Binding mechanism of riproximin - The Journal of ...Binding mechanism of riproximin 3 fined synthetic carbohydrates as bovine serum albumin (BSA) or human serum albumin (HSA) conjugates,

Binding mechanism of riproximin

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Evaluation of Riproximin Binding Properties Reveals a Novel Mechanism for Cellular Targeting *

Helene Bayer 1, Katharina Essig 1, Sven Stanzel 2, Martin Frank 3, Jeffrey C. Gildersleeve 4, Martin R. Berger 1, and Cristina Voss 5

1 Toxicology and Chemotherapy Unit, German Cancer Research Center,

Im Neuenheimer Feld 581, 69120 Heidelberg, Germany 2 Department of Biostatistics, German Cancer Research Center, Im Neuenheimer Feld 581,

69120 Heidelberg, Germany 3 BIOGNOS AB, Generatorsgatan 1, 41705 Goeteborg, Sweden

4 Chemical Biology Laboratory, National Cancer Institute, 376 Boyles Street, Frederick, MD 21702, USA

5 Department of Biochemistry, Heidelberg Pharma GmbH, Schriesheimer Strasse 101, 68526 Ladenburg, Germany

* Running title: Binding mechanism of riproximin

To whom correspondence should be addressed: Dr. Cristina Voss, Department of Biochemistry, Heidel-berg Pharma GmbH, Schriesheimer Strasse 101, 68526 Ladenburg, Germany; Phone: +49 6203 1009 29; Fax: +49 6203 1009 764; E-mail: [email protected] Keywords: riproximin, glycans, receptor, glycan microarray, Tn antigen, NA Background: Riproximin is a cytotoxic lectin from Ximenia americana showing tumor selecti-vity. Results: Riproximin selectively binds to two types of glycoconjugates present on glycoproteins, crosslinking them by its two binding sites. Conclusion: Riproximin's biologic activity is de-termined by specific and dynamic interactions with multivalent, cancer related glycan targets. Significance: Riproximin's selectivity for cancer cells relies on its unique targeting mechanism. SUMMARY

Riproximin is a cytotoxic type II ribosome inactivating protein showing high selectivity for tumor cell lines. Its binding to cell surface gly-cans is crucial for subsequent internalization and cytotoxicity. In this paper, we describe a unique mechanism of interaction and discuss its implications for riproximin's cellular targeting and cytotoxicity. On a carbohydrate micro-array, riproximin specifically bound to two types of asialo-glycans, namely to bi- and tri-antennary complex N-glycan structures (NA2/ NA3) and to repetitive N-acetyl-D-galactosa-mine (GalNAc), the so-called clustered Tn anti-gen, a cancer-specific O-glycan on mucins. Two glycoproteins showing high riproximin binding,

the NA3-presenting asialofetuin (ASF) and the clustered Tn-rich asialo bovine submaxillary mucin (aBSM), were subsequently chosen as model to mimic riproximin's binding interac-tions with the two types of glycans. ELISA analyses were used to relate the two binding specificities of riproximin to its two sugar binding sites. Riproximin's ability to crosslink the two model proteins revealed that binding of the two types of glycoconjugates occurs within different binding sites. The biological implica-tions of these binding properties were analyzed in cellular assays. The cytotoxicity of riproxi-min was found to depend on its specific and concomitant interaction with the two glycocon-jugates, as well as on dynamic avidity effects typical for lectins binding to multivalent glyco-proteins. The presence of definite, cancer re-lated structures on the cells to be targeted de-termines the therapeutic potency of riproximin. Due to its crosslinking ability, riproximin is expected to show a high degree of specificity for cells exposing both NA2/NA3 and clustered Tn structures.

Riproximin is a lectin with potent antineo-plastic activity in vitro and in vivo (1,2). It was originally identified as the active component of a powdered plant material used in African traditional

http://www.jbc.org/cgi/doi/10.1074/jbc.M112.368548The latest version is at JBC Papers in Press. Published on August 7, 2012 as Manuscript M112.368548

Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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medicine for treating cancer and has subsequently been isolated from the fruit kernels of Ximenia americana. Riproximin is selectively cytotoxic to cancer cell lines with IC50 in nM range. For exam-ple, the breast cancer MCF7 cells showed >500-fold higher sensitivity than the non-tumorigenic breast epithelium MCF10A cells (3). The remar-kable potency and selectivity prompted us to ex-amine the molecular mechanisms of cell targeting in more detail.

Riproximin belongs to type II of the cytotoxic ribosome inactivating proteins (RIPs) (4). Several members of this family were investigated as po-tential antineoplastic agents, most prominently ricin. The mechanism of action of the RIP family of proteins involves two key steps represented by the A- and B-chains of the protein. First, the B-chain, a lectin, binds to cell surface glycans re-sulting in internalization of the RIP. Within the cell, the A-chain, an rRNA N-glycosidase, depuri-nates the 28S RNA leading to transcriptional arrest and eventually cell death (5). Recently, it was shown that the mode of action also involves the induction of the unfolded protein response (6).

The cellular patterns of cytotoxicity and in vivo toxicity of a particular RIP are primarily deter-mined by its binding and internalization efficiency (7). Several types of biomolecules, such as glyco-proteins, glycosphingolipids, proteoglycans, or glycosyl-phosphatidylinositol-linked proteins, con-tribute to a cell surface glycosylation pattern (8,9) and are potential riproximin targets.

Investigations of the glycan structures from malignant tissues showed that the tumor-associated glycosylation significantly differs from that of nor-mal tissues. Mucins showing aberrant O-glycosyl-ation are a typical feature of epithelia-derived can-cer (8). N-glycans are significantly altered in can-cer, too (10). The presence of immature (11) or highly branched, core-fucosylated structures (12-15) has been related to various types of tumors.

The first attempts to use ricin as a cancer thera-peutic were based on its higher cytotoxicity in transformed cells (16), a mechanism that today would be called "targeted towards cancer related glycostructures". Its development, however, failed due to unexpectedly high toxicity (17). Riproximin showed potent antineoplastic activity that might be derived from its glycan binding profile. The aim of this study was to identify the glycans that function as binding receptors for riproximin, and are thus responsible for its tumor-specific cytotoxicity.

Using a glycan microarray, riproximin was found to specifically bind to two types of glycans, the N-glycan structures NA2/NA3 and the Tn anti-gen, a prominent cancer-related O-glycan (for abbreviations see Table 1). The mechanism of binding of lectins to multivalent globular and li-near glycoproteins was recently elucidated (18). Both the interaction of Gal-binding galectins with multiple NA3 structures on ASF (19), as well as that of GalNAc-binding soybean agglutinin with Tn on Tn-rich porcine submaxillary mucin (Tn-PSM) (20) were described to depend on bind-and-jump and negative cooperativity effects. Using similar model glycoproteins, the mechanism of riproximin’s interaction with its glycotargets and implications for its cytotoxicity were investigated. EXPERIMENTAL PROCEDURES

Riproximin purification and labeling−Riproxi-min was purified from Ximenia americana fruit kernels as described before (3). In short, the purifi-cation procedure included an initial aqueous ex-traction of proteins from the crude kernel material, removal of lipids with chloroform and subsequent chromatographic purification on a strong anion exchange resin and lactosyl-Sepharose.

For detection, riproximin was fluorescently la-beled with an amine-reactive, N-hydroxysuccin-imide ester activated dye (DyLight 549, Pierce, Rockford, USA) as described by the manufacturer. Protein containing fractions were pooled, washed and concentrated (molecular weight cut off, MWCO, 10,000 Da) in 20 mM Tris-HCl-buffer, pH 7.5 with 200 mM NaCl.

