Ahmed S A, Byrne M P, Jensen M, Hines H B, Brueggemann E, Smith L A
Department of Immunology and Molecular Biology, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702-5011, USA.
J Protein Chem. 2001 Apr;20(3):221-31. doi: 10.1023/a:1010952025677.
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.
肉毒杆菌神经毒素 A 型的高度纯化重组锌内肽酶轻链经历了自催化蛋白水解加工和片段化过程。在不添加锌的情况下，最初从 448 个氨基酸残基的蛋白质的 C 末端切割掉 10 - 28 个残基，随后出现 SDS 稳定的二聚体，最后在分子中部附近发生片段化。在添加锌的情况下，片段化速率加快，但可切割键的特异性发生了变化，这表明锌在轻链中具有结构作用。通过向轻链中添加金属螯合剂 TPEN，C 末端的蛋白水解加工减少，分子中部附近的片段化受到抑制。同样，添加轻链催化活性的竞争性肽抑制剂（CRATKML）也大大减少了蛋白水解。基于这些结果，我们首次提供了明确的证据，证明 C 末端肽的丢失和轻链的片段化是酶促和自催化的。通过分离大小肽段，我们通过埃德曼降解和电喷雾离子化质谱对它们进行了测序，并确定了蛋白水解位点。我们还发现，除了先前报道的 Y250 - Y251 和 K438 - T439 键外，在 F266 - G267、F419 - T420、F423 - E424、R432 - G433 和 C430 - V431 键处也发生了蛋白水解。
