characteristic (phenomenological) changes in the cell's glycosylation but will also come up with the localization of epitopes with potential in biorecognition. It is for this purpose that we have set up a panel of 16 markers (plant lectins and a carbohydrate-specific antibody). The selection met two criteria: a) to be able to detect the common constituents of natural glycans; and b) to place emphasis on detection of neutral carbohydrate units at the spatially accessible branch ends of glycan chains, which are known to be active as ligands for endogenous lectins in situ. Next, we incorporated recent insights into the importance of epitope clustering to turn less abundant oligosaccharides into potent ligands into our study design. To be able to focus on such high-affinity sites, we performed systematic titration studies aimed at defining the probe concentration at which carbohydrate-independent background staining is minimal while still yielding a clear signal. These requirements were met by marker concentrations of 1.25-2.5 microg/ml. Under these conditions, we defined cell-type- and differentiation-dependent changes in bovine testis. Sertoli cells lacked reactivity, whereas gonocytes were differentially reactive with the tested markers. The extent of staining intensity was subject to developmental changes, preferentially for Gal/GalNAc presentation and in this group most prominently with the galactoside-specific lectin from Viscum album L. (mistletoe). Of interest in this context, this lectin is known as a potent mitogen and signal inductor as well as haemagglutinin. The Gal/GalNAc-dependent signals decreased markedly in the course of development and staining was completely lost in the case of mistletoe lectin 12 weeks after gestation. Spermatids of adult testis presented respective glycan epitopes. In contrast to this developmental course of staining, endothelial cells either maintained a constant signal intensity or revealed a signal increase during development for Gal/GalNAc-specific lectins. Their binding of concanavalin A and the two phyto-haemagglutinins (PHA-E/L), which were not or only weakly reactive for gonocytes, served as inherent activity control. Based on lectin blot analysis with the mistletoe lectin as the marker which detected the most prominent change, the glycoprotein patterns from fetal and adult tissue specimens were qualitatively different, rendering changes in expression of the protein part of glycoproteins more likely than remodeling a glycoprotein's glycan chains. Methodologically, results of this procedure were compared to data obtained with lectin affinity chromatography and the combination of the two procedures. Differences in the profiles were discovered that can be assigned to the disparate ways to process the detergent extracts. When access to sample quantity is limited, as is possible in the case of fetal tissue, direct lectin blotting is recommended.     

Biochemical, histochemical and cell biological investigations on the actions of mistletoe lectins I, II and III with human breast cancer cell lines

Cytotoxicity of the mistletoe lectins I, II and III towards six human breast cancer cell lines was assessed using the Mossman assay. In addition, binding of the three mistletoe lectins to the separated membrane glycoproteins of these cell lines, the binding and uptake of these lectins into the cells in tissue culture and the binding of the lectins to histological preparations of these cell lines were analysed. The results indicate that there are quantitative differences concerning the toxicity of these three lectins towards the different cell lines. Furthermore, the lectin binding pattern in the cell lines differed. In Western blots, several membrane glycoproteins were labelled by the lectins. Our results indicate subtle differences between the three lectins with regard to the parameters mentioned above; however, the toxicity of all three lectins from mistletoe is so similar that they all seem suitable for the construction of immunotoxins.Dedication: This work is dedicated to one of the discoverers (amongst many other important contributions) ofHelix pomatia agglutinin, which plays an important role in metastasis research, Professor Dr G. Uhlenbruck on the occasion of his 65th birthday.     

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.     

Seasonal lectin binding variations of thumb pad in the frog (Pelophylax ridibundus)

The thumb pad is one of the most common secondary sexual characteristics in frogs. Although it is known that amphibian skin has affinity for several lectins, there is no report regarding lectin‐binding affinity of the thumb pad or its structural components. This study investigated localization and seasonal variation of specific carbohydrate moieties of glycoconjugates in both the epidermal and dermal components of the frog thumb pad at the light microscopic level using lectin histochemistry. The study consisted of four seasonal groups of the frog species, Pelophylax ridibundus (Synonym of Rana ridibunda): active, prehibernating, hibernating and posthibernating. Four horseradish peroxidase conjugated lectins were employed. It was found that dolichos biflorus agglutinin (DBA), wheat germ agglutinin (WGA), and ulex europaeus (UEAI) gave positive reactions in both epidermal layers and breeding glands. These three lectins bound specific secretory cells in the breeding glands, and the distribution of the cells and epithelial lectin reactions exhibited seasonal changes. In addition, UEA‐I and peanut agglutinin (PNA) showed an affinity in granular glands and the granular zone of mixed glands. Generally, epidermal lectin binding showed dense affinity during the posthibernation period. DBA, UEA‐I, and WGA‐specific cells in the mucous gland decreased gradually until the posthibernation period. These findings suggest that differences of lectin binding in the thumb pad may be related to functional activities and, thus, seasonal adaptations. Moreover, the presence of specific lectin‐binding cells in the breeding glands indicated that they consisted of heterogeneous secretory cell composition or that the cells were at different secretory stages. J. Morphol. 275:76–86, 2014. © 2013 Wiley Periodicals, Inc.     