Fluorescently labeled riproximin was addition-ally purified on a lactosyl-Sepharose column (Lac-tosyl Sepharose 4 Fast Flow, GE Healthcare, Upp-sala, Sweden) as described before (3) to exclude all conjugates with blocked binding sites. The elu-ate fractions were concentrated (MWCO 10,000 Da) and the protein content was determined by the absorbance at 280 nm.

Integrity and biological activity of the labeled riproximin were controlled by SDS-PAGE and by cell viability assay with HeLa cells, respectively (see below). The dye payload was determined from the UV/Vis spectrum.

Binding analysis of riproximin in a carbohy-drate microarray−The carbohydrate microarrays were prepared as previously described, with the following modifications (21,22). The array con-tained 157 components including 97 different de-


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fined synthetic carbohydrates as bovine serum albumin (BSA) or human serum albumin (HSA) conjugates, 28 synthetic glycopeptides and 32 nat-ural glycoproteins. For a list of the carbohydrate structures see Table 1 of the Supplemental Materi-als. Samples were printed in duplicate on Super-Epoxy 2 Protein glass slides (TeleChem Interna-tional, Inc., Sunnyvale, CA) using a Biorobotics MicroGrid II microarrayer (Genomic Solutions, Ann Arbor, MI) fitted with Stealth pins (Telechem International; #SMP3, which produce ~100 µm spots). Relative humidity within the printing chamber was maintained at > 50%. Due to the small footprint of a single array (4.5 x 4.5 mm), sixteen copies of the full array could be printed onto each glass slide. Printed glass slides were stored at -20 °C until use.

Riproximin binding was evaluated using minor modifications of the previously reported protocol (21,23). Briefly, slides were fitted with a sixteen well-module (Grace Bio-Labs) to physically sepa-rate the sixteen printed copies of the array. Each well was blocked with 3% BSA (200 µl/well; im-munoglobulin-free BSA; Sigma-Aldrich) in PBS for 1 h. DyLight-labeled riproximin was diluted 1:25 in 3% BSA/PBS and then incubated on the array (75 µl/well) for 2 h at room temperature in the dark. After washing 6 times with PBS, the well-module was disassembled and the slide was incubated in PBS for 5 min. The slide was centri-fuged at 453 x g for 5 min, and then scanned using a GenePix Scanner 4000A (Molecular Devices Corporation, Union City, CA). Slides were scanned at 10 μm resolution and image analysis was carried out with Genepix Pro 6.0 analysis software (Molecular Devices Corporation). The fluorescent spots were defined as circular regions of interest (ROIs) with a diameter of 100 μm. ROIs were allowed to be adapted to the actual feature size by ± 30 μm. After local background subtrac-tion the median pixel intensity of ROIs was used to obtain a single fluorescence value for each spot. The mean value of the two replicate spots was used as the final value for each array component.

To investigate the competitive binding of riproximin with Tn3 structures, riproximin was incubated with 60 µg/ml Tn3-BSA (15 Tn3 mole-cules per BSA molecule) in 3% BSA/PBS for 1 h prior to the incubation of the mixture on the carbo-hydrate microarray.

Desialylation of bovine submaxillary mucin− Asialo bovine submaxillary mucin (aBSM) was

prepared by incubating 9 mg/ml of BSM with 28 mU/mg neuraminidase (Roche Diagnostics, Mannheim, Germany) in 50 mM Na-acetate-buffer, pH 5.0 for 3 h at 37 °C. As a control, the same amount of BSM was incubated in buffer without neuraminidase. The degree of desialylation was monitored by dot blot analysis using biotinyl-ated wheat germ agglutinin (WGA, Vector labor-atories, Peterborough, UK). For cell culture ex-periments, aBSM and BSM control samples were filter sterilized (Ø 0.45 µm). BSM sample concen-trations were determined using the Glycoprotein Carbohydrate Estimation Kit (Thermo Scientific, Pierce, Rockford, USA) and a BSM standard curve. The BSM sample concentration was also used to estimate the aBSM concentrations, since the desialylation procedure created additional re-ducing ends, which interfered with the measure-ment.

Enzymatic deglycosylation of asialofetuin−To deglycosylate ASF, 6 µg/µl of the glycoprotein were incubated for 5 min at 100 °C in denaturing buffer containing 50 mM sodium-phosphate-buffer, pH 7.5, 0.1% SDS and 50 mM β-mercap-toethanol. The mixture was cooled on ice and 0.75% (v/v) Triton X-100 as well as 1 μl N-glyco-sidase F (5000U/ml; Sigma Aldrich, Steinheim, Germany) were added subsequently. The control reaction contained no N-glycosidase. Reaction mixtures were incubated overnight at 37 °C. Pro-tein deglycosylation was monitored by SDS-PAGE.

Dot blot analysis−Glycoproteins were serially diluted in PBS and 1 µl of each dilution was spot-ted onto a nitrocellulose transfer membrane (Whatman Protran, Dassel, Germany). The mem-brane was dried at room temperature and blocked with 5% BSA. The membrane was probed with riproximin (30 µg/ml in 5% BSA) and after wash-ing incubated with an anti-riproximin monoclonal mouse antibody (mRpx-Ab #62). HRP-linked, human pre-adsorbed goat-anti-mouse antibody (Santa Cruz, Heidelberg, Germany) was used for detection.

Isolation of N-glycans from glycoproteins−The isolation of N-glycans from asialofetuin and fetuin was performed according to Karg et al., 2009 with some modifications (24). 100 mg/ml glycoprotein was digested with 110 mU/µl pepsin (Roche Diag-nostics, Mannheim, Germany) in 10 mM HCl for 48 h at 37 °C. Pepsin was inactivated by rising the pH above 5.0 with NaOH. The samples were buf-


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fered with Na-acetate buffer, pH 5.2 (final con-centration 100 mM). PNGase F (Sigma Aldrich, Steinheim, Germany) was subsequently added and the sample incubated for 24 h at 37 °C. Free N-glycans and smaller peptides were separated by ultrafiltration on membranes with a MWCO of 30,000 Da.

To remove the peptides, the glycans were addi-tionally purified on C18 resin (Waters Corpora-tion, Milford, USA) spin columns, which were prepared using 30 µl resin per column pre-washed with ethanol followed by H2O. The N-glycan-pep-tide mixtures were added to the spin columns, in-cubated for 5 min and centrifuged. The eluates were subsequently desalted on a cation exchange resin (AG 50W-X8, hydrogen form, 100-200 mesh; BioRad Laboratories, Hercules, USA). For this step, the resin was washed with H2O before transferring 0.6 ml to an empty spin column and drying the resin by centrifugation. The N-glycan- containing eluates were loaded onto the resin, in-cubated at room temperature for 10 min and eluted by centrifugation. The N-glycan samples were dried in a speed-vac and stored at 4 °C.

Enzyme-linked immunosorbent assay (ELISA)−Glycoprotein binding assays were per-formed using Immuno 96 MicroWell Solid Plates (Maxisorp, NUNC, Langenselbold, Germany) coated with 1 µg/ml ASF or aBSM in PBS, re-spectively. After blocking with 5% BSA/PBS, a one hour pre-incubated mixture of riproximin and serially diluted glycoprotein was added. Riproxi-min was detected using the monoclonal riproximin antibody Rpx-mAb #62 followed by a HRP-cou-pled, human pre-adsorbed anti-mouse antibody (SantaCruz, Heidelberg, Germany). Binding was colorimetrically measured using 3,3′, 5,5′-tetra-methylbenzidine (TMB). When ASF binding was assessed, 5% milk/PBS was used for blocking and an ovine anti-bovine asialofetuin IgG (AbD Sero-tec, Düsseldorf, Germany) followed by HRP-linked anti-sheep-antibody were used for detection.

