Kurazono H, Mochida S, Binz T, Eisel U, Quanz M, Grebenstein O, Wernars K, Poulain B, Tauc L, Niemann H
Institute for Microbiology, Federal Research Center for Virus Diseases of Animals, Tübingen, Federal Republic of Germany.
J Biol Chem. 1992 Jul 25;267(21):14721-9.
To define conserved domains within the light (L) chains of clostridial neurotoxins, we determined the sequence of botulinum neurotoxin type B (BoNT/B) and aligned it with those of tetanus toxin (TeTx) and BoNT/A, BoNT/C1, BoNT/D, and BoNT/E. The L chains of BoNT/B and TeTx share 51.6% identical amino acid residues whereas the degree of identity to other clostridial neurotoxins does not exceed 36.5%. Each of the L chains contains a conserved motif, HExxHxxH, characteristic for metalloproteases. We then generated specific 5'- and 3'-deletion mutants of the L chain genes of TeTx and BoNT/A and tested the biological properties of the gene products by microinjection of the corresponding mRNAs into identified presynaptic cholinergic neurons of the buccal ganglia of Aplysia californica. Toxicity was determined by measurement of neurotransmitter release, as detected by depression of postsynaptic responses to presynaptic stimuli (Mochida, S., Poulain, B., Eisel, U., Binz, T., Kurazono, H., Niemann, H., and Tauc, L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7844-7848). Our studies allow the following conclusions. 1) Residues Cys439 of TeTx and Cys430 of BoNT/A, both of which participate in the interchain disulfide bond, play no role in the toxification reaction. 2) Derivatives of TeTx that lacked either 8 amino- or 65 carboxyl-terminal residues are still toxic, whereas those lacking 10 amino- or 68 carboxyl-terminal residues are nontoxic. 3) For BoNT/A, toxicity could be demonstrated only in the presence of added nontoxic heavy (H) chain. A deletion of 8 amino-terminal or 32 carboxyl-terminal residues from the L chain had no effect on toxicity, whereas a removal of 10 amino-terminal or 57 carboxyl-terminal amino acids abolished toxicity. 4) The synergistic effect mediated by the H chain is linked to the carboxyl-terminal portion of the H chain, as demonstrated by injection of HC-specific mRNA into neurons containing the L chain. This finding suggests that the HC domain of the H chain becomes exposed to the cytosol during or after the putative translocation step of the L chain.
为了确定梭菌神经毒素轻链（L链）中的保守结构域，我们测定了B型肉毒杆菌神经毒素（BoNT/B）的序列，并将其与破伤风毒素（TeTx）以及BoNT/A、BoNT/C1、BoNT/D和BoNT/E的序列进行比对。BoNT/B和TeTx的L链有51.6%的氨基酸残基相同，而与其他梭菌神经毒素的相同程度不超过36.5%。每条L链都含有一个保守基序HExxHxxH，这是金属蛋白酶的特征性基序。然后我们构建了TeTx和BoNT/A的L链基因的特异性5'端和3'端缺失突变体，并通过将相应的mRNA显微注射到加州海兔颊神经节中已鉴定的突触前胆碱能神经元中来测试基因产物的生物学特性。通过测量神经递质释放来确定毒性，这可通过突触后对突触前刺激的反应减弱来检测（Mochida, S., Poulain, B., Eisel, U., Binz, T., Kurazono, H., Niemann, H., and Tauc, L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7844 - 7848）。我们的研究得出以下结论。1）TeTx的Cys439残基和BoNT/A的Cys430残基都参与链间二硫键形成，但在毒性反应中不起作用。2）缺失8个氨基端或65个羧基端残基的TeTx衍生物仍具有毒性，而缺失10个氨基端或68个羧基端残基的则无毒。3）对于BoNT/A，只有在添加无毒重链（H链）的情况下才能证明其毒性。L链缺失8个氨基端或32个羧基端残基对毒性没有影响，而缺失10个氨基端或57个羧基端氨基酸则消除了毒性。4）如将HC特异性mRNA注射到含有L链的神经元中所证明的，H链介导的协同效应与H链的羧基端部分相关。这一发现表明，在L链假定的转运步骤期间或之后，H链的HC结构域暴露于细胞质中。
