Cloning and characterization of the genes encoding toxic lectins in mistletoe (Viscum album L)

Leaves of mistletoe (Viscum album L) contain three toxic lectins (type 2 ribosome-inactivating proteins) MLI, MLII, and MLIII, differing in molecular mass and carbohydrate specificity. Clones, containing sequences of three gene variants designated ml1p, ml2p, and ml3p, were obtained using PCR amplification from cDNA and from mistletoe genomic DNA. The quantitative ratio of the ml1p, ml2p, and ml3p genes in genomic DNA was found to be 1.5 : 1 : 4, respectively, whereas the ratio of their mRNA was 50 : 10 : 1. The quantitative prevalence of the ml1p transcript correlates well with the observation that MLI is quantitatively dominant over MLII and MLIII in the mistletoe extract. The sequences of the proteins encoded by the ml1p, ml2p, and ml3p genes are identical to MLI by 98, 88, and 77%, respectively. The similarity to MLI of the amino acid sequence encoded by the gene ml1p, the quantitative prevalent of its mRNA, as well as structural properties of the B-chain indicate that the gene, ml1p, corresponds to MLI. Western blot analysis of recombinant A-chains encoded by the three variants of mlp genes with the monoclonal antibody MNA4 having differential affinity to MLI, MLII and MLIII A-chains suggests that the ml2p and ml3p genes correspond to MLII and MLIII, respectively. Structural differences in the carbohydrate-binding sites of the B-subunits of ML1p, ML2p, and ML3p probably explain the difference in sugar specificity of MLI, MLII and MLIII.