Binding signals of 2-3 replicates were averaged and plotted. Concentration-response curves were fitted using the log-logistic model. To directly refer the potential inhibition effects of the glyco-proteins to the relative number of carbohydrate structures, the number of N-acetyl-D-lactosamine (LacNAc) and Tn (GalNAc) residues per ASF and aBSM was estimated as follows. For ASF, a mo-lecular weight of 48 kDa and three tri-antennary NA-glycans resulting in nine terminal LacNAc

residues were used for calculation. For aBSM, a molecular weight of 400 kDa was assumed (25,26). 920 GalNAc residues per molecule were estimated for aBSM based on data available for PSM, which possess a molecular mass of 106 Da with ~2300 GalNAc residues per molecule (20).

Desialylation of cells with neuraminidase−For desialylation of the cell surface, MDA-MB-231, MCF7 or HeLa cells were seeded into microplates and allowed to settle down overnight. Neuramini-dase (Roche Diagnostics, Mannheim, Germany) was diluted into media without FCS to a concen-tration of 1 mU in 50 µl. FCS containing media was removed from the cells and replaced by the neuraminidase containing, FCS free media. Con-trol cells received FCS free media without neu-raminidase. The plates were incubated for 1 h at 37 °C. Subsequently, neuraminidase containing and control media were replaced by fresh FCS containing media and the cells were treated with serial dilutions of riproximin. Cell viability was tested with the MTT assay after 72 h incubation (see below). Three independent experiments were performed for cells in which an effect of neuram-inidase exposure was observed on riproximin cy-totoxicity.

For the analysis, neuraminidase pre-treated cells (Neu+/Rpx) and control cells (Neu-/Rpx) were analyzed as two separate groups. For each experiment, antiproliferative activity was meas-ured in triplicate for each riproximin concentration as well as for cells treated with solvent control. Observed antiproliferative activity response values were normalized to mean values of solvent control cells and averaged per riproximin concentration. For each treatment group, the four-parameter log-logistic model (27) was fitted to the averaged nor-malized antiproliferative activity values. From each of the two fitted antiproliferative activity curves, the IC50 value, defining the concentration that produces 50% of the responsive maximal cy-totoxic effect of riproximin, was estimated.

The ratio of the two IC50 estimates, i.e. (IC50 Neu-/Rpx)/ (IC50 Neu+/Rpx), and the according 95% confidence interval were computed to assess statistical significance. The IC50 shift was consid-ered significant when the IC50 ratio explicitly dif-fered from the value “1” and the respective 95% confidence interval did not include the value “1”.

Competitive MTT cell viability assay−The cy-totoxic activity of riproximin was assessed using the MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphe-


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nyl tetrazolium bromide) cell viability assay on HeLa, MCF7 and MDA-MB-231 tumor cell lines. Cells were propagated in a humid atmosphere containing 5% CO2 at 37 °C in media that was supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. For the assay, the cells were seeded into micro-plates (2500 cells/well for HeLa, 3000 cells/well for MDA-MB-231, 3500 cells/well for MCF7) and allowed to settle down overnight. To analyze the influence of single glycoproteins, serial dilutions of the glycoproteins ASF, Fet, aBSM and BSM were added to the cells prior to the addition of riproximin in a concentration corresponding to its IC50 (HeLa: 0.14 ng/ml, MCF7: 0.10 ng/ml, MDA-MB-231: 0.75 ng/ml). Plates were incubated for 72 h and the cell growth (CG) was determined by the MTT method.

To investigate the inhibitory effect of the gly-coprotein derived N-glycans alone, the HeLa cells were treated with N-glycans obtained from ASF and Fet by PNGase F treatment (see above). The glycan effect on the cytotoxicity of riproximin was referred to the originating protein amount that had been used for deglycosylation.

For data analysis, the inhibitory effect of the single glycoproteins (GP) on riproximin’s (Rpx) cytotoxicity (inhibition of riproximin’s cytotoxi-city, IRC (%)) was calculated as (CGGP/Rpx – CGRpx) / (100 – CGRpx) · 100. Both values, the CGGP/Rpx (average cell growth with glycoprotein and riproximin) and CGRpx (average cell growth with riproximin alone), were normalized to the respective control without riproximin treatment. Computed IRC values were averaged for every glycoprotein concentration. Inverse cell viability curves were determined for each single glycopro-tein, by fitting the four-parameter log-logistic model (27). The IRC50 value determined from the curve depicts the glycoprotein concentration that inhibits 50% of the riproximin’s cytotoxicity. Ac-cordingly, IRC25 and IRC75 values describe the concentrations inhibiting 25% and 75% of the riproximin cytotoxicity.

Statistical two-way analysis of variance (ANOVA) with interaction was conducted to as-sess significance. The effects of the independent factors ‘group’ (i.e. native versus analogical desialylated glycoprotein) and ‘concentration’ (i.e. the concentration of the glycoprotein) as well as the interaction effect between group and concen-tration on calculated average IRC values (depen-

dent model variable) were studied by performing according global F-tests. ANOVA was carried out on a significance level of 5%, i.e. p-values of p ≤ 0.05 obtained in the F-tests were regarded as statistically significant.

Combination cell viability assays−For the in-vestigation of ASF and aBSM in combination, different concentrations of these glycoproteins were combined according to the inhibiting effect of the single proteins. Concentrations correspond-ding to the IRC25, IRC50 and IRC75 of ASF were combined with aBSM concentrations correspond-ding to IRC75, IRC50 and IRC25, respectively, resul-ting in the initial concentration for each combina-tion. Serial dilutions of all three combinations were added to the cells prior to the treatment of the cells with riproximin, as described above.

Four MTT viability experiments were per-formed for each of the three combinations. Data analysis was performed as for the experiments with the single glycoproteins (see above). To cir-cumvent potential problems due to negative or > 100% IRC, the model parameters of the lower and upper curve asymptotes were constrained to be ≥ 0 and ≤ 100, respectively. Combination indices (CIs) were used to assess the effect of each combi-nation of ASF and aBSM on riproximin’s cytoto-xicity. CIs and the corresponding pointwise 95% confidence bounds were computed for theoretical IRC values ranging between 0.01 and 0.99 (i.e. between 1% and 99%), with a step size of Δ = 0.005 between two neighboring theoretical IRC values, as described by Lee and Kong 2009 (27). For each of the three examined combinations of ASF and aBSM, estimated CIs and their 95% confidence bounds were plotted against the theo-retical IRC values. Combination effects of ASF/ aBSM combinations were characterized as syner-gistic for CI < 1, as additive for CI = 1, and as an-tagonistic for CI > 1.

