Pepsin fragmentation of botulinum type E neurotoxin: isolation and characterization of 112, 48, 46, and 16 kD fragments.

Controlled digestion of approximately 150 kD single chain botulinum type E neurotoxin with pepsin at pH 6.0 produced 112, 48, 46, and 16 kD fragments. These were chromatographically purified; their locations in the approximately 1300 amino acid residue long neurotoxin were determined by identifying the amino terminal 10 residues of 112 and 48 kD fragments, 50 residues of 46 kD fragment, and 59 residues of 16 kD fragment. The 48 and 112 kD fragments contain the N-terminal segment of the neurotoxin (i.e., residue no. 1 to approximately 425 and 1 to approximately 990, respectively), the 46 kD fragment corresponds to approximately 407 residues of the C-terminal region, and the 16 kD fragment contains the approximately 140 residues from a segment nearer to the C-terminus. The 48 kD fragment is similar to the approximately 50 kD N-terminal light chain of the approximately 150 kD dichain neurotoxin, which is generated by tryptic cleavage of the approximately 150 kD single chain neurotoxin, and is separated from the approximately 100 kD C-terminal heavy chain by dithiothreitol (DTT) reduction of an intrachain disulfide bond in the presence of 2 M urea (Sathyamoorthy and DasGupta, J. Biol. Chem. 260, 10461, 1985). The pepsin-generated 48 kD fragment, unlike the light chain, was isolated without exposure to DTT and urea. The single chain 112 kD fragment following trypsin digestion yielded 48 and 60 kD fragments that were separable after DTT reduction of the intrachain disulfide which links them. The N-terminal residues of the smaller fragment were identical to that of the single chain 150 kD neurotoxin; the single chain 112 kD fragment is therefore the neurotoxin minus the approximately 50 kD C-terminal half of the heavy chain. The biological activities of the 48 and 112 kD fragments can be demonstrated in permeabilized PC12 cells (Lomneth et al., J. Neurochem. 57, 1413, 1991); they inhibit norepinephrine release.