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

Abstract

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., Mo Èckel, 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 mm). 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 IC 50 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. Mistletoe lectins type I, II and III are a group of glycosylated, 56±64 kDa cytotoxic proteins, which consist of two covalently cystine linked subunits. The A-chain mediates the enzymatic inactivation of eucaryotic ribosomes and the B-chain is responsible for carbohydrate binding [17] and therefore mistletoe lectin belongs to the type II ribosome inactivating proteins (RIP II) [18]. Interestingly, the specificity of carbohydrate binding varies between the three species with ML I being mainly specific for d-galactose and ML III for N-acetyl-d-galactosamine [19,20]. Both, ML I and ML III mediated cytotoxicity can be strongly reduced by b-d-lactose as a competitor [21]. ML III, ho...

Recombinant mistletoe lectin induces p53-independent apoptosis in tumour cells and cooperates with ionising radiation

“…Mistletoe lectins consist of a toxic A-chain, the site-specific ribosome-inactivating N-glycosidase, and a carbohydrate-binding-subunit B. The B-chain is important for cellular lectin uptake, internalisation of the A-chain and is partially involved in the induction of cytokine synthesis (Hajto et al, 1990), but does not induce apoptosis on its own (Hostanska et al, 1996;Eck et al, 1999b). On the other hand, the cytotoxic and apoptosisinducing effect of ML is exerted by the ribosome inactivating A-chain (Hostanska et al, 1996;Langer et al, 1999).…”

Pharmacokinetics of natural mistletoe lectins after subcutaneous injection

“…This may cause different uptake and distribution in blood and tissue and could explain the longer detectability of nML in serum. Cytotoxicity of nML and rML were similar in MOLT-4 cell cultures [17]. In vitro studies using human peripheral blood mononuclear cells, however, significant differences of rML and nML on cell viability and immunomodulation were found [18].…”

“…In contrast to rML, nML is glycosylated and has different kinetics of association and dissociation with glycoconjugates. Furthermore, B-chains of rML and nML differ in binding specifity to carbohydrates [17]. This may cause different uptake and distribution in blood and tissue and could explain the longer detectability of nML in serum.…”

Difficulties and perspectives of immunomodulatory therapy with mistletoe lectins and standardized mistletoe extracts in evidence based medicine

“…However, plant MLs exhibit a heterogeneity that most likely results from the post-translational processing of ML-I to the isoforms ML-II and ML-III [37]. Only a small difference was found in their primary structures.…”