Viscotoxin A1

Mistletoe (Viscum album L.) is a hemiparasitic plant that absorbs water and nutrients from the host tree. Mistletoe contains two groups of cytotoxic, immunomodulatory and antitumor proteins, viscotoxins and lectins. This study evaluated the quantity and quality of viscotoxins and total lectins in the stems with leaves (foliage) and fruit of mistletoe on Parrotia persica and Carpinus betulus in September with immature green berries and in December with mature white berries. Viscum album L. plants were harvested from host species located in the Hyrcanian forests of Iran in 2019. The highest level of viscotoxins was detected in the December foliage of V. album settled on C. betulus (9.25 mg/g dry weight [DW]), and the highest content of lectins was found in the December foliage of V. album settled on P. persica (0.79 mg/g DW) and C. betulus (0.73 mg/g DW) respectively. The immature green berries of V. album from both host species contained much higher concentrations of viscotoxins and lectins than the mature white berries. Four isoforms of viscotoxins, viscotoxin A1, A2, A3 and B could be identified in all samples of both host species. Viscotoxin A3 was the predominant viscotoxin isoform followed by viscotoxin A1.

The high-resolution three-dimensional structure of the plant toxin viscotoxin A3, from Viscum album L., has been determined in solution by (1)H NMR spectroscopy at pH 3.6 and 12 degrees C (the structure has been deposited in the Protein Data Bank under the id. code 1ED0). Experimentally derived restraints including 734 interproton distances from nuclear Overhauser effect measurements, 22 hydrogen bonds, 32 φ angle restraints from J coupling measurements, together with three disulphide bridge constraints were used as input in restrained molecular dynamics, followed by minimization, using DYANA and Discover. Backbone and heavy atom root-mean-square deviations were 0.47+/-0.11 A (1 A=10(-10) m) and 0.85+/-0.13 A respectively. Viscotoxin A3 consists of two alpha-helices connected by a turn and a short stretch of antiparallel beta-sheet. This fold is similar to that found in other thionins, such as crambin, hordothionin-alpha and -beta, phoratoxin A and purothionin-alpha and -beta. The difference in the observed biological activity for thionins of known structure is discussed in terms of the differences in the calculated surface potential distribution, playing an important role in their function through disruption of cell membranes. In addition, the possible role in DNA binding of the helix-turn-helix motif of viscotoxin A3 is discussed.

Crystals of the cytotoxic thionin proteins viscotoxins A1 and B2 extracted from mistletoe diffracted to high resolution (1.25 and 1.05 A, respectively) and are excellent candidates for testing crystallographic methods. Ab initio direct methods were only successful in solving the viscotoxin B2 structure, which with 861 unique non-H atoms is one of the largest unknown structures without an atom heavier than sulfur to be solved in this way, but sulfur-SAD phasing provided a convincing solution for viscotoxin A1. Both proteins form dimers in the crystal and viscotoxin B2 (net charge +4 per monomer), but not viscotoxin A1 (net charge +6), is coordinated by sulfate or phosphate anions. The viscotoxin A1 crystal has a higher solvent content than the viscotoxin B2 crystal (49% as opposed to 28%) with solvent channels along the crystallographic 4(3) axes.