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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 1, 2004 as doi:10.1096/fj.04-2494fje. |
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2-6Galß1-4GlcNAc-residues are receptors for the anticancer drug rViscumin
**
* Institute for Medical Physics and Biophysics, University of Münster, Münster, Germany;
Institute for Immunology, University of Dresden, Dresden, Germany;
Department of General Surgery, Münster University Hospital, Münster, Germany;
Clinic of Poultry, Hannover School of Veterinary Medicine, Hannover, Germany; and
|| VISCUM AG, Bergisch Gladbach, Germany
1Correspondence: Institute for Medical Physics and Biophysics, University of Münster, Münster 48149, Germany. E-mail: jm{at}uni-muenster.de
SPECIFIC AIMS
The sialic acid specific ribosome-inactivating protein (RIP) rViscumin is currently under clinical development as an anticancer drug. This study was designed to characterize the fine specificity of rViscumin toward gangliosides, to identify its receptor gangliosides on gastrointestinal tumors, and to investigate whether the identified receptor gangliosides show an enhanced expression in tumor tissues compared with unaffected tissues. To further characterize the distinct specificities of rViscumin and ricin, an rViscumin homologous but galactoside-specific RIP, solid-phase binding assays were performed with reference glycoproteins varying in type and degree of sialylation.
PRINCIPAL FINDINGS
1. rViscumin is a lectin with CD75s-specific binding characteristics
CD75s is a carbohydrate epitope with the sialylated lactosaminyl group Neu5Ac
2-6Galß1-4GlcNAc-R. Comparative TLC binding assays with CD75s-specific monoclonal antibodies (mAbs) and a CD75s-specific polyclonal antibody revealed rViscumin to be a CD75s-specific RIP. The binding pattern of two representative CD75s-categorized mAbs, HB6 and J3-89 that are reactive toward 2-6-sialylated neolacto series gangliosides (CD75s-gangliosides; for structures, see Fig. 3
) within a human granulocyte ganglioside (HGG) fraction, were found to be identical with those obtained by rViscumin and the polyclonal antibody AB2-6. mAb HB6 is known to preferentially bind to the medium polar 2-6-sialylated neolacto series ganglioside VI6Neu5Ac-nLc6Cer, whereas mAb J3-89 exhibited no distinct preference for 2-6-sialylated neolacto series gangliosides with a certain carbohydrate chain length. Polyclonal antibody AB2-6 and rViscumin showed equivalent binding to IV6Neu5Ac-nLc4Cer and VI6Neu5Ac-nLc6Cer, and the representative stain of AB2-6 is shown in Fig. 1
(b lanes). rViscumin was classified as a lectin with CD75s-specific binding characteristics from comparative binding studies.
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2. Enhanced expression of CD75s-gangliosides in tumors of the gastrointestinal tract
To investigate the clinical potential of the rViscumin targets, CD75s-gangliosides were identified with antibody AB2-6 in a random collection of four different types of gastrointestinal tumors: hepatocellular, gastric, pancreas, and colon. The majority of the tumors showed an enhanced expression of CD75s-gangliosides compared with unaffected tissues. Figure 1A
shows TLC overlay assays of lipid extracts from one hepatocellular and one gastric cancer tissue with corresponding tumor-free tissue counterparts. Figure 1B
shows one pancreas and one colon cancer tissue with corresponding tumor-free tissue counterparts. The most pronounced increased expression of CD75s-gangliosides was found in pancreas (Fig. 1B
, lane d) and hepatocellular tumors (Fig. 1A
, lane d) compared with extremely low abundance of these tumor-associated gangliosides in the control tissues from the same patients. High and moderate enhancement in CD75s-ganglioside expression was detected for colon (Fig. 1B
, lane f) and gastric tumors (Fig. 1A
, lane f) compared with the absence of these structures in healthy tissue samples. As a positive control, HGG were used in all TLC overlay assays. Corresponding orcinol stain and AB2-6 binding are shown in Fig. 1A, B
, lanes a, b, respectively.
3. Structural characterization of the CD75s-ganglioside VIII6Neu5Ac-nLc8Cer
Within a HPLC-purified ganglioside fraction named HGG4, which exclusively contains 2-6-sialylated neolacto series gangliosides, a faint AB2-6-positive band, separating below VI6Neu5Ac-nLc6Cer in TLC binding assays, was found to represent two CD75s-gangliosides with nLc8Cer core structure. Two low abundant molecular ions obtained from the HGG4 fraction at m/z 2247.41 and 2357.53, respectively, were analyzed by ESI-QTOF-MS, selected for low-energy CID experiments, and identified as VIII6Neu5Ac-nLc8Cer (d18:1, C16:0) and VIII6Neu5Ac-nLc8Cer (d18:1, C24:1). Their structures could be elucidated by a full Y-type ion series, supported by a few B-type ions, and fragment ions generated by ring cleavages and double cleavages (data not shown). This newly characterized ganglioside with nLc8Cer-core represents the third CD75s-ganglioside with rViscumin receptor binding specificity (in addition to the gangliosides IV6Neu5Ac-nLc4Cer and VI6Neu5Ac-nLc6Cer).
