Abstract
Tetanus toxin (TeTX) is a dichain protein that blocks neuroexocytosis, an action attributed previously to Zn(2+)-dependent proteolysis of synaptobrevin (Sbr) by its light chain (LC). Herein, its cleavage of Sbr in rat cerebrocortical synaptosomes was shown to be minimized by captopril, an inhibitor of certain metalloendoproteases, whereas this agent only marginally antagonized the inhibition of noradrenaline release, implicating a second action of the toxin. This hypothesis was proven by preparing three mutants (H233A, E234A, H237A) of the LC lacking the ability to cleave Sbr and reconstituting them with native heavy chain. The resultant dichains were found to block synaptosomal transmitter release, albeit with lower potency than that made from wild type LC; as expected, captopril attenuated only the inhibition caused by the protease-active wild type toxin. Moreover, these protease-inactive toxins or their LCs blocked evoked quantal release of transmitter when micro-injected inside Aplysia neurons. TeTX was known to stimulate in vitro a Ca(2+)-dependent transglutaminase (TGase) (Facchiano, F., and Luini, A. (1992) J. Biol. Chem. 267, 13267-13271), an affect found here to be reduced by an inhibitor of this enzyme, monodansylcadaverine. Accordingly, treatment of synaptosomes with the latter antagonized the inhibition of noradrenaline release by TeTX while not affecting Sbr cleavage. This drug also attenuated the inhibitory action of all the mutants. Hence, it is concluded that TeTX inhibits neurotransmitter release by proteolysis of Sbr and a protease-independent activation of a neuronal TGase.
Highlights
Tetanus toxin (TeTX)[1] is a 150-kDa protein produced by Clostridium tetani that causes tetanus by and irreversibly blocking transmitter release (1)
TeTx-induced Blockade of Synaptosomal Neurotransmitter Release Persists after the Toxin’s Cleavage of Sbr Is Minimized by Captopril—Treatment of synaptosomes with TeTX gave a concentration-dependent reduction of depolarization-evoked Ca2ϩ-dependent NA release, but this was only marginally attenuated by captopril (Fig. 1A), an inhibitor of certain metalloproteases that gives partial antagonism of the neuroparalytic action of TeTX at the mammalian neuromuscular junction (12)
The ability of protease-deficient TeTX mutants to inhibit exocytosis was shown for synaptosomal NA secretion and ACh release from Aplysia neurons
Summary
Rat cerebrocortical synaptosomes were prepared and release of tritiated noradrenaline (NA) measured as outlined previously (12). Native or recombinant LC were incubated with synaptic vesicles (0.5 mg/ml; purified by differential centrifugation from bovine cortex (see Ref. 14)) for 90 min at 37 °C in 50 mM Hepes, 400 mM NaCl, 5 mM dithiothreitol, 2 M ZnSO4 (pH 7.4) and subjected to SDS-PAGE/Western blotting, as above. Free maltose was removed by extensive dialysis, and the dichain preparations were separated from the released MBP by a further chromatographic step on amylose resin. The amount of Zn2ϩ associated with each LC was calculated by subtracting the content for MBP alone, treated from that of MBP-LC fusion proteins
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