Characterization of nicking of the nontoxic-nonhemagglutinin components of Clostridium botulinum types C and D progenitor toxin.
Clostridium botulinum C and D strains produce two types of progenitor toxins, 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 around the nicking sites in the NTNHAs of the M toxins produced by C. botulinum type C and D strains by analysis of their C-terminal and N-terminal sequences and mass spectrometry. The C-terminus of the 15-kDa fragments was identified as Lys127 from these strains, indicating that a bacterial trypsin-like protease is responsible for the nicking. The 115-kDa fragment had mixtures of three different N-terminal amino acid sequences beginning with Leu135, Val139, and Ser141, indicating that 7-13 amino acid residues were deleted from the nicking site. The sequence beginning with Leu135 would also suggest cleavage by a trypsin-like protease, while the other two N-terminal amino acid sequences beginning with Val139 and Ser141 would imply proteolysis by an unknown protease. The nicked NTNHA forms a binary complex of two fragments that could not be separated without sodium dodecyl sulfate.
Department of Food Science, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Japan.
Y
Y
Sagane
T
T
Watanabe
H
H
Kouguchi
H
H
Sunagawa
K
K
Inoue
Y
Y
Fujinaga
K
K
Oguma
T
T
Ohyama
D000595:Amino Acid Sequence; D001665:Binding Sites; D001905:Botulinum Toxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003014:Clostridium botulinum / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006388:Hemagglutinins / Q000737:chemistry / Q000302:isolation & purification; D037102:Lectins; D009498:Neurotoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C405516:Clostridium botulinum L toxin; C080139:Clostridium botulinum M toxin; D006388:Hemagglutinins; D037102:Lectins; D009498:Neurotoxins; D010446:Peptide Fragments; C096325:botulinum toxin type D; D010450:Endopeptidases; D001905:Botulinum Toxins; C096324:botulinum toxin type C