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Site-Specific Mutagenesis of Mistletoe Lectin: The Role of RIP Activity in Apoptosis

https://doi.org/10.1006/bbrc.1999.1610Get rights and content

Abstract

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.

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    Abbreviations used: rMLA, recombinant mistletoe lectin A-chain; rMLA E166Q R169Q, recombinant mistletoe lectin A-chain with modified active site; rML, recombinant mistletoe lectin heterodimer; rML E166Q R169Q, recombinant mistletoe lectin heterodimer with modified active site; pML, plant-derived mistletoe lectin I; MOLT-4, human T-cell leukemia line (ECACC No. 85011413); RIP, ribosome inactivating protein

    1

    To whom correspondence should be addressed at BRAIN GmbH, Darmstädter Str. 34, D-64673 Zwingenberg, Germany. Fax: ++49 6251 9331-11. E-mail: [email protected].

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