Neurophysiological action of centrally-acting spider toxin polypeptides derived from Hadronyche versuta and Tegenaria agrestis venoms.

Established dogma concerning the action of insecticidal arthropod-derived peptides (e.g., scorpion toxins), was that they acted on the peripheral nervous system and were excluded from the central nervous system (CNS) by barrier systems. Initial evidence for a CNS-directed toxicological effect following parenteral administration was for a novel peptide from the Hobo spider, Tegeneria agrestis. This toxin was inactive on peripheral sensory and motor nerves, but had a potent excitatory effect on the CNS of larval Musca domestica. Recently, a commercialized formulation of GS-omega/kappa-Hxtx-Hv1a (HXTX), derived from the venom of the Australian blue mountain funnel web spider (Hadronyche versuta) was introduced for use in agriculture by Vestaron Corp. Its primary mode of action was found to be central neuroexcitation via positive allosteric modulation of nicotinic acetylcholine receptors (nAchR) of cockroach neurons. In the present study, this peptide showed hyperexcitation followed by a decrease in firing of the Drosophila melanogaster larval CNS that was prevented by co-exposure to 100 nM α-bungarotoxin (α-BGTX), a classical nAchR noncompetitive antagonist. This effect was mirrored in isobologram analysis, which showed clear antagonism between the two toxins when injected into adult houseflies. Interestingly, U1-agatoxin-Ta1b-QA derived from Tegeneria agrestis (VST-7304) had a similar biphasic action, but showed increased nerve discharge when co-exposed with 100 nM α-BGTX, and had additive effects when injected together with α-BGTX in isobologram analyses. Binary mixtures of HXTX or VST-7304 with 30 nM nicotine showed clear evidence of synergized nerve block, which was also observed for mixtures of HXTX and VST-7304. Taken together, these data suggest that HXTX and VST-7304 have somewhat different and complementary modes of action.

Structural and functional analysis of the Pig-a protein that is mutated in paroxysmal nocturnal hemoglobinuria.

There is now convincing evidence that the Pig-a gene is mutated in patients with paroxysmal nocturnal hemoglobinuria (PNH), a disease in which one or more clones of hematopoietic cells have incomplete assembly of glycosylphosphatidylinositol (GPI) anchors and absence of GPI-linked protein expression on the cell surface. Little is known, however, about the Pig-a protein product that is necessary for GPI anchor bioassembly. Relatively few substitution (missense) Pig-a gene mutations have been identified, but we noted two apparent clusters at codons 128-129 and 151-156 and hypothesized that these might represent critical regions of the Pig-a protein. We therefore used site-directed mutagenesis to create conservative mutations in the Pig-a protein, then performed structural and functional analysis. Each Pig-a mutation generated a Pig-a protein of normal size and stability, but certain mutations had substantial deleterious effects on protein function. Conservative mutation of codons histidine 128 (H128), serine 129 (S129), and serine 155 (S155) had greatly diminished function, while mutations of flanking residues had no effect on function. Our results represent the first structure/function analysis of the Pig-a protein, and suggest that codons H128, S129, and S155 represent critical regions of the Pig-a protein. Our results also suggest a means by which transgenic mice with a "partial knock-out" of Pig-a function could be generated, which would allow investigation of PNH in an animal model.