Statistical software−Data analysis of the carbo-hydrate microarray, ELISA and cell viability ex-periments was performed with Microsoft Excel. For further analysis the open-source statistical software environment R, version 2.8.0 (http://www.R-project.org) was used. Specifically, the R application package ‘drc’ was applied for fitting cell viability curves and for estimation of IC50 values from the calculated curves, as well as for computation of the corresponding IC50 ratio with its 95% confidence interval. The same pack-age was used for fitting inverse cell viability


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curves and for estimation of IRC25, IRC50 and IRC75 values from the computed curves. Combi-nation indices and according pointwise 95% confi-dence bounds were computed using a specific R function that was written and provided online by Lee and Kong (http://biostatistics.mdanderson.org/Software Download/) (27). RESULTS

Carbohydrate microarray analysis−Fluoro-phore-labeled riproximin was investigated on a glycan microarray with 97 carbohydrates, 28 gly-coproteins and 32 glycopeptides. Glycan structures with riproximin binding signals of > 100 relative fluorescence units (RFU) are shown in Figure 1A. For fluorescence intensity data of all 157 glycan structures see Table 2 of the Supplemental Materi-als. From these carbohydrates, riproximin bound selectively to NA2, NA3 and Tn3 structures. The NA structures designate bi-antennary (NA2) and tri-antennary (NA3) complex N-glycans with ter-minal Galβ1-4GlcNAc- structures. The Tn3 glycan consists of three consecutive O-linked GalNAcα- serine residues that form a short mucin-like poly-peptide backbone, a type of structure also referred to as clustered Tn. The binding signal of riproxi-min to NA3 was the strongest (> 20,000 RFU) fol-lowed by NA2 (> 10,000 RFU). The binding signal of riproximin to Tn3 was about 5-fold lower than to NA3 but still significant. Binding was strongest at highest Tn3 density (Ac-Tn3-3 << Ac-Tn3-15 << Ac-Tn3-27).

From the group of the glycoproteins, riproximin showed strong binding to asialofetuin (ASF, > 15,000 RFU), a glycoprotein with NA2 or NA3 N-glycan structures and weaker binding (< 5,000 RFU) to the desialylated glycoproteins bovine (aBSM) and ovine (aOSM) submaxillary mucin, both glycoproteins with massive O-glycosylation and a high proportion of Tn antigens. Riproximin also showed significant binding to the carcinoem-bryonic antigen (CEA) that is reported to contain N-glycan structures (Figure 1A).

Taken together, riproximin’s binding profile on the array was very narrow and confined to two types of glycans, the NA2/NA3 structures from the group of complex N-glycans and the O-glycan Tn3 structures representing the mucin-type clustered Tn.

To further investigate the relationship between the two binding activities, competitive binding

experiments were performed. The pre-incubation of riproximin with Tn3 linked to BSA led to a de-crease of riproximin binding to O-glycan rich structures, such as Tn3 or the mucins aBSM and aOSM. In contrast, the N-glycan structures NA2 and NA3 and the glycoprotein CEA showed no decrease in the binding signal. However, the signal of ASF declined to approximately 50% suggesting the presence of a second riproximin binding site with lower affinity (Figure 1A).

Binding analyses−For validation of the micro-array binding results, selected glycoproteins were investigated by dot blot analysis. ASF, Fetuin (Fet), N-deglycosylated ASF (ASFdeg), aBSM and BSM were immobilized on a nitrocellulose mem-brane and subsequently probed with riproximin. As expected, riproximin showed distinctly stronger binding to the desialylated glycoproteins than to their native forms. N-deglycosylation of ASF re-sulted in a significant loss of riproximin binding (Figure 1B), confirming that riproximin binding is confined to the sugar component of this glycopro-tein.

The two glycoproteins ASF and aBSM were thus chosen as model proteins to represent the two types of glycans NA3 and clustered Tn for further characterization of riproximin’s binding mecha-nism and its biological significance.

Riproximin’s binding affinity for the two model proteins was measured using the Microscale Thermophoresis method. For ASF, two Kd values of 11 nM and 7 µM were determined. The second affinity constant is in line with the assumption that riproximin also binds to a non-NA3 glycoconju-gate on the ASF molecule. Riproximin bound to aBSM with a Kd of > 50 µM. Two similar Kd val-ues of 48 nM and 1.1 µM were also detected in a preliminary Isothermal Titration Calorimetry (ITC) experiment, when riproximin was titrated with ASF and the two sites binding model was applied for the analysis of the data. However, due to the high microdiversity of the binding partners, the interactions measured in this experiment turned out to be too complex to be covered by the available models and no reasonable stoichiometry data were obtained. The complete thermophoresis and ITC data are presented in Figures 2 and 3 of the Sup-plemental Materials, respectively.

Competitive binding analyses of ASF and aBSM in ELISA−To further characterize the bind-ing of riproximin to the two classes of glycans, ELISA analyses were performed with riproximin


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alone or under competitive conditions. On an ASF coat, the pre-incubation of riproximin with ASF or aBSM resulted in a decrease of the signal with in-creasing ASF or aBSM concentrations (Figure 2A). On each ASF molecule, three NA3 and thus nine LacNAc structures have been reported. Mu-cins such as aBSM, however, show a much higher GalNAc density of > 1000 per molecule. The com-petitive effect of both glycoproteins in the ELISA was therefore also related to the absolute number of the respective carbohydrate structure, LacNAc for ASF and GalNAc for aBSM (Figure 2B). The resulting curves indicate that the aBSM competi-tion was only effective at very high Tn concentra-tions, as expected from the higher affinity of riproximin for LacNAc structures. On aBSM coat, however, pre-incubation with ASF did not reduce the riproximin signal, despite the higher affinity of riproximin for ASF (Figure 2C).

To test whether the lower signal at high aBSM concentrations was not an experimental artifact, plates were either coated with ASF followed by incubation with different riproximin concentra-tions, or directly coated with different riproximin concentrations under identical conditions. Upon aBSM incubation, detection of riproximin de-creased with rising aBSM concentrations for both ASF-bound and directly coated riproximin, but steeper for the ASF coat (Figure 2D). Moreover, aBSM abolished the riproximin signal only for ASF bound riproximin completely.

A riproximin signal increased to more than 100% was detected on the ASF coat at low aBSM concentrations (Figure 2A and 2D). This finding indicates that riproximin may have crosslinked riproximin-loaded aBSM to the ASF coat. To fur-ther investigate crosslinking of ASF and aBSM by riproximin, an antibody against ASF was used. Coated aBSM was incubated with a serial dilution of riproximin in the presence of 10 or 50 µg/ml ASF. The results showed significant binding of ASF to aBSM that was dependent on the ripro-ximin but not the ASF concentration (Figure 2E). As shown in Figure 2C, aBSM crosslinking to itself was not observed. The riproximin signal remained ≤ 100% even when lower aBSM con-centrations were tested (data not shown). Con-versely and consistent with the second binding constant observed for ASF, riproximin showed some ASF crosslinking to itself at high riproximin to ASF ratios (Figure 2F).

Influence of different glycoproteins and sugar moieties on riproximin cytotoxicity−To investigate the biological significance of riproximin’s binding to the identified glycostructures, the chosen model glycoproteins ASF and aBSM and their sialylated counterparts Fet and BSM were tested in three different tumor cell lines (HeLa, MCF7 and MDA-MB-231) to evaluate their effect on riproximin cytotoxicity.

The neutral glycoproteins ASF and aBSM in-hibited riproximin’s cytotoxicity significantly stronger than their sialylated counterparts BSM and Fet in all three cell lines tested (all p < 0.01; F-test for factor ‘group’). In addition, the statistical analysis revealed that this effect was concentration dependent, because with increasing concentrations the difference between the inhibitory effect of the neutral and sialylated glycoproteins also increased (all p < 0.01; F-tests for factor ‘concentration’ and for the interaction between ‘group‘ and ‘concen-tration‘).

The relative inhibition of riproximin cytotoxi-city achieved by ASF or aBSM, however, varied between the cell lines. While ASF and aBSM were both able to completely abolish riproximin’s cyto-toxicity in HeLa cells (Figure 3A+B), only 70% maximal inhibition could be achieved in MCF7 cells (Figure 3D+E). A particularly intriguing finding was that the inhibitory effect of aBSM decreased at high aBSM concentrations in MDA-MB-231 cells (Figure 3G).

Cytotoxic activity of riproximin was also re-duced in HeLa cells following co-treatment with N-glycans that were obtained by deglycosylation of ASF with PNGase F. This finding demonstrated that the inhibitory effects of glycoproteins on riproximin cytotoxicity were glycan but not core-protein dependent (Figure 3C).

Influence of the sialyl caps on riproximin’s cy-totoxicity−To investigate the relationship between riproximin cytotoxicity and the abundance of sialyl groups on the cell surface, HeLa, MCF7 and MDA-MB-231 cells were treated with neuramini-dase and subsequently exposed to riproximin in a cellular viability assay. A detectable increase in riproximin’s cytotoxicity was observed only in the MDA-MB-231 cells. For untreated MDA-MB-231 cells, an IC50 value of 0.22 ng/ml (95% confidence interval, CI: 0.192-0.250) was estimated for ri-proximin. After treatment with neuraminidase, the IC50 value decreased significantly (IC50 = 0.08 ng/ml, 95% CI: 0.074-0.094). The ratio of the IC50


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values with and without neuraminidase treatment of 2.64 (95% CI: 2.256-3.081) demonstrated that the enzymatic removal of the sialic acid caps from glycans on the cell surface led to significantly en-hanced cytotoxicity of riproximin in MDA-MB-231 cells (Figure 3H). In contrast, neuraminidase treatment did not influence the cytotoxic potency of riproximin in MCF7 or HeLa cells (data not shown). It is conceivable that the removal of addi-tional sialic acids in the most sensitive, sialyl-poor MCF7 cells could not further increase its riproxi-min response. Furthermore, the fact that neura-minidase treatment did not influence the sensitivity of HeLa cells would indicate a low cell surface sia-lyl content.

Competitive influence of ASF and aBSM on riproximin activity−To investigate the functional effects of the interactions between riproximin and the two different types of glycans that were found to be its binding partners, the model proteins ASF and aBSM were added in combination to compete with riproximin in cytotoxicity assays. Because of the high inhibitory effect of both glycoproteins, HeLa cells were chosen for the competitive inves-tigation. ASF and aBSM concentrations were combined in three different proportions (ASF/aBSM: IRC25/IRC75, IRC50/IRC50, IRC75/IRC25) according to the degree of riproximin cytotoxicity inhibition each of them had achieved alone (IRC values in Table 2). The three different combinations were serially diluted resulting in three concentration ranges (Table 3 of the Supple-mental Materials) and added in combination with riproximin to HeLa cells. The respective inverse cell viability curves depicting the cytotoxicity in-hibition effects of the three combinations are shown in Figure 4A-C. The theoretical IRC values calculated for each combination model were ex-amined for additive, synergistic or antagonistic effects (Figure 4D-F).

Combinations of the IRC25 of ASF with the IRC75 of aBSM contained a lower ASF, i.e. NA-type active component, proportion and showed an additive effect that did not depend on the overall cytotoxicity inhibition (Figure 4D). The other combinations (ASF with IRC50 + aBSM with IRC50 as well as ASF with IRC75 + aBSM with IRC25) showed an effect that was dependent on the overall cytotoxicity inhibition degree achieved (Figure 4E+F). At low riproximin cytotoxicity inhibition, <27% for ASF50/aBSM50 or <6% for ASF75/aBSM25, both combinations showed a

synergistic effect. For inhibition degrees of 26-79% (ASF50/ aBSM50) or 6-71% (ASF75/ aBSM25), an additive effect was observed. At overall inhibition rates over 79%, the combination effect turned into an antagonistic one (Figure 4E+F).

In summary, the mode of inhibition of riproxi-min cytotoxicity was dependent on the proportion of each glycoprotein i.e. glycan type in the combi-nation, as well as on the cumulated glycoprotein amount. For most combinations, the effect was additive, which implies that ASF and aBSM were able to inhibit riproximin in an independent man-ner. DISCUSSION

NA2/NA3 and cancer-related, clustered Tn gly-costructures are specifically bound by riproximin− Riproximin, a type II ribosome inactivating protein (RIP), was purified as a galactose/lactose specific protein (3). At first, the lactose binding and elution suggested broad galactose specificity similar to that of ricin and abrin (28,29), both very toxic RIPs that render Ricinus communis and Abrus precatorius seeds lethal on ingestion. However, riproximin’s low peroral toxicity indicated that its binding profile would be narrower. Indeed, analy-sis in a carbohydrate microarray revealed that riproximin preferentially binds to two groups of glycoconjugates, the branched bi- and tri-anten-nary N-glycan structures NA2 and NA3 and the O-glycan structure known as clustered Tn, especially when present at high density. The terminal residue of NA2/NA3 structures is a Galβ that is connected to a GlcNAc of each antenna. Clustered Tn con-sists of a series of single GalNAcα residues (single Tn) bound to adjacent amino acids in protein re-gions rich in Ser/Thr repeats. They are typical for the extracellular domains of mucins. Tn and par-ticularly clustered Tn is an established tumor-spe-cific antigen present on many adenocarcinomas (30-32).

Riproximin’s specificity for the clustered Tn antigen is remarkable and could explain its tumor specificity previously described (1,2). Prominent targets for riproximin on cancer cells could there-fore be the cancer-associated mucins (MUCs). MUC 6, for example, was shown to be responsible for the high density of clustered Tn on the surface of the highly sensitive MCF7 cells (33). Cancer-specific MUC 1 and MUC 2 were also described as Tn-rich glycoproteins (34,35). However,


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riproximin showed its highest binding for bi- and tri-antennary complex N-glycans (NA2 and NA3 structures). No direct link between the presence of NA2 or NA3 and cancer has been established so far. However, N-glycans from cancer cells show higher branching resulting in a higher NA3 density (15,36,37) that would provide additional riproxi-min binding sites. On the other hand, it cannot be excluded that these structures are also present on normal cells, since 17% of the IgG molecules in human serum are NA2 glycosylated (10).

Riproximin is specific for asialo glycostruc-tures−Riproximin showed a clear preference for desialylated glycan structures. Most cancer cells and cancer-related glycoproteins show abnormal sialylation, but its investigation revealed contro-versial results. For many tumors, increased sialyl-Lewis X expression was described (15,38-40). Conversely, N-glycans of the prostate-specific antigen (PSA) showed a tumor-dependent decrease of sialylation (41-43). While cancer derived O-glycans were often described as highly sialylated, they also contain the sialyl-free, cancer specific Tn and T antigens (44). The sialidase Neu 3, which is located in the plasma membrane and leads to si-alyl-cap removal, is up regulated during carcino-genesis (45).

An increase in riproximin cytotoxicity was ob-served upon neuraminidase treatment for the strongly sialylated MDA-MB-231 cells but not for the sialyl-poor MCF7 (46), the most sensitive cell line within the panel tested (2). This experiment demonstrated a direct relationship between the biological activity of riproximin and the extent of sialylation found on cell surfaces.

Riproximin displays a narrow binding profile− Riproximin demonstrated remarkable selectivity on the glycan array. Despite the presence of many other sialyl-free glycan structures with terminal Galβ or GalNAcα on the array, such as LNnT (Galβ1-4GlcNAcβ1-3Galβ-) or the tumor related Adi (GalNAcα1-3 Galβ-) (47), Forssman antigen (GalNAcα1-3GalNAcβ-) (48) and TF antigen (Galβ1-3GalNAcα-) (49), no binding of riproximin to these structures was detected. In comparison, other commonly studied Gal and GalNAc binding lectins, such as RCA (Ricinus communis aggluti-nin), SBA (soybean agglutinin), HPA (Helix pomatia agglutinin), jacalin, and BPL (Bauhinia purpurea lectin) recognize a wide range of glycans on the array. For example, the Gal-binding type II RIP RCA significantly bound to almost any struc-

ture with a terminal LacNAc and lactose (50). Thus, riproximin showed a very narrow binding profile that was not only dependent on the nature of the terminal sugars but also on their amount and constellation.

The preference of riproximin for trimeric structures such as NA3 and Tn3 was also remarka-ble. The three-fold sugar specificity might reflect the structure of the typical RIP B-chain binding domains, which are both trimers of an ancient lec-tin motif. However, molecular modeling of riproximin B-chain interaction with NA3 struc-tures revealed that the terminal Gal residues on NA3 cannot span the distance between two sub-domains, but could interact with other aromatic residues that are frequently present in the closer neighborhood. A multivalent binding of riproximin to NA2 or NA3 structures could explain the ob-served high specificity, which is unusual for a lec-tin and comparable to that of a monoclonal anti-body.

On the other hand, it cannot be completely ruled out that the stronger binding signals on NA3 result from stoichiometric and/or bind-and-jump effects. Binding of two or three ricin B-chains to a single NA3 structure has been described (51). However, the fact that the presence of an addi-tional terminal Galβ in NA4 structures did not improve, but significantly reduced the binding signal, contradicts this hypothesis.

Glycoproteins as riproximin’s binding counter-parts−Accordingly, the NA2/NA3 containing gly-coproteins asialofetuin (ASF) (52) and carci-noembryonic antigen (CEA) (53) as well as the Tn-rich glycoproteins aOSM and aBSM (54) on the array showed strong riproximin binding. ASF and aBSM were thus chosen as model proteins to mimic the effects of the NA2/NA3 and Tn-struc-tures, respectively, in cellular experiments.

Both asialo-glycoproteins showed significant inhibition of riproximin cytotoxicity in HeLa, MCF7 and MDA-MB-231 cells. Moreover, degly-cosylation of ASF resulted in loss of its riproximin binding, while the ASF derived N-glycans signifi-cantly inhibited riproximin’s cytotoxicity. These findings demonstrate that specific binding of riproximin to NA2/NA3- and/or Tn-glycostruc-tures on the cellular surface is a prerequisite for cytotoxicity.

The degree of inhibition, however, strongly de-pended on the cell line (i.e. the cell surface glyco-sylation). For example, >10-fold higher glycopro-


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tein concentrations were required in the particu-larly Tn-rich MCF7 cells (33) to reduce riproximin sensitivity by 50% as compared to HeLa cells, although HeLa and MCF7 are equally sensitive to riproximin. It must therefore be assumed that the abundance and surface distribution of glycostruc-tures on a particular cell play a crucial role in its sensitivity to riproximin. Moreover, it cannot be excluded that additional riproximin glycotargets exist on the cell surface, which have not been identified yet.

Two different binding sites are associated with different specificities of riproximin−The specific binding of riproximin to both Galβ within NA2/NA3 and GalNAcα within Tn structures re-lated to the presence of two binding domains in the B-chain of riproximin. The structures of the clus-tered Tn antigen and NA2/NA3 are significantly different, and it is uncommon for a lectin or mono-clonal antibody to bind both. For example, Tn spe-cific antibodies have never shown binding to NA2/NA3 (55). The very narrow binding profile of riproximin makes the recognition of both clustered Tn and NA2/NA3 in the same binding site very unlikely. We thus hypothesized that riproximin binds the two sugar structures with different bind-ing sites corresponding to the two sugar binding sites of the B-chain.

To evaluate this hypothesis, competitive anal-yses were performed. In array experiments, pre-incubation of riproximin with Tn3 decreased the binding of riproximin to immobilized Tn3 struc-tures and aBSM but did not influence the NA binding. However, since the NA affinity of riproximin is significantly higher than its Tn3 af-finity, it cannot be excluded that the Tn3 concen-trations used in the experiment was too low to affect NA binding.

The strongest argument supporting the exist-ence of two different binding specificities is based on the finding that riproximin was able to crosslink the two proteins ASF (NA2/NA3 structures) and aBSM (Tn structures). On the other hand, even at low aBSM concentrations no crosslinking of aBSM with itself could be detected. However, at very low ASF to riproximin ratios, a significant amount of ASF-crosslinking was observed, which probably results from the interaction of the riproximin GalNAc-binding site with an addi-tional, probable non-NA3 sugar of ASF. This finding is consistent with several other observa-tions regarding the interaction of riproximin with

ASF, including the partial competition of ASF binding by Tn3 structures observed in the array, as well as the second affinity constant determined for riproximin and ASF. For fetuin, the presence of an O-linked glycoconjugate has been described (56) and it is conceivable that the desialylated structure would be present on ASF.

Riproximin’s affinity for glycostructures de-pends on avidity effects−In the microarray experi-ments, riproximin showed a distinctly stronger binding signal to NA2/NA3 than to Tn glyco-structures. In microscale thermophoresis experi-ments, the high affinity of riproximin for ASF was >1000-fold higher than for aBSM. In the ELISA experiments, however, aBSM was able to compe-titively inhibit the binding of riproximin to ASF at high concentrations, but not vice versa. Moreover, in cellular experiments, aBSM was the stronger cytotoxicity competitor. Even when the high num-ber of Tn structures on aBSM was considered, the concentration needed to achieve the same inhi-bition was lower for the aBSM-derived Tn than for the ASF-derived LacNAc. These observations demonstrate that the interaction of riproximin with a particular glycostructure is not confined to a strict lock-and-key correlation but is strongly in-fluenced by dynamic interactions similar to those described by Dam and Brewer (18).

Riproximin primarily interacts with terminal Gal and GalNAc, respectively, via its two binding sites, with low but significant affinity. The purifi-cation of riproximin on Gal-exposing resins was based on this primary affinity. Sequence analysis and molecular modeling data revealed that the typical type II RIP Gal-binding activity is retained for both α1 and γ2 subdomains of the riproximin B-chain (2). However, the highly specific interac-tions of riproximin with glycoconjugates present-ing two or three moieties of the preferred terminal sugars Gal (NA2/NA3) or GalNAc (Tn3) point towards additional glycan-protein interactions.

When the interactions of riproximin with each of the proteins ASF and aBSM is considered, the main features of the mechanism of lectins binding to multivalent targets apply (19,20). On multiva-lent structures several equivalent epitopes are available for the so-called bind-and-jump effects; the lectin can be recaptured by each of the re-maining free epitopes leading to decreased dissoci-ation. On the other hand, negative cooperativity results from decreasing functional valence when more lectin molecules occupy additional epitopes.


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The affinity of each consecutive binding step frac-tionally decreases due to the decreased number of free epitopes available for recapturing.

Based on the hypothesis of the two binding sites specific for NA2/NA3 and Tn3, respectively, riproximin would be able to interact with three NA3 structures of ASF and with >1000 Tn clusters of aBSM, respectively. There is probably a second, O-linked glycan of ASF that would be able to in-teract with the Tn3 - binding site of riproximin. These interactions and their effect on riproximin binding and detection in ELISA experiments are schematically depicted in Figure 5A-D. In particu-lar, the high epitope density on aBSM is expected to allow broad dynamic interactions with the Tn3-binding site of riproximin.

The increasing affinity of riproximin for BSA bearing Tn3 at increasing density (Tn3-03 << Tn3-15 << Tn3-27) and its high affinity for mucins bearing up to thousands of Tn per molecule reflect the bind-and-jump effects that were described by Dam et al. for the interaction of soybean agglutinin with porcine submaxillary mucin (57). Together with the high Tn concentration of aBSM, this dy-namic increase in affinity explains the high com-petitive potency of aBSM in ELISA or cell expe-riments. On the other hand, since the NA2/NA3 structures are only present at comparatively low density, they did not provide enough binding sites for a detectable bind-and-jump - related increase in specificity.

