Ab 123456 7 89 10

Figure 1 Electrophoretic separation pattern of the ML by SDS-PAGE under non-reducing (lanes 1-6, A, B) and reducing conditions (8-10), respectively. Samples representing lanes 4-6 were blocked prior to separation by iodine acetamide. Lane B indicates the chitin binding lectin (CBL) from Viscum album L., VisalbCBL. The ML B chains exhibit a higher molecular weight (about 32 to 36 kDa) as compared to the A chains (about 28.5 to 31 kDa). Molecular weight marker (MM) is represented by lanes A (for CBL) and 7.

Figure 2 Recognition pattern of a complex glycoconjugate (asialofetuin) by ML I, ML II, ML III and VisalbCBL as demonstrated by surface plasmon resonance spectroscopy (BIA, biochemical interaction analysis). The lectins were applied to asialofetuin immobilised on sensorchip CM5 (Pharmacia) in equal concentrations. While the binding pattern of ML I and ML III are strongly related to each other and show only some differences in the dissociation step (D), ML II and VisalbCBL reveal different kinetics in the association (A) and dissociation (D) step of ligand (ASF)—analyte (lectins at 2.5 Mg/ml HEPES-buffered saline) interaction. As compared to ML I or ML III, ML II has considerably lower binding constants for glycoconjugates, and core glucNAc residues are obviously less efficient targets for the VisalbCBL.

Figure 2 Recognition pattern of a complex glycoconjugate (asialofetuin) by ML I, ML II, ML III and VisalbCBL as demonstrated by surface plasmon resonance spectroscopy (BIA, biochemical interaction analysis). The lectins were applied to asialofetuin immobilised on sensorchip CM5 (Pharmacia) in equal concentrations. While the binding pattern of ML I and ML III are strongly related to each other and show only some differences in the dissociation step (D), ML II and VisalbCBL reveal different kinetics in the association (A) and dissociation (D) step of ligand (ASF)—analyte (lectins at 2.5 Mg/ml HEPES-buffered saline) interaction. As compared to ML I or ML III, ML II has considerably lower binding constants for glycoconjugates, and core glucNAc residues are obviously less efficient targets for the VisalbCBL.

on different deciduous trees contain mainly ML I-1. The preparative isolation and separation of single isoforms belonging to ML I, ML II and ML III is possible by chromatofocusing using FPLC-technique.

The differences in molecular mass, electrophoretic and chromatographic behaviour between mistletoe isolectin groups are mainly caused by the glycosylation degree and possibly, to some extent, by the amino acid sequence. All three lectins are glycosylated and carry plant typical mannose- and complex-type sugar side chains (Debray et al., 1992, 1994; Zimmermann et al., 1996) (Figure 4). A glycosylation of the ML III A chain of ML III isolated from ML III from different mistletoes (regarding to host tree, season and localisation) was never observed (Pfuller and Eifler, unpublished results).The glycoconjugate nature of ML allows also "passive" interactions of ML with mannose-glucNac- and fucose- recognising lectins on tissue structures which are able to bind ML in different quantities reflecting the sugar composition of the glycan part.

Recently the amino acid sequences of ML I A chain and B chain were published, demonstrating high conservation of structure when compared to other type 2 RIP toxins (Eschenburg et al., 1998, Soler et al., 1998; Krauspenhaar et al., 1999). The X-ray structure of ML I was estimated to a degree of resolution of 3.0 A (Figure 5), underlying high degree of structural identity with other known RIP 2 molecules. Noticeable, the dimeric character of ML demonstrated by gel permeation

Figure 3 Seasonal variations in the levels of ML isolectins (ML I, ML II and ML III) as measured in the leaves from Viscum album L. grown on host tree Malus sylvestris. Leaves (1-2 years old) were harvested from January (1) to December (12). The amount of different ML (mg/100 g fresh plant weight, FW) was determined by FPLC and ELISA techniques. ML II, however, shows a peak in May, while the concentration of ML I and ML III strongly decreased during summer and showed the highest level during winter time.

chromatography (Ziska et al., 1978) and by electron microscopy (Lutsch et al., 1984) was confirmed by X-ray investigations (Sweeney et al., 1993; Krauspenhaar et al., 1999). Depending on concentration and composition of buffer solutions, only ML I but not ML II or ML III shows dimerisation (Franz et al., 1981; Lutsch et al., 1984).

Independently of high structural identity between ML I and ricin, no cross reactivity between both polyclonal and monoclonal anti-ricin and anti-ML antibodies was detected (Jäggy et al. 1995; Tonevitsky et al., 1995, 1999). Anti-ML antibodies are important tools to describe structural features of ML and allow their detection in plant extracts. Hybridomas producing monoclonal antibodies (mab) against ML and their A and B subunits have been obtained by Jäggy et al. (1995) and Tonevitsky et al. (1995, 1999). Three groups of mAb displaying different affinities to ML and recombinant ML I A-chain were generated: (1) mAb against ML I-A and ML II-A, (2) mAb against ML II-A and ML III-A, and (3) mAb against A-subunits of ML I, ML II and ML III. Antigenic determinants of ML recognised by mAb MNA4, MNA9 and MTC12 contain no carbohydrate side chains (Tonevitsky et al., 1999).

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