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Tetanus toxin: biochemical and pharmacological comparison between its protoxin and some isotoxins obtained by limited
U Weller1, F Mauler, E Habermann
1Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Abstract:
Single-chain tetanus toxin (toxin S) was prepared from short-term cultures by lysis under protection with protease inhibitors, precipitation with 40% ammonium sulfate, gel filtration, and chromatography on DEAE ion exchanger. Its limited proteolysis by trypsin, post-arginine cleaving enzyme from mouse submaxillary gland and clostripain led to bichainal derivatives (BT, BA, BCl) consisting of a heavy chain and a larger version of the light chain. The latter was then converted by trypsin into a small version which comigrated with the light chain of bichainal extracellular toxin (BE). The light chain produced by chymotrypsin (BC) and elastase (BE1) was of intermediate size. The nick region serves as substrate for all esteroproteases investigated and comprises between one and two kDa. Limited proteolysis increased the hydrophilicity (BT greater than BE greater than S) in hydrophobic interaction HPLC, and anionic behaviour (BC greater than BE greater than BT greater than S) in DEAE ion exchanger HPLC. The bichainal toxins assessed (BC, BE or BT) were about two times more toxic than toxin S (LD50, mouse s.c. 2 ng/kg vs. 4 ng/kg). They were five to twelve times more potent than toxin S in three in vitro assays measuring the prevention of neurotransmitter release, i.e. on the phrenic nerve-hemidiaphragm preparation of the mouse (acetylcholine, with toxin BE and BT), on primary brain cell cultures from the mouse ([3H]noradrenaline, with toxin BE and BT), and on brain homogenate from rats ([3H]noradrenaline, with toxin BA, BC, BE and BT). Thus single-chain toxin is a less potent precursor of, or protoxin for, various bichainal isotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Single-chain tetanus toxin (toxin S) is a less potent precursor that can be converted into more toxic bichainal forms. These derivatives show increased hydrophilicity and potency in preventing neurotransmitter release.
Area of Science:
- Neuroscience
- Biochemistry
- Toxicology
Background:
- Single-chain tetanus toxin (toxin S) is a precursor molecule.
- Understanding its conversion to active forms is crucial for toxin research.
Purpose of the Study:
- To investigate the limited proteolysis of single-chain tetanus toxin.
- To characterize the resulting bichainal derivatives and their biological activity.
Main Methods:
- Preparation of toxin S via lysis, precipitation, and chromatography.
- Limited proteolysis using trypsin, mouse submaxillary gland enzyme, clostripain, chymotrypsin, and elastase.
- Analysis using hydrophobic interaction HPLC and DEAE ion exchanger HPLC.
- Toxicity assessment via LD50 and in vitro neurotransmitter release assays.
Main Results:
- Limited proteolysis generated various bichainal tetanus toxin derivatives (BT, BA, BCl, BC, BE, BE1).
- Proteolysis altered toxin hydrophilicity and anionic behavior.
- Bichainal toxins were approximately twice as toxic and 5-12 times more potent in inhibiting neurotransmitter release compared to toxin S.
Conclusions:
- Single-chain tetanus toxin acts as a less potent protoxin.
- Limited proteolysis yields more potent bichainal isotoxins with enhanced neuroinhibitory activity.