The simultaneous interaction of riproximin with both proteins therefore strongly depends on the relative amounts of riproximin, NA2/NA3 and Tn3 epitopes. In the presence of both structures, riproximin crosslinked the NA2/NA3 and Tn3 epitopes via its two binding sites (Figure 5D). It is very likely that crosslinking via riproximin also occurs on the cell surface. Crosslinking is known to be important for internalization of several cell surface receptors (58,59). Since ASF alone was able to inhibit the cytotoxicity of riproximin by up to 50% in MCF7 cells that are particularly Tn rich (33) but have < 5% NA2/NA3 (60), it is very probable that the crosslinking is also important for the internalization and cytotoxicity of riproximin.

In the presence of high aBSM concentrations, however, the interaction of riproximin with the high density of Tn antigens masked its epitopes. A large cell surface mucin molecule might therefore be able to recruit riproximin and concomitantly

mask its second, NA-specific binding site, thereby inhibiting its crosslinking potential (Figure 5C).

Riproximin’s cytotoxicity was influenced by its dynamic interaction with ASF and aBSM−To in-vestigate the interdependence of binding, cross-linking and cytotoxicity within the complex envi-ronment of a cell, viability experiments were per-formed in which both ASF (NA3) and aBSM (clustered Tn) were allowed to compete for riproximin binding and thus inhibit its toxicity. The results of these experiments revealed that the interaction patterns of the mixtures containing a high aBSM proportion clearly differed from those of mixtures containing equal active amounts of aBSM and ASF or low aBSM proportions. More-over, the combinatory effects of the latter two combinations were strongly dependent on the total glycoprotein concentration. The interactions ex-pected to be responsible for these effects are sche-matically depicted in Figure 4 of the Supplemental Materials.

Mixtures with a high proportion of aBSM ex-hibited a broad additivity, thus supporting the hy-pothesis that both binding sites of riproximin are necessary for its cytotoxic activity. In these mix-tures, the riproximin to aBSM molecular ratio is particularly low. Increased internal diffusion and bind-and-jump effects lead to an enhanced aBSM affinity resulting in riproximin sequestration and masking of its ASF binding site. Very little cross-linking occurs under these circumstances, so that the entire ASF fraction would be available to bind to free riproximin leading to an additive effect.

Conversely, mixtures containing ≤50% of aBSM showed synergistic effects at low global concentrations that turned into additivity and eventually antagonism at high concentrations. Due to the higher riproximin to aBSM ratio, more riproximin molecules are available to bind to the same aBSM molecule. Less Tn3 binding sites are available for bind-and-jump effects leading to a lower apparent affinity and less masking. At high overall glycoprotein concentrations, ASF would bind to unmasked riproximin molecules that are also bound to aBSM; crosslinking would occur. Since a significant amount of glycoproteins bind the same riproximin molecule, antagonism would be observed. At low global concentration of both glycoproteins the chance for crosslinks decreases, so that ASF and aBSM would bind different riproximin molecules. The synergistic effects ob-served under these circumstances strongly suggest


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that blocking of a single riproximin binding site significantly interferes with the activity of ripro-ximin. A schematic of the interactions of riproxi-min with the two glycoproteins in each of the three different cases discussed is presented in Figure 4 of the Supplemental Materials. Overall, these fin-dings indicate that crosslinking is part of the mechanism responsible for riproximin’s internali-zation and cytotoxicity (Figure 5E).

In summary, even in the cellular context of a single cell type, the interaction pattern of riproxi-min with its glycotargets was very complex and depended on dynamic effects. At low riproximin concentrations, the internal diffusion along large glycoproteins predominated. At high concentra-tions, negative cooperativity opposed the bind-and-jump effects and favored crosslinking. Cellular experiments revealed that in spite of the lower affinity of aBSM to riproximin in direct binding experiments, in cellular assays it had the stronger impact on the cytotoxicity of riproximin.

Conclusions−In summary, the antineoplastic-active type II RIP riproximin was shown to spe-cifically bind to two types of glycostructures, the N-linked NA2/NA3 and the O-linked clustered Tn tumor-specific antigen. The two specificities were related to the two binding sites present on the riproximin B-chain. The sugar interactions of ri-proximin were shown to combine high specificity with dynamic interactions that are typical for lec-tins interacting with multivalent binding targets.

Understanding the mechanism of riproximin targeting is particularly important, since its thera-

peutic potency strongly depends on the presence of definite, cancer related structures on the cells to be targeted. Crosslinking of the two structures NA2/ NA3 and Tn3 might confer on riproximin an en-hanced selectivity for cells exposing both struc-tures. However, a relation between the parallel occurrence of these two structures and cancer has not been established yet. The ideal riproximin tar-get cell would contain surface glycostructures with high Tn densities and sialyl-free NA3 structures. On the other hand, due to the broad range of dy-namic interactions riproximin can get involved in, it is conceivable that the biological activity of riproximin might be modulated by the addition of particular glycan structures such as glycopolymers with high riproximin binding capacity but low affinity. Such a polymer could function as a "car-rier" for riproximin, preventing it from binding to low affinity structures and delivering it specifically to tumor cells where it would find high avidity targets.

Since the field of glycobiology is rapidly ex-panding, the availability of synthetic complex gly-can structures and the development of novel tools will support further investigations. The detailed characterization of the binding properties of riproximin reported here provides a model for functional lectin studies. Moreover, riproximin can be included in the panel of lectins with well-cha-racterized properties that currently find a broad usage in glycobiology.

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Acknowledgements - We thank NanoTemper Technologies GmbH for kindly providing the Monolith in-strument for the affinity measurement and the calculations of the affinity constants. We also thank Dr. Vladimir Rybin from the Protein Expression and Purification Core Facility at the European Molecular Biology Laboratory for his support in performing the Isothermal Titration Calorimetry experiment. FOOTNOTES * This research was supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, USA. Helene Bayer was funded by grants from the German Federal Ministry of Economics and Technology (Pro Inno II, KF0425101UL6 and ZIM, KF2301002AJ0). 5 To whom correspondence should be addressed: Dr. Cristina Voss, Department of Biochemistry, Heidel-berg Pharma AG, Schriesheimer Strasse 101, 68526 Ladenburg, Germany; Phone: +49 6203 1009 29; Fax: +49 6203 1009 764; E-mail: [email protected] 6 For frequently used abbreviations see Table 1. FIGURE LEGENDS FIGURE 1: Binding profile of riproximin. A. Fluorophore-labelled riproximin (Rpx, 27 µg/ml) was applied to the carbohydrate microarray. For the competition experiment, riproximin was pre-incubated with 60 µg/ml BSA carrying 15 Tn3 moieties (Rpx + Tn3). Carbohydrate structures with signals >100 RFU are shown. For carbohydrate abbreviations and the numerical data of all 157 glycan structures see Table 1 and 2 of the Supplemental Materials, respec-tively. The number after carbohydrate abbreviation refers to the average number of carbohydrates per molecule BSA (e.g. Ac-Tn3-27 has 27 Tn3 per BSA molecule). Riproximin significantly bound to the bi- and tri-antennary structures NA3 and NA2 as well as to Tn3 structures at high glycan density. Within the group of the glycoproteins, riproximin significantly bound to asialofetuin (ASF), to the carcinoembryonic antigen (CEA) as well as to the desialylated bovine (aBSM) and ovine (aOSM) submaxillary mucins. B. Dot blots comparing the binding of riproximin to serial dilutions of desialylated or deglycosylated gly-coproteins vs. their unprocessed counterparts. ASF: asialofetuin, Fet: fetuin, ASF deg: N-deglycosylated ASF, BSM: bovine submaxillary mucin, aBSM: asialo BSM. Riproximin preferentially bound to the asi-alo-glycoprotein forms. N-deglycosylation of ASF significantly reduced its Rpx binding capacity. FIGURE 2: Enzyme-linked immunosorbent assay (ELISA) binding analysis. A. Plates coated with ASF were incubated with riproximin/ASF or -/aBSM mixtures. Riproximin was detected by Rpx antibody. B. Binding curves from (A) were referred to the numbers of glycans present on each glycoprotein (LacNAc for ASF and GalNAc for aBSM). C. Plates coated with aBSM were incubated with riproximin/ASF or -/aBSM mixtures. Riproximin was detected by Rpx antibody. D. Plates were ei-ther coated with ASF and incubated with 5 µg/ml riproximin (ASF coat + Rpx (5)) or directly coated with 5 µg/ml riproximin (Rpx (5) coat). Serial dilutions of aBSM were subsequently applied. Riproximin was detected by Rpx antibody. E. Plates coated with aBSM were incubated with serial dilutions of riproximin in the presence of 10 or 50 µg/ml ASF. Binding of ASF was detected by an anti-bovine ASF IgG. F. Plates coated with decreasing ASF concentrations were incubated with 50 µg/ml ASF alone or mixed with 10 or 30 µg/ml riproximin. Each plot shows one representative experiment. Data are represented as mean +/- standard deviation from 2-3 replicates. Negative values due to background subtraction in plot 2D were set to zero. Concentration-response curves were fitted using the log-logistic model.