Ligand binding characteristics of the major mistletoe lectin.

Carbohydrate binding specificity of the galactose-specific, major lectin of mistletoe extract (ML-1) was studied by an inhibition assay using monosaccharides, monosaccharide derivatives, disaccharides, and compounds containing multiple galactosyl terminals. The results indicate that 1) both alpha- and beta-galactosyl residues are recognized equally well; 2) each of the hydroxyl groups of galactose contributes to varying degrees to the binding process, the 4-OH being the most important and the 6-OH the least important hydroxyl group; 3) disaccharide sequences of Gal beta 2Gal and Gal beta 3Gal have much higher affinity than galactose, whereas affinity of all other Gal-disaccharides is only slightly better than galactose; 4) macromolecular ligands having 10 or more terminal galactosyl residues have 500-fold higher affinity than Gal; and 5) a group on ML-1 with pK alpha of 4.8 appears to be involved in the binding of ligand.     

A method for the fine-structural demonstration of lectin receptors

A new two-step method using an Fc-fragment/ferritin conjugate as a marker for the visualization of lectin-binding sites on neuronal and other cell membranes is described. In this study of rat synaptosomes, three lectins were tested: concanavalin A, mistletoe lectin I and wheat germ agglutinin. The specificity of the method was proved by control experiments.     

Fine sugar specificity of the mistletoe (Viscum album) lectin I

The behaviour ofN-acetyllactosamine-type oligosaccharides and glycopeptides on a column of mistletoe lectin I (MLI) immobilized on Sepharose 4B was examined. The immobilized lectin does not show any affinity for asialo-N-glycosylpeptides and related oligosaccharides, which possess one to four unmaskedN-acetyllactosamine sequences. However, substitution of at least one of theN-acetyllactosamine sequences by sialic acid residues, either at O-3 or O-6 of galactose, slightly enhances the affinity of the lectin. Such sialylatedN-glycosylpeptides or oligosaccharides are eluted from the lectin column by the starting buffer as retarded fractions. Surprisingly, the affinity of the immobilized MLI is higher for P1 antigen-containing glycopeptide isolated from turtle-dove ovomucoid and for glycopeptides from bovine thyroglobulin containing terminal non-reducing Gal1–3Gal sequences. These structures are strongly bound on the lectin column and their elution is obtained with 0.15M galactose in the starting buffer.In memory of Hartmut Franz.     

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.     

Comparative analysis of membrane glycoproteins of normal and chronic lymphatic leukemia B lymphocytes by lectins

Eight plant lectins were used to investigate membrane alterations in lymphocytes from patients with chronic lymphocytic leukemia (CLL). By rosetting with lectins attached to latex particles, the cell percentages with the abundance of each lectin receptor were compared in B normal and leukemic lymphocytes. Comparing these data with the number of lectin molecules bound to each cell and the affinity, which are values calculated with 125I-labeled lectins, it was possible to deduce differences in the composition of glycoproteins in B normal and B-CLL lymphocytes membrane. Compared to B normal, B-CLL lymphocytes had fewer receptors for WGA and more for Lens culinaris, SBA and Tetragonolobus purpureus lectins. Receptors for Concanavalin A, Pisum sativum, PHA and Tetragonolobus purpureus showed a higher affinity with B normal lymphocytes, while the other lectins assayed showed more affinity with B-CLL lymphocytes. So, it is possible to establish a comparative analysis about the plasma membrane glycoproteins in the B normal and CLL lymphocytes by lectin binding studies.     

A method for the fine-structural demonstration of lectin receptors

Summary A new two-step method using an Fc-fragment/férritin conjugate as a marker for the visualization of lectin-binding sites on neuronal and other cell membranes is described. In this study of rat synaptosomes, three lectins were tested: concanavalin A, mistletoe lectin I and wheat germ agglutinin. The specificity of the method was proved by control experiments.     

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.