Site-specific mutagenesis of mistletoe lectin: the role of RIP activity in apoptosis.
Recombinant mistletoe lectin (rML) belongs to the class of type II ribosome-inactivating proteins (RIP) composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding properties. To investigate the contribution of the enzymatic activity of the rML A-chain to the observed cytotoxic and apoptotic effects, an rMLA E166Q R169Q molecule was developed by means of site-specific mutagenesis. Following heterologous expression, the activity of mutant rMLA was measured in a cell-free assay for rRNA-N-glycosidase activity. Moreover, after generation of heterodimer, the activities of mutant rML E166Q R169Q and rML wild type were determined in a cytotoxicity and apoptosis assay. Although the reduction of activity as measured in the cell-free RIP assay was more pronounced (factor 237) than in both cellular assays (factors 20-22), the data clearly indicate a close correlation between cytotoxicity, apoptosis, and the enzymatic activity of the rML A-chain. Thus, RIP activity is an essential feature of rML and therefore a prerequisite for its biological function as an anticancer agent.
Langer
M
M
BRAIN GmbH, Zwingenberg, 64673, Germany. ml@brain-biotech.de
Möckel
B
B
Eck
J
J
Zinke
H
H
Lentzen
H
H
D017209:Apoptosis / Q000235:genetics*; D037102:Lectins / Q000235:genetics* / Q000378:metabolism; D014784:Mistletoe; D016297:Mutagenesis, Site-Directed; D037121:Plant Lectins; D028321:Plant Preparations; D010940:Plant Proteins; D010946:Plants, Medicinal; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012263:Ribonucleosides / Q000235:genetics / Q000378:metabolism; D054790:Ribosome Inactivating Proteins, Type 2; D012270:Ribosomes; D014118:Toxins, Biological / Q000235:genetics* / Q000378:metabolism
D037102:Lectins; D037121:Plant Lectins; D028321:Plant Preparations; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D012263:Ribonucleosides; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C009674:ribosome inactivating protein, Viscum