Botulinum Neurotoxin Type A: Limited Proteolysis by Endoproteinase Glu-C and α-Chymotrypsin Enhanced Following Reduction; Identification of the Cleaved Sites and Fragments

Botulinum neurotoxin (NT) serotype A is a ∼150-kDa dichain protein. Posttranslational nicking of the single-chain NT (residues Pro 1–Leu 1295) by the protease(s) endogenous to Clostridium botulinum excises 10 residues, leaving Pro 1–Lys 437 and Ala 448–Leu 1295 in the ∼50-kDa light (L) and ∼100-kDa heavy (H) chains, respectively, connected by a Cys 429–Cys 453 disulfide and noncovalent bonds [Krieglstein et al. (1994), J. Protein Chem. 13, 49–57]. The L chain is a metalloprotease, while the amino- and carboxy-terminal halves of the H chain have channel-forming and receptor-binding activities, respectively [Montecucco and Schiavo (1995), Q. Rev. Biophys. 28, 423–472]. Endoproteinase Glu-C and α-chymotrypsin were used for controlled digestion at pH 7.4 of the ∼150-kDa dichain NT and the isolated ∼100-kDa H chain (i.e., freed from the L chain) in order to map the cleavage sites and isolate the proteolytic fragments. The dichain NT appeared more resistant to cleavage by endoproteinase Glu-C than the isolated H chain. In contrast, the NT with its disulfide(s) reduced showed rapid digestion of both chains, including a cleavage between Glu 251 and Met 252 (resulting in ∼30- and ∼20-kDa fragments of the L chain) which was not noted unless the NT was reduced. Interestingly, an adjacent bond, Tyr 249–Tyr 250, was noted earlier [DasGupta and Foley (1989), Biochimie 71, 1193–1200] to undergo “self-cleavage” following reductive separation of the L chain from the H chain. The site Tyr–Tyr–Glu–Met (residues 249–252) appears to become exposed following reduction of Cys 429–Cys 453 disulfide. Identification of Glu 669–Ile 670 and Tyr 683–Ile 684 as protease-susceptible sites demonstrated for the first time that at least two peptide bonds in the segment of the H chain (residues 659–684), part of which (residues 659–681) is thought to interact with the endosomal membranes and forms channels [Oblatt-Montal et al., (1995), Protein Sci. 4, 1490–1497], are exposed on the surface of the NT. Two of the fragments of the H chain we generated and purified by chromatography are suitable for structure–function studies; the ∼85- and ∼45-kDa fragments beginning at residue Leu 544 and Ser 884, respectively (both extend presumably to Leu 1295) contain the channel-forming segment and receptor-binding segments, respectively. In determining partial amino acid sequences of 10 fragments, a total of 149 amino acids in the 1275-residue NT were chemically identified.