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FIGURE 3: Cytotoxicity experiments. ASF vs. Fet (A, D, F) and aBSM vs. BSM (B, E, G) were added in combination with riproximin to HeLa (A, B), MCF7 (D, E) and MDA-MB-231 (F, G) cells and incubated for 72 h at 37°C. The asialo glyco-proteins showed a significantly higher degree of inhibition of riproximin’s cytotoxicity. C. N-glycans (N-Gly) derived from ASF or Fet were used to compete for riproximin’s cytotoxicity in HeLa cells as in (A). The effect of the N-glycans was related to the original protein amount that had been deglycosylated. D. Neuraminidase pre-treated (Neu+/Rpx) and control (Neu-/Rpx) MDA-MB-231 cells were treated with riproximin for 72 h at 37°C. Vertical lines represent estimated IC50 values. Cell viability was determined by MTT-assay. For each graph, mean values from 2-4 independent experi-ments were used and fitted using the four-parameter log-logistic model. For the inhibition effect of aBSM/BSM on MDA-MB-231, a five-parametric Brain-Cousens hormesis model was used for fitting (61). FIGURE 4: Analysis of the combined competitive inhibitory effect of ASF and aBSM on the cyto-toxicity of riproximin. ASF and aBSM were applied in cellular viability assay with riproximin in HeLa cells. Three glycoprotein combinations with following ASF-to-aBSM active component proportion were analyzed: IRC25/IRC75 (A, D), IRC50/IRC50 (B, E) and IRC75/IRC25 (C, F). The cells were incubated with riproximin and a serially diluted glycoprotein combination for 72 h at 37°C. Cell viability was determined by MTT-assay. For each combination of ASF/aBSM, average IRC-values of four independent experiments and the respective in-verse cell viability curves were fitted by using the four-parameter log-logistic model (A, B, C). The com-bination effect of the glycoproteins was characterized by the combination indices (CI). For all theoretical IRC-values of each ASF/aBSM combination, CI values and the respective confidence bounds were calcu-lated and plotted (D, E, F). Combination effects were characterized as synergistic for CI < 1, as additive for CI = 1, and as antagonistic for CI > 1. FIGURE 5: Schematic of riproximin’s interactions with its two binding counterparts. A. Schematic representation of the riproximin molecule showing its two different binding sites (BS) and its possible interactions with ASF and aBSM, respectively, as described by Dam et al. 2005 for galectin and ASF (19) and by Dam et al. 2007 for SBA and aPSM (20). At low riproximin concentrations, several binding epitopes are available on each glycoprotein molecule and an increased affinity would be observed due to bind-and-jump entropic effects. At high riproximin concentrations, most binding sites on the glyco-proteins have already been occupied. Due to negative cooperativity effects, the binding affinity is lower for each subsequent binding step. B-D. Schematic representation of riproximin interacting with both its counterparts as observed in ELISA experiments. On coated ASF at low aBSM concentrations, crosslink-ing of aBSM to the plate occurs. Due to the high riproximin concentrations in the mixture, several other riproximin molecules are bound to the crosslinked aBSM molecule, resulting in more detectable ripro-ximin (B). At higher aBSM concentrations, competition and masking effects predominate (C). Riproximin crosslinks ASF to the coated aBSM (D). E. Proposed model for the selective targeting of NA3 and Tn3 presenting tumor cells. The cooperative binding to the two different glycoconjugates confers riproximin an increased affinity for cells exposing both NA2/NA3 and cancer-related clustered Tn structures, result-ing in selectivity over cells presenting NA2/NA3 structures only. Moreover, crosslinking of the two structures on the cell surface would result in increased internalization efficiency and thus cytotoxicity.


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TABLES TABLE 1: Abbreviations and names of relevant glycostructures and reagents Abbreviation Name aBSM aOSM ASF ASFdeg BSM Fet Gal GalNAc GlcNAc LacNAc NA2 NA3 OSM PSM Tn Tn3 RIP WGA

asialo bovine submaxillary mucin asialo ovine submaxillary mucin asialofetuin N-deglycosylated asialofetuin bovine submaxillary mucin fetuin D-galactose N-acetyl-D-galactosamine N-acetyl-D-glucosamine N-acetyl-D-lactosamine bi-antennary complex N-glycans tri-antennary complex N-glycans ovine submaxillary mucin porcine submaxillary mucin single O-linked GalNAc three consecutive O-linked GalNAc ribosome inactivating protein wheat germ agglutinin

TABLE 2: Inhibition of riproximin cytotoxicity (IRC) concentration values as calculated from the fitted in-verse cell viability curves and estimation of the related carbohydrate concentration a)

HeLa MCF7 MDA-MB-231

IRC25 IRC50 IRC75 IRC50 IRC50

ASF Protein (µg/ml)

16 74 340 ~1000 b) 59

Protein c) (nM)

333 1540 7080 19900 1200

LacNAc d) (µM)

3 14 64 179 11

aBSM Protein (µg/ml)

0.1 0.4 1.6 6 0.3

Protein c) (nM)

0.3 1 4 14 0.8

GalNAc d) (µM)

0.3 0.9 3.7 13 0.7

a) Inhibition of riproximin’s cytotoxicity by 25% (IRC25), 50% (IRC50) or 75% (IRC75). b) The real relative IRC50 value is >1000 µg/ml. c) Molar concentrations were calculated using molecular weights of 48 kDa for ASF and 400 kDa for aBSM. d) The amount of the carbohydrate residues was estimated based on nine terminal LacNAc residues for an ASF and 920 GalNAc residues for an aBSM molecule.


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Figure 1


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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Martin R. Berger and Cristina VossHelene Bayer, Katharina Essig, Sven Stanzel, Martin Frank, Jeffrey C. Gildersleeve,

cellular targetingEvaluation of Riproximin binding properties reveals a novel mechanism for

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