SAfETY Assessment of ANTifuNGAL Proteins

Antifungal proteins (AFPs) of the plant defensin class have been assessed by both academic researchers and the biotechnology industry for their ability to control a wide range of fungal pathogens in nonfood (cotton) and food crops (potato and wheat). Key targets for control include Verticillium in potato and Fusarium in wheat.6162 Plant defensins are small (~5 kD), basic, cysteine-rich proteins that are members of a phylogenetically diverse class of structurally related proteins that share a scorpion-fold motif. Plant defensins have a series of eight conserved cys-teines that form four disulfide bridges,63 conferring significant structural stability to these proteins. Plant antifungal proteins are a ubiquitous class of proteins that inhibit fungal hyphae growth at the low ppm level in in vitro fungal inhibition assays.64 They form part of the innate immunity of plants and are expressed in various plant tissues. Because they are ubiquitous in plants, AFPs have a history of consumption in the human diet and are present in very familiar plant species such as corn, wheat, and potatoes.

AFPs and scorpion toxins share structural similarities. Both contain the cysteine stabilized a-helix motif -CxxxC- and share primary sequence similarity. Nuclear magnetic resonance spectroscopy (NMR) observations reveal similar but noniden-tical topography. The AFPs and scorpion toxins have been shown to modify cell membranes (AFPs, fungal hyphae; scorpion toxins, neurons) via interaction with ion transport proteins and/or formation of ion channels.65,66 These changes in intracel-lular ion concentrations lead to perturbations of cell signaling pathways that ultimately cause cell death. AFPs are also homologous to plant "sweet proteins" that may interact directly with taste receptors (neurons). It was hypothesized that AFPs and scorpion toxins may have similar functional mechanisms, but with highly divergent specificities based on protein-protein interactions. Furthermore, AFPs were shown to be resistant to digestion with in in vitro SGF assays.

Since AFPs' mode of action, AFPs' stability to digestion, and bioinformatic analyses all suggest that these proteins may have a potential effects on human health, protein-specific studies were performed. Perturbation of neural viability, steady-state electromembrane potentials, and sodium, potassium, and calcium channel function were examined and compared with purified AFP protein (alfALP, isolated from the seeds of M. sativa), scorpion toxin proteins (Csev3 from Centruroides sculpturatus Ewing venom), control neurotoxins, and ordinary dietary proteins such as Rubisco.

The AFP (0.1 to 100 pM) had no effect on rat neocortical cell viability as measured by lactose dehydrogenase activity (leakage) in a 24-hour assay period. The scorpion toxin, however, showed a significant dose-dependent effect on cell leakage at all concentrations tested (0.1 to 100 pM). The AFP was shown to have no effect on resting membrane potential and action potential. The AFP had a possible effect on sodium channels by increasing the current duration. However, the electrophysiological significance of this observation is unclear. One possibility is that the effect is due to nonspecific protein-protein interactions at high protein concentrations (effect seen at 10 mM). To determine the specificity of this effect, a greater range of "noninteract-ing" proteins could be tested.

Additional studies to assess potential allergenicity of the protein were conducted. A structural homologue of alfAFPs was purified from wheat (g-thionin) and directly tested for allergenicity using IgE from wheat allergic patients. Sera from 14 wheat-allergic patients were used for IgE blotting experiments. A population of 14 patients is considered sufficient to provide a 95% chance of identifying a major allergen.67 IgE blotting experiments showed no significant binding to g-thionin. The plant defensin, g-thionin, was therefore not implicated as a major allergen in wheat.

AFP proteins have not been introduced into any food crop due to a limited efficacy. It is clear, however, that additional protein-specific safety data would need to be generated in order to make scientifically sound decisions about their safety and, hence, potential to be introduced into the food supply via plant biotechnology.

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