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A method was established to isolate and quantify small amounts of chitin-binding mistletoe lectin (cbML) from extracts of the mistletoe (Viscum album L.) by affinity and reverse phase high performance liquid chromatography. A validation, according to ICH guidelines, of this analytical method was carried out and showed that specificity, robustness and precision are guaranteed. In addition, linearity is ensured for a content between 0.6 and 4.1 microg/ml of cbML in the extracts and recovery was calculated to be in the range of 94 to 100%. So, accuracy of the method is guaranteed as well. As far as the range of the analytical method is concerned, a minimum of 1.2 microg and a maximum of 8.2 microg cbML can be incubated with the affinity material. Detection and quantitation limits were calculated to be 0.13 and 0.46 microg/ml cbML, respectively.

The primary structure of the B chain of the N-acetyl-D-galactosamine-recognizing mistletoe lectin-3 (ML-3B) has been deduced from proteolytic digest peptides of the purified glycoprotein, their HPLC-separation and Edman degradation and confirmation of the peptide sequences by MALDI-MS. ML-3B consists of 262 amino acid residues including 10 cysteine moieties. The structure and linkage of the carbohydrate side chains, connected to two N-glycosylation sites at positions Asn(95) and Asn(135) of the lectin, were determined by a combination of glycosidase treatment and MALDI-MS of corresponding glycopeptide fragments. The sequence alignment reveals a high homology with other B chains of type-II RIPs, although there are remarkable differences in the D-galactose-specific mistletoe lectin-1B chain. The recently published primary structure of the mistletoe lectin-3A chain1 and the now available primary sequence of the 3B chain allowed the construction of a preliminary homology model of ML-3. The model demonstrates, unequivocally, that ML-3 is a member of the type-II RIP family with rigid conservation of the enzymatic active site of the A chain and an identical overall protein fold. Specific amino acid residue exchanges and the different glycosylation pattern in comparison with ML-1 are discussed and related to the properties of the two glycoproteins. The knowledge of the complete primary structure of mistletoe lectin-3 is a major contribution towards more insight into the mechanism of the biological activity of commercial mistletoe preparations.

The complete primary structure of the A chain of mistletoe lectin III (ML3A), a type II ribosome-inactivating protein, was determined using proteolytic digests of ML3A, HPLC separation of the peptides, Edman degration and MALDI-MS. Based on our results, ML3A consists of 254 amino acid residues, showing a high homology to the A chain of isolectin ML1 with only 24 amino acid residue exchanges. A striking important structural difference compared with ML1A is the lack of the single N-glycosylation site in ML3A due to an amino acid exchange at position 112 (ML1A: NL112GS ==> ML3A: T112GS). The alignment of ML3A with the A chains of ML1, isoabrins, ricin D, Ricinus communis agglutinin and three lectins, identified from the Korean mistletoe Viscum album ssp. coloratum, demonstrates the rigid conservation of all amino acid residues, responsible for the RNA-N-glycosidase activity as reported for ricin D. In addition, the fully determined primary structure of ML3A will give further information about the biological mechanism of mistletoe lectin therapy.

The crystal structure of the ribosome-inactivating protein (RIP) mistletoe lectin I (ML-I) from Viscum album has been solved by molecular replacement techniques. The structure has been refined to a crystallographic R-factor of 24.5% using X-ray diffraction data to 2.8 A resolution. The heterodimeric 63-kDa protein consists of a toxic A subunit which exhibits RNA-glycosidase activity and a galactose-specific lectin B subunit. The overall protein fold is similar to that of ricin from Ricinus communis; however, unlike ricin, ML-I is already medically applied as a component of a commercially available misteltoe extract with immunostimulating potency and for the treatment of human cancer. The three-dimensional structure reported here revealed structural details of this pharmaceutically important protein. The comparison to the structure of ricin gives more insights into the functional mechanism of this protein, provides structural details for further protein engineering studies, and may lead to the development of more effective therapeutic RIPs.

The structures of mistletoe lectin I (ML-I) from Viscum album complexed with lactose and galactose have been determined at 2.3 A resolution and refined to R factors of 20.9% (Rfree = 23.6%) and 20.9 (Rfree = 24.6%), respectively. ML-I is a heterodimer and belongs to the class of ribosome-inactivating proteins of type II, which consist of two chains. The A-chain has rRNA N-glycosidase activity and irreversibly inhibits eukaryotic ribosomes. The B-chain is a lectin and preferentially binds to galactose-terminated glycolipids and glycoproteins on cell membranes. Saccharide binding is performed by two binding sites in subdomains alpha1 and gamma2 of the ML-I B-chain separated by approximately 62 A from each other. The favoured binding of galactose in subdomain alpha1 is achieved via hydrogen bonds connecting the 4-hydroxyl and 3-hydroxyl groups of the sugar moiety with the side chains of Asp23B, Gln36B and Lys41B and the main chain of 26B. The aromatic ring of Trp38B on top of the preferred binding pocket supports van der Waals packing of the apolar face of galactose and stabilizes the sugar-lectin complex. In the galactose-binding site II of subdomain gamma2, Tyr249B provides the hydrophobic stacking and the side chains of Asp235B, Gln238B and Asn256B are hydrogen-bonding partners for galactose. In the case of the galactose-binding site I, the 2-hydroxyl group also stabilizes the sugar-protein complex, an interaction thus far rarely detected in galactose-specific lectins. Finally, a potential third low-affinity galactose-binding site in subunit beta1 was identified in the present ML-I structures, in which a glycerol molecule from the cryoprotectant buffer has bound, mimicking the sugar compound.