Botulinum neurotoxins heavy chains

Brunger, A.T., Briedenbach, M.A., Jin, R., Fischer, A., Santos, J.S., Montal, M. (2007). Botulinum neurotoxin heavy chain belt as an intramolecular chaperone for the light chain. FLOS Path. 3 1191. ... 

Koriazova, L.K., Mental, M. (2003). Translocation of botulinum neurotoxin light chain protease through the heavy chain channel. Nat. Struct. Biol. 10 13-18. 

The most ingenious exocytosis toxins, however, come from the anaerobic bacteria Clostridium botulinum and Clostridium tetani. The former produces the seven botulinum neurotoxins (BoNTs) A-G the latter produces tetanus neurotoxin (TeNT). All eight toxins consist of a heavy (H) chain and a light (L) chain that are associated by an interchain S-S bond. The L-chains enter the cytosol of axon terminals. Importantly, BoNT L-chains mainly enter peripheral cholinergic terminals, whereas the TeNT L-chain mainly enters cerebral and spinal cord GABAergic and glycinergic terminals. The L-chains are the active domains of the toxins. They are zinc-endopeptidases and specifically split the three core proteins of exocytosis, i.e. the SNAREs (Fig. 1 inset). Each ofthe eight toxins splits a... 

Poulain, B., Mochida, S., Weller, U., Hogy, B., Habermann, E., Wadsworth, J. D., Shone, C.C., Dolly, J.O. and Tauc, L., Heterologous combinations of heavy and light chains from botulinum neurotoxin A and tetanus toxin inhibit neurotransmitter release in Aplysia, J. Biol. Chem., 266, 9580-9585, 1991. 

Daniels-Holgate, P., Dolly, J. (1996). Productive and nonproductive binding of botulinum neurotoxin A to motor nerve endings are distinguished by its heavy chain. J. Neurosci. Res. 44 263-71. 

Maruta, T., Dolimbek, B.Z., Aoki, K.R., Steward, L.E., Atassi, M.Z. (2004). Mapping of the synaptosome-binding regions on the heavy chain of botulinum neurotoxin A by synthetic overlapping peptides encompassing the entire chain. Protein J. 23 539-52. 

Matsuda M, Lei DL, Sugimoto N, Ozutsumi K, OkabeT (1989) Isolation, purification and characterization of fragment B, the NH2-terminal half of the heavy chain of tetanus toxin. In Infect. Immun. 57 3588-93 Matthews BW (1988) Structural basis of the action of thermolysin and related zinc peptidases.In Acc. Chem. Res. 21 333-40 Mclnnes C, Dolly JO (1990) Ca -dependent noradrenaline release from perme-abilised PC 12 cells is blocked by botulinum neurotoxin A or its light chain. In FEBS Lett. 261 323-6... 

Kohda, T., Ihara, H., Seto, Y., and Tsutsuki, H. 2007. Differential contribution of the residues in C-terminal half of the heavy chain of botulinum neurotoxin type B to its binding to the ganglioside GT lb and the synaptotagmin 2/GTlb complex. Microb. Pathog. 42 72-79. 

Fig. 4. Organization and fragmentation of botulinum neurotoxin. Botulinum neurotoxin is a protein with a molecular weight of 150,000 to 160,000. It is synthesized as a single chain polypeptide in the Clostridium bacteria. It is nicked by endogenous protease to yield a dichain molecule linked by a disulfide bond. This nicking is usually associated with activation of its toxicity. The nicked molecule is separated into heavy and light chains by reduction of the disulfide bond.

Blaustein, R. O., Germann, W. J., Finkelstein, A., and DasGupta, B. R., 1987, The N-terminal half of the heavy chain of botulinum type A neurotoxin forms of channels in planar phospholipid bilayers, FEBS Lett. 226 1 15-120. 

Therefore, we developed a drug delivery vehicle (DDV) comprising the nontoxic recombinant heavy chain of BoNT-A coupled to a 10-kDa amino dextran via the heterobifunctional linker 3-(2-pyridylthio)-propionyl hydrazide. The heavy chain served to target botulinum neurotoxin-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted cells. 

BoNT Botulinum neurotoxin LC light chain HC heavy chain DDV drug delivery vehicle. 

Botulinum neurotoxins (seven serotypes, A-G) are relatively large water soluble proteins (150 kDa) produced by the Clostridium botulinum. Each protein has two polypeptide chains (a 100 kDa heavy chain and a 50 kDa light chain) linked through a disulfide bond (Fig. 2). In the proposed mode of action of botulinum and tetanus neurotoxins (Simpson, 1986, 1989), the C-terminal half of the heavy chain binds to the nerve membrane leading to internalization of the neurotoxin in the nerve cell through endocytosis. Subsequently, the pH of the endosome is lowered causing the heavy chain to get integrated in the membrane... 

Figure 2. Schematic diagram of botulinum neurotoxin showing its light and heavy chains. The two different domains of the heavy chain shaded with different patterns indicate the N-terminal and C-terminal halves (about 50 kDa each). These two domains are believed to play different functional roles during the intoxication process. The light chain has been shown to contain the toxic site.

It is also possible that a combination of both modes suggested above is involved in the formation of membrane channel for the translocation botulinum neurotoxin. The main assumptions of the hypothesis are (i) The presence of amphiphilic and transmembrane segments in the light and heavy chains as predicted by the hydrophobic moment analysis, (ii) Existence of botulinum neurotoxin in oligomeric form. 

Low pH is apparently required for the strong channel formation activity of botulinum and tetanus neurotoxins and their heavy chains (Boquet and Duflot, 1982 Hoch et al., 1985). A pH of 5.0 or lower induces the channel formation. Two possible effects of a low pH are... 

Figure 8. Schematic model of an oligomeric membrane channel structure by botulinum or tetanus neurotoxin. The model shows a trimer of the neurotoxin with smaller cylinders representing light chain whereas larger lobe representing heavy chain.

Singh, B. R. and DasGupta, B. R., 1989d, Changes in the molecular topography of the light and heavy chains of type A botulinum neurotoxin following their separation. Biophysical Chem. 34 259-267. 

Strains of C. botulinum produce seven antigenetically distinct neurotoxins designated as serotypes A through G. All seven serotypes have a similar structure and molecular weight, consisting of a heavy (H) chain and a light (L) chain joined by a disulfide bond. They all interfere with neural transmission by blocking the release of ACh (see Chapter 14), which is the principal neurotransmitter at the neuromuscular junction. 

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