Characterization of nicking of the nontoxic-nonhemagglutinin components ofClostridium botulinum types C and D progenitor toxin

Clostridium botulinum C and D strains produce two types of progenitor toxin s, M and L. Previously we reported that a 130-kDa nontoxic-nonhemagglutinin (NTNHA) component of the M toxin produced by type D strain CB16 was nicked at a unique site, leading to a 15-kDa N-terminal fragment and a 115-kDa C- terminal fragment. In this study, we identified the amino acid sequences ar ound the nicking sites in the NTNHAs of the M toxins produced by C. botulin um type C and D strains by analysis of their C-terminal and N-terminal sequ ences and mass spectrometry. The C-terminus of the 15-kDa fragments was ide ntified as Lys127 from these strains, indicating that a bacterial trypsin-l ike protease is responsible for the nicking. The 115-kDa fragment had mixtu res of three different N-terminal amino acid sequences beginning with Leu13 5, Val139, and Ser141, indicating that 7-13 amino acid residues were delete d from the nicking site. The sequence beginning with Leu135 would also sugg est cleavage by a trypsin-like protease, while the other two N-terminal ami no acid sequences beginning with Val139 and Ser141 would imply proteolysis by an unknown protease. The nicked NTNHA forms a binary complex of two frag ments that could not be separated without sodium dodecyl sulfate.