Glycobiology, 2002, Vol. 12, No. 8 485-497
© 2002 Oxford University Press
Preferential binding of the anticancer drug rViscumin (recombinant mistletoe lectin) to terminally 
2-6-sialylated neolacto-series gangliosides
2 Institute for Medical Physics and Biophysics, University of Münster, Robert-Koch-Str. 31, D-48149 Münster, Germany; 3 Institute of Cell Culture Technology, University of Bielefeld, D-33501 Bielefeld, Germany; 4 VISCUM AG, D-64673 Zwingenberg, Germany; 5 Clinic of Poultry of the Hannover School of Veterinary Medicine, D-30559 Hannover, Germany; and 6 Biotechnology Research and Information Network (BRAIN) AG, D-64673 Zwingenberg, Germany
Production of biochemically defined recombinant mistletoe lectin was achieved by cloning and separate expression of the single catalytically active A-chain and the B-chain with carbohydrate binding properties in Escherichia coli, yielding an active heterodimeric protein named rViscumin (Eck et al. [1999] Eur. J. Biochem., 265, 788–797). Employing solid phase binding assays, rViscumin was shown to preferentially bind to terminally 
2-6-sialylated neolacto-series gangliosides IV6Neu5Ac-nLc4Cer, VI6Neu5Ac-nLc6Cer, and VIII6Neu5Ac-nLc8Cer isolated from human granulocytes. Only marginal binding of rViscumin to galactose-terminated neutral GSLs was determined, whereas reinvestigation of ricin specificity demonstrated this lectin as a galactose-binding protein. Human promyelotic HL-60 cells exhibited an IC50 value (half maximum cytotoxicity) of 1.16 pM and human bladder carcinoma 5637 cells of 12.1 pM rViscumin; CHO-K1 cells were resistant to rViscumin treatment up to a concentration of 5.26 nM tested. Quantification of the predominant receptor ganglioside IV6Neu5Ac-nLc4Cer by means of a specific anti-Neu5Ac2-6Galß1-4GlcNAc-R antibody revealed 3.68 x 106 and 1.54 x 106 receptor molecules per HL-60 and 5637 cell, respectively; CHO-K1 cells were negative, lacking 2-6-sialylated gangliosides. The data imply a direct correlation of rViscumin cytotoxicity and the expression of receptor ganglioside. Moreover, CHO-K1 cells were rendered susceptible toward rViscumin cytotoxicity after exogenous application of human granulocyte gangliosides. Thus, (1) rViscumin has to be considered as a sialic acid–specific rather than a galactose-specific type II ribosome-inactivating protein, and (2) neolacto-series gangliosides with Neu5Ac2-6Galß1-4GlcNAc-terminus are true functional and physiologically relevant rViscumin receptors.
1 To whom correspondence should be addressed; E-mail: jm@uni-muenster.de
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