C. Botulinum neurotoxin types A and E: isolated light chain breaks down into two fragments. Comparison of their amino acid sequences with tetanus neurotoxin

Abstract

The flaccid paralysis in the neuromuscular disease botulism appears to depend on the coordinated roles of the ∼50 kDa light and ∼100 kDa heavy chain subunits of the ∼150 kDa neurotoxic protein produced by Clostridium botulinum ( J. Biol. Chem. (1987) 262, 2660 and Eur. J. Biochem. (1988) 177, 683). We observed that the light chain after separation from its conjugate heavy chain in the presence of dithiothreitol and 2 M urea, begins to split into ∼28 and ∼18 kDa fragments. The other subunit—the ∼100 kDa heavy chain following its isolation—and the parent ∼150 kDa dichain neurotoxin do not break down under comparable conditions. This cleavage was examined in the neurotoxin serotypes A and E. The cleavage does not appear to be due to a protease. Partial amino acid sequences established that: i) the ∼28-kDa and ∼18-kDa fragments comprise the N- and C-terminal regions of the light chain, respectively; ii) the light chain of the neurotoxin serotypes A and E break down at precise peptide bonds; iii) the peptide bonds cleaved in serotypes A and E are five residues apart; and iv)_the portions of the ∼18 kDa fragments of serotype A and E neurotoxin sequenced so far are highly homologous to the corresponding region of tetanus neurotoxin produced by Clostridium ietani. The partial N-terminal sequence of the ∼28 kDa fragment matches with the N-terminal sequence of the intact L chain. The 47 residues of the ∼18-kDa fragment of type A sequenced from its N-terminal are: — Y. E.M.S.G.L.E.V. S. F. E.E.L. R. T.F.G.G. H. D.A. K.F. I. D.S.L.Q.E. N. E. F.R.L.Y.Y.Y. N. K.F. K. D. I.A. S.T. L. —. These align with those of tetanus neurotoxin beginning at its residue #259 (Tyr); The 18 underlined residues of the above 47 residues ( 1.e 38%) are identical in positions between the two proteins. The 41 residues sequenced from the ⊥18kDa fragment of type E botulinum neurotoxin are: —K.G. I N.I. E.E. F.L. T.F.G. N.N. D. L. N. I. I. T.V.A.Q.Y. N.D. I. Y. T.N.L. L.N.D.Y. R.K. I.A. X. K . L.—. These align with tetanus neurotoxin, beginning at its residue #264 (Tyr); the 20 underlined residues ( i.e. 49%) are positionally identical to tetanus neurotoxin. Also the 13 overlined residues of type E ( i.e. 32%) are positionally identical to type A botulinum neurotoxin.