Enzymatic autocatalysis of botulinum A neurotoxin light chain.
Highly purified recombinant zinc-endopeptidase light chain of the botulinum neurotoxin serotype A underwent autocatalytic proteolytic processing and fragmentation. In the absence of added zinc, initially 10-28 residues were cleaved from the C-terminal end of the 448-residue protein followed by the appearance of an SDS-stable dimer and finally fragmentation near the middle of the molecule. In the presence of added zinc, the rate of fragmentation was accelerated but the specificity of the cleavable bond changed, suggesting a structural role for zinc in the light chain. The C-terminal proteolytic processing was reduced, and fragmentation near the middle of the molecule was prevented by adding the metal chelator TPEN to the light chain. Similarly, adding a competitive peptide inhibitor (CRATKML) of the light-chain catalytic activity also greatly reduced the proteolysis. With these results, for the first time, we provide clear evidence that the loss of C-terminal peptides and fragmentation of the light chain are enzymatic and autocatalytic. By isolating both the large and small peptides, we sequenced them by Edman degradation and ESIMS-MS, and mapped the sites of proteolysis. We also found that proteolysis occurred at F266-G267, F419-T420, F423-E424, R432-G433, and C430-V431 bonds in addition to the previously reported Y250-Y251 and K438-T439 bonds.
Ahmed
S A
SA
Department of Immunology and Molecular Biology, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702-5011, USA. syed.ahmed@amedd.army.mil
Byrne
M P
MP
Jensen
M
M
Hines
H B
HB
Brueggemann
E
E
Smith
L A
LA
D000595:Amino Acid Sequence; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D002614:Chelating Agents / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005029:Ethylenediamines / Q000378:metabolism; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D002614:Chelating Agents; D005029:Ethylenediamines; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010450:Endopeptidases; D019274:Botulinum Toxins, Type A; D015032:Zinc; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine