Neurotoxic mechanism of botulinum neurotoxin type B: Stimulation of tissue transglutaminase

Abstract

The clostridial neurotoxins have been known to inhibit neuroexocytosis via Zn-dependent protease activity. It was recently suggested that tissue transglutaminase is stimulated by the tetanus toxin and finally affect neurotransmitter release by crosslinking synaptic vesicles to cytoskeleton through a nerve ending substrate like synapsin $I^(1,2)$. When botulinum toxin type B and transglutaminase were mixed, the resulting fluorescence markedly increased over the arithmatic sum calculated from each fluorescence of the pure proteins. When the recombinant light chain and transglutaminase were mixed the rusult was same. The results indicate that botulinum neurotoxin type B specifically interacts with transglutaminase. The native toxin and the recombinant light chain stimulated liver transglutaminase respectively and futhermore the endogenous transglutaminase of the synaptic vesicle was also stimulated in a dose dependent manner. The stimulatory effects of recombinant light chain on transglutaminase activity was similar within 4 hours after 30 minutes (about 50-60% over control). However, the heat-killed botulinum toxin type B showed very low stimulatory effects on transglutaminase activity, and on the other hand, dithiothreitol reducing disulfide bond of botulinum neurotoxin reduced the hinrance effects of heavy chain to the interaction with transglutaminase. Futhermore the recombinant light of botulinum neurotoxin type B was twice effective on transglutaminase stimulation than native toxin. It is thus supposed that the light chain plays a major role in the stimulatory effect. However, the change of transglutaminase activity was not made by the proteolytic activity of botulinum toxin type B converting an inactive precursor to an active product. The stimulation of transglutaminase was noticeable especially at high calcium concentration, which implicates thet botulinum toxin may be more poisonous at the excited presynaptic nerve endings. Furthermorein the cellular sy...