Preferential binding of the anticancer drug rViscumin (recombinant mistletoe lectin) to terminally alpha2-6-sialylated neolacto-series gangliosides.
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 alpha2-6-sialylated neolacto-series gangliosides IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-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 IC(50) 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 IV(6)Neu5Ac-nLc4Cer by means of a specific anti-Neu5Acalpha2-6Galbeta1-4GlcNAc-R antibody revealed 3.68 x 10(6) and 1.54 x 10(6) receptor molecules per HL-60 and 5637 cell, respectively; CHO-K1 cells were negative, lacking alpha2-6-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 Neu5Acalpha2-6Galbeta1-4GlcNAc-terminus are true functional and physiologically relevant rViscumin receptors.
Müthing
Johannes
J
Institute for Medical Physics and Biophysics, University of Münster, Robert-Koch-Str. 31, D-48149 Münster, Germany. jm@uni-muenster.de
Burg
Monika
M
Möckel
Babette
B
Langer
Martin
M
Metelmann-Strupat
Wolfgang
W
Werner
Andreas
A
Neumann
Ulrich
U
Peter-Katalinic
Jasna
J
Eck
Jürgen
J
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D005732:Gangliosides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000493:pharmacokinetics; D006028:Glycosphingolipids / Q000737:chemistry / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D037121:Plant Lectins / Q000737:chemistry / Q000378:metabolism*; D028321:Plant Preparations / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010940:Plant Proteins; D010946:Plants, Medicinal; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D054790:Ribosome Inactivating Proteins, Type 2; D012276:Ricin / Q000378:metabolism; D012794:Sialic Acids / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014118:Toxins, Biological / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D005732:Gangliosides; D006028:Glycosphingolipids; D037121:Plant Lectins; D028321:Plant Preparations; D010940:Plant Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D054790:Ribosome Inactivating Proteins, Type 2; D012794:Sialic Acids; D014118:Toxins, Biological; C009674:ribosome inactivating protein, Viscum; D012276:Ricin