Botulinum Neurotoxin Types A, B, and E: Fragmentations by Autoproteolysis and Other Mechanisms Including by O-Phenanthroline–Dithiothreitol, and Association of the Dinucleotides NAD+/NADH with the Heavy Chain of the Three Neurotoxins

Abstract

The first evidence of autoproteolytic activity of the approximately 50-kDa light chain of the clostridial neurotoxins (NT) is traceable to the observations that the light chains of botulinum NT serotypes A and E, separated from their approximately 100-kDa heavy chain conjugate, were found cleaved at the amino side of Tyr250 and Arg244, respectively [DasGupta and Foley (1989). Biochimie 71: 1183-1200]. Specific cleavages of the recombinant light chain of NT type A, including at Tyr249-Tyr250, firmly established that the cleavages reported earlier were due to autoproteolysis [Ahmed et al. (2001). J. Protein Chem. 20: 221-231; Ahmed et al. (2003). Biochemistry 42:12539-12549] and not by contaminating proteases or non-enzymatic. We now report many cleavages in the NT types A, B and E and also in their separated light and heavy chains, and identification of several of the peptide bonds cleaved. None of the identified cleaved bonds (-P1-P1' -) in one serotype (except Asp-Pro) was found common in other serotypes or cleaved within itself at a second site. After separation from the heavy chain self-cleavages of the light chains of type A, B and E at Tyr249-Tyr250, Gln258-Ser259 and Ile243-Arg244, respectively indicate an intriguing feature (in the aligned sequences these bonds of type A and B are 2 and type A and E are 4 peptide bonds apart) that may have some role in the NT's structure-function relationship yet to be understood. We point out that autoproteolysis of a single peptide bond (Phe418-Thr419 or Phe422-Glu423) in NT type A reported by Ahmed et al. (2001) can potentially generate proteolytically active light chain freed of the heavy chain; this is an efficient pathway, that by-passes nicking by a trypsin-like protease(s) inside the intrachain disulfide bridge and its reductive cleavage. We offer probable explanations for the observed cleavages such as acid- and metal-mediated (non-catalytic and non-stoichiometric) reactions in addition to autoproteolysis but cannot predict which mechanism(s) of cleavage occur or prevail following NT's entry in the body as poison or therapeutic agent. The metal chelator O-phenanthroline (above critical miceller concentration) in the presence of dithiothreitol cleaved type E NT at limited sites generating discrete 114-, 87-, 49-, 42-, and 31-kDa fragments but degraded NTs type A and B extensively. The limited cleavage of type E NT was dependent on the presence of metal ion(s) bound to the protein and its native (urea sensitive) conformation. The self-cleavage of the NTs at specific sites prompted us to search for specific binding sites on the NTs analogous to SNARE-motifs-the 9-residuelong motifs present on the NT's natural substrates (SNAP-25, syntaxin, VAMP/synaptobrevin); such putative binding motifs (sites) noted on all clostridial NTs are reported here. Their relationship to the observed autoproteolysis remains to be determined experimentally. The dinucleotide NAD(+)/NADH associated with the NTs type A, B and E (2-3 NADH per protein molecule) via their H-chains, and a portion ...

Light Chain Separated from the Rest of the Type A Botulinum Neurotoxin Molecule Is the Most Catalytically Active Form

“…Our results extended the earlier observations by DasGupta [34], [35] that BoNT/A does not undergo autocatalysis with an implication that shielding of the active site by the belt in these constructs prevents this unwanted reaction. Our results prove that complete removal of the belt is necessary for the autocatalytic reaction to occur.…”

The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site

“…1) (23,24), but the rate of the autocatalytic reaction was much slower than the rate of the catalytic reaction (25). Thus, a C-terminal sequence comprising these sites might act as a competitive inhibitor of LcA activity, and the extent of inhibition should correspond to its length for effective interactions with the LcA molecule.…”

Botulinum neurotoxins: Perspective on their existence and as polyproteins harboring viral proteases

“…This fusion brought about two developments: i) The payload (the protease) became coupled to a delivery vehicle with aiming ability which (two-thirds of the 150-kDa NT) becomes, as believed, a throwaway piece after the protease reaches the target neuronal cytosol. ii) The protease with autoproteolysis activity (Ahmed et al, 2001(Ahmed et al, , 2003DasGupta et al, 2005) became refractory to self-digestion by the presence of the H-chain. The probability of this imagined scenario of the past is demonstrable: the ϳ50-kDa protease domain by itself and also separately the protease domain fused to its adjacent neighbor the ϳ50-kDa transmembrane (translocation) domain, both recombinant products (the ϳ50-and ϳ100-kDa, respectively), are proteolytically active (Ahmed et al, 2001;Chaddock et al, 2002).…”

“…The large extracellular region of the a-neurexin (for a schematic model see Fig. 1 in Littleton and Sheng, 2003) could be conjectured cleavable by a NT protease (or its variant) under any of the three circumstances by being accessible to the i) extracellular 150-kDa NT (prior to internalization by endocytosis) while bound to its receptors on the presynaptic membrane or ii) the ϳ50-kDa L-chain released in an extracellular milieu from the ϳ100-kDa H-chain following rupture of the interchain -S-S-bridge (Bhattacharya et al, 1988) or following probable autoproteolytic cleavage outside the -S-S-bridge (DasGupta et al, 2005), or iii) the ϳ50-kDa protease (the conjectured viral protease before it became fused to the transmembrane and receptor domains; noted above). Whether the a-neurexins have determinants of NT cleavage (binding and cleavage sites) is not yet known.…”

“…ii) A protein (cellubrevin) found in non-neuronal cells is cleaved; consequences of the cleavages are becoming known (see below). iii) The light chain of the NT is autoproteolyzed (Ahmed et al, 2001(Ahmed et al, , 2003DasGupta et al, 2005), implications of which are not yet clear. In addition a speculated feature is a protein (hypothetical) that is cleaved in vivo but the effect is benign and/or manifestation of the cleavage consequence is not acute (see below, e.g., Rab3A).…”