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 active principle in extract preparations from mistletoe, commonly used as immunomodulator in adjuvant tumour therapy. The heterodimeric disulfide-linked cytotoxic protein is classified as type II ribosome inactivating protein (RIP). Recently, the sequence coding for the mistletoe lectin prepro-protein was identified and the existence of a single intron-free gene was shown [Eck, J., Langer, M., Möckel, B., Baur, A., Rothe, M., Zinke, H. & Lentzen, H. (1999) Eur. J. Biochem. 264, 775-784]. The aim of this study was to prepare pure and homogeneous rMLB-chain as well as rML heterodimer for studying the carbohydrate binding specificity of recombinant versus natural protein and its contribution to the observed cytotoxic effect. Expression in E. coli resulted in the production of insoluble protein (inclusion bodies). A procedure for generating correctly folded, biochemically and biologically active rMLB was established starting from the insoluble single chain. Carbohydrate binding and specificity of pMLB and rMLB were analysed by a competitive enzyme linked lectin assay (ELLA). Asialofetuin was able to compete with binding of both chains (50% at 0.8 microM). The specificity of the B-chains to lactose was more distinct with halfmaximal competition at 4.9 mM (pMLB) and > 90 mM (rMLB), respectively. Furthermore, in a coassociation process rMLA- and rMLB inclusion bodies were associated in one step by defined dilution yielding active rML-heterodimer. The activities of recombinant (rML) and plant derived mistletoe lectin (pML) were compared. Cytotoxicity was determined using MOLT-4 cells and enzymatic rRNA N-glycosidase activity was measured in a coupled transcription/translation assay. The IC50 values of the two heterodimers were similar in both assays; rMLB-chain did not show any cytotoxic effect. In the ELLA with lactose as a competitor 50% competition of binding to asialofetuin was achieved 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 activity and the rRNA N-glycosidase activity, are prerequisites for the cytotoxic effects on target cells.
Eck
J
J
Biotechnology Research and Information Network (BRAIN) GmbH, Zwingenberg, Germany.
Langer
M
M
Möckel
B
B
Witthohn
K
K
Zinke
H
H
Lentzen
H
H
D001212:Asialoglycoproteins / Q000737:chemistry; D001667:Binding, Competitive; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli; D060748:Fetuins; D007785:Lactose / Q000378:metabolism; D037102:Lectins / Q000737:chemistry* / Q000235:genetics; D014784:Mistletoe / Q000737:chemistry*; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D037121:Plant Lectins; D010940:Plant Proteins / Q000737:chemistry*; D010946:Plants, Medicinal; D011485:Protein Binding; D017510:Protein Folding; D020033:Protein Isoforms; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D054788:Ribosome Inactivating Proteins; D000509:alpha-Fetoproteins / Q000737:chemistry
D001212:Asialoglycoproteins; D060748:Fetuins; D037102:Lectins; D037121:Plant Lectins; D010940:Plant Proteins; D020033:Protein Isoforms; D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins; D007785:Lactose