Characterization of recombinant and plant-derived mistletoe lectin and their B-chains

Mistletoe lectin I(pML) and its isoforms ML II and III constitute the activ e principle in extract preparations from mistletoe. commonly used as immuno modulator in adjuvant tumour therapy. The heterodimeric disulfide-linked cy totoxic protein is classified as type II ribosome inactivating protein (RIP ). Recently, the sequence coding for the mistletoe lectin prepro-protein wa s identified and the existence of a single intron-free gene was shown [Eck, J., Langer, M., Mockel, B., Baur, A., Rothe, M., Zinke, H. & Lentzen, PI. (1999) flip: J. Biochem. 264, 775-784]. The aim of this study was to prepare pure and homogeneous rMLB-chain as wel l as rML heterodimer for studying the carbohydrate binding specificity of r ecombinant versus natural protein and its contribution to the observed cyto toxic effect. Expression in E. coli resulted in the production of insoluble protein (inclusion bodies). A procedure for generating correctly folded, b iochemically and biologically active rMLB was established starting from the insoluble single chain. Carbohydrate binding and specificity of pMLB and r MLB were analysed by a competitive enzyme linked lectin assay (ELLA). Asial ofetuin was able to compete with binding of both chains (50% at 0.8 mu M). The specificity of the B-chains to lactose was mon distinct with halfmaxima l competition at 4.9 mM (pMLB) and > 90 mM (rMLB), respectively. Furthermore, in a coassociation process rMLA- and rMLB inclusion bodies wer e associated in one step by defined dilution yielding active rML-heterodime r. The activities of recombinant (rML) and plant derived mistletoe lectin ( pML) were compared. Cytotoxicity was determined using MOLT-4 cells and enzy matic rRNA N-glycosidase activity was measured in a coupled transcription/t ranslation assay. The IC50 values of the two heterodimers were similar in b oth assays: rMLB-chain did not show any cytotoxic effect. In the ELLA with lactose as a competitor 50% competition of binding to asialofetuin was achi eved at 1.6 mM (rML) and 1.8 mM (pML). Hence, using three different assays we found no significant differences between the recombinant protein and the glycosylated form of ML. Comparing the biological activities of the single chains with those of the heterodimer we conclude, that both, lectin activi ty and the rRNA N-glycosidase activity, are prerequisites for the cytotoxic effects on target cells.