Preferential binding of the anticancer drug rViscumin (recombinant mistletoe lectin) to terminally α2-6-sialylated neolacto-series gangliosides

Abstract

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 α2-6-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-Neu5Acα2-6Galβ1-4GIcNAc-R antibody revealed 3.68 × 106 and 1.54 × 106 receptor molecules per HL-60 and 5637 cell, respectively; CHO-K1 cells were negative, lacking α2-6-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, Neu5Acα2-6Galβ1-4GlcNAc-terminus are true functional and physiologically relevant rViscumin receptors.