4. rViscumin binds to glycoproteins with CD75s-determinants and ricin to desialylated glycans
The rViscumin binding specificity was further investigated with reference glycoproteins carrying sialylated and desialylated type II N-glycans, namely human transferrin, fetuin, haptoglobin and 1-acid glycoprotein, bovine fetuin and asialofetuin, and human recombinant erythropoietin (EPO) produced with hamster CHO cells. Comparative Western blots of rViscumin and ricin, an rViscumin homologous but galactoside-specific RIP, revealed specific recognition of type II N-glycans with CD75s-determinants by rViscumin, whereas ricin failed to react with terminally sialylated oligosaccharides such as CD75s-motifs and others (data not shown; examples of biantennary type II N-glycans are shown in Fig. 3
).
To investigate the influence of the epitope presentation upon lectin binding (i.e., recognition of oligosaccharides presented on denatured glycoproteins (membrane fixed glycoproteins after SDS-PAGE under reducing conditions) or exposed on the native species (adsorbed to the surface of microtiter plates)), the same set of glycoproteins was used for microwell adsorption assays with rViscumin, ricin, and the originally plant-derived viscumin. As shown in Fig. 2
A, B, both rViscumin and viscumin exhibited nearly identical binding profiles in the microwell assays. Strong positive reactions were observed for glycoproteins predominantly carrying 2-6-sialylated structures, such as human haptoglobin and human fetuin (Fig. 2A, B
, columns e and b, respectively). In general, all glycoproteins with 2-6-sialylated N-glycans showed more or less intensive reactions (bovine fetuin, human 1-acid glycoprotein, and human transferrin, Fig. 2A, B
, columns c, f, and a, respectively). The positive reaction of rViscumin and viscumin toward bovine fetuin (Fig. 2A, B
, column c) was almost completely diminished after desialylation, indicated by the marginal binding with asialofetuin (Fig. 2A, B
, column d). No detectable reactions were observed for recombinant EPO (Fig. 2A, B
, column g), characterized by exclusive presence of 2-3-sialylated N-glycans.
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The ricin binding pattern was basically different from those of rViscumin and viscumin. The highest ricin reactivity toward the employed reference glycoproteins was observed for asialofetuin (Fig. 2C
, column d). However, similar relative binding intensities were observed for human haptoglobin and bovine fetuin (Fig. 2C
, columns e, c, respectively), referring to a high content of asialo glycans of these proteins. Ricin showed no detectable interaction with recombinant EPO oligosaccharides, indicating full sialylation of this glycoprotein.
5. No correlation of rViscumin and ricin binding specificities
For comparative statistical analysis, ELISA measurements for rViscumin, viscumin, and ricin toward reference glycoproteins were assigned ranks according to their binding intensities as indicated in Fig. 2A-C
. The comparison of ranks of rViscumin (Fig. 2A
) with viscumin (Fig. 2B
) revealed a rank correlation coefficient of rS= 1.0, indicating a maximum correlation of the binding specificities of rViscumin and viscumin toward the different reference glycoproteins. A low rank correlation coefficient of rS = 0.3571 was calculated for rViscumin (or viscumin) (Fig. 2A, B
) and ricin (Fig. 2C
). At the 1% significance level, rS values < 0.893 (n=7) and even at the 5% significance level, rS values < 0.714 (n=7) indicate that a statistically significant relationship concerning the binding characteristics does not exist for either Neu5Ac2-6Galß1-4GlcNAc-R specific rViscumin and viscumin or the galactose binding ricin. ELISA binding assays in conjunction with nonparametric statistical calculation provided additional and unequivocal evidence that the investigated lectins are clearly distinguishable by their specific binding patterns using native solid-phase fixed-glycoproteins for the binding studies.
CONCLUSIONS AND SIGNIFICANCE
Early animal and human studies have shown that viscumin causes a significant increase and activation of natural killer cells and enhances phagocytotic activity of granulocytes and monocytes, although its mechanism of action is largely unknown. An important activity of viscumin includes its cytostatic and cytotoxic effect on different tumor cells, most likely mediated by induction of apoptosis (a highly conserved mechanism of programmed cell death). To elucidate the potential role of CD75s-gangliosides as tumor-associated antigens, careful investigation of tumor tissues must be performed with regard to their specific expression of CD75s-epitopes. Preliminary data of random samples taken from the four types of gastrointestinal tumors shown in this study revealed enhanced expression of CD75s-gangliosides in more than half of the tumors investigated. The suggested combined action of rViscumin (i.e., the induction of apoptosis in tumor cells with enhanced expression of CD75s-gangliosides in conjunction with the stimulation of the immune system) makes this recombinant lectin an interesting and promising candidate for oncological applications.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2494fje;
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