Botulinum neurotoxin type A: sequence of amino acids at the N-terminus and around the nicking site.
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DasGupta BR, Dekleva ML
Botulinum neurotoxin type A: sequence of amino acids at the N-terminus and around the nicking site.
Biochimie. 1990 Sep;72(9):661-4.
- PubMed ID
- 2126206 [ View in PubMed]
- Abstract
Clostridium botulinum synthesizes the type A botulinum neurotoxin (NT) as a approximately 150 kDa single chain protein. Post-translational proteolytic processing yields a approximately 150 kDa dichain protein composed of a approximately 50 kDa light and approximately 100 kDa heavy chain, which has higher toxicity. Trypsin's action mimics the endogenous proteolytic processing. The proteolytic cleavages could occur at 4 sites. We have examined 2 such sites and defined the peptide sequences before and after proteolytic processing. The N-terminal residues of the newly synthesized approximately 150 kDa single chain NT, Pro-Phe-Val-Asn-Lys-, remain intact at the N-terminus of the approximately 50 kDa light chain generated either in the clostridial culture or in vitro with trypsin or with a protease purified from the homologous bacterial culture. The clostridial protease cleaves the single chain NT in vitro, at 1/3 the distance from its N-terminus, on the amino side of Gly of the sequence -Gly-Tyr-Asn-Lys-Ala-Leu-Asn-Asp-Leu- before cleaving the bond Lys-Ala at a slower rate. The data indicate that the dichain NT is formed in the bacterial culture in at least 2 steps. Cleavage at X-Gly produces a approximately 100 kDa heavy chain-like fragment which is then truncated; cleavage 4 residues downstream at Lys-Ala, and excision of the tetrapeptide Gly-Tyr-Asn-Lys, generates the mature heavy chain with Ala as its N-terminal residue. The approximately 100 kDa heavy chain generated in vitro, by nicking the single chain NT with trypsin, also has Ala-Leu-Asn- as the N-terminal residues.