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.

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