In Vitro Reconstitution of the Clostridium botulinum Type D Progenitor Toxin
Copyright policy )In Vitro Reconstitution of the Clostridium botulinum Type D Progenitor Toxin
Clostridium botulinum type D strain 4947 produces two different sizes of progenitor toxins (M and L) as intact forms without proteolytic processing. The M toxin is composed of neurotoxin (NT) and nontoxic-nonhemagglutinin (NTNHA), whereas the L toxin is composed of the M toxin and hemagglutinin (HA) subcomponents (HA-70, HA-17, and HA-33). The HA-70 subcomponent and the HA-33/17 complex were isolated from the L toxin to near homogeneity by chromatography in the presence of denaturing agents. We were able to demonstrate, for the first time, in vitro reconstitution of the L toxin formed by mixing purified M toxin, HA-70, and HA-33/17. The properties of reconstituted and native L toxins are indistinguishable with respect to their gel filtration profiles, native-PAGE profiles, hemagglutination activity, binding activity to erythrocytes, and oral toxicity to mice. M toxin, which contained nicked NTNHA prepared by treatment with trypsin, could no longer be reconstituted to the L toxin with HA subcomponents, whereas the L toxin treated with proteases was not degraded into M toxin and HA subcomponents. We conclude that the M toxin forms first by assembly of NT with NTNHA and is subsequently converted to the L toxin by assembly with HA-70 and HA-33/17.
Chromatography, Botulinum Toxins, Erythrocytes, Transcription, Genetic, Molecular Sequence Data, Models, Biological, Protein Structure, Tertiary, Mice, Hemagglutinins, Protein Biosynthesis, Endopeptidases, Chromatography, Gel, Animals, Electrophoresis, Polyacrylamide Gel, Trypsin, Amino Acid Sequence, Amino Acids, Protein Precursors, Protein Binding
Chromatography, Botulinum Toxins, Erythrocytes, Transcription, Genetic, Molecular Sequence Data, Models, Biological, Protein Structure, Tertiary, Mice, Hemagglutinins, Protein Biosynthesis, Endopeptidases, Chromatography, Gel, Animals, Electrophoresis, Polyacrylamide Gel, Trypsin, Amino Acid Sequence, Amino Acids, Protein Precursors, Protein Binding
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