BOTULINUM NEUROTOXIN TYPE-A - SEQUENCE OF AMINO-ACIDS AT THE N-TERMINUS AND AROUND THE NICKING SITE

Clostridium botulinum synthesizes the type A botulinum neurotoxin (NT) as a ∼ 150 kDa single chain protein. Post-translational proteolytic processing yields a ∼ 150 kDa dichain protein composed of a ∼ 50 kDa light and ∼ 100 kDa heavy chain, which has higher toxicity. Trypsin's action mimics the endogenous proteolytic processing [12]. 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 ∼ 150 kDa single chain NT, ProPheValAsnLys-, remain intact at the N-terminus of the ∼ 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 [10]. 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 -GlyTyrAsnLysAlaLeuAsnAspLeu- before cleaving the bond LysAla at a slower rate. The data indicate that the dichain NT is formed in the bacterial culture in at least 2 steps. Cleavage at XGly produces a ∼ 100 kDa heavy chain-like fragment which is then truncated; cleavage 4 residues downstream at LysAla, and excision of the tetrapeptide GlyTyrAsnLys, generates the mature heavy chain with Ala as its N-terminal residue. The ∼ 100 kDa heavy chain generated in vitro, by nicking the single chain NT with trypsin, also has AlaLeuAsn- as the N-terminal residues. © 1990.