Expression and Purification of the Light Chain of Botulinum Neurotoxin A: A Single Mutation Abolishes Its Cleavage of SNAP-25 and Neurotoxicity after Reconstitution with the Heavy Chain

Abstract

Botulinum neurotoxin type A (BoNT/A) selectively and irreversibly inhibits acetylcholine release from peripheral nerve endings. While the toxin's heavy (H) chain contributes to neuronal binding and internalization, its light (L) chain is a Zn(2+)-dependent endoprotease that intracellularly cleaves synaptosomal-associated protein of M(r) = 25 kDa (SNAP-25). For research and clinical exploitation of this uniquely-acting neurotoxin, recombinant wild-type L chain was produced together with a mutant in which His227 in the Zn(2+)-binding motif was substituted by Tyr. The PCR-amplified wild-type and mutant L chain genes were cloned, fused to the gene for maltose-binding protein, and expressed at high levels in Escherichia coli. The soluble fusion proteins were purified using amylose affinity chromatography, and, after factor Xa cleavage, the free L chains were isolated. The wild-type was shown to proteolyze SNAP-25 at a rate approaching that of the native chain while the mutant was inactive. Reconstitution of the pure wild-type L chain with native homogeneous H chain yielded a disulfide-linked dichain form that inhibited neuromuscular transmission in vitro and produced the symptoms of botulism in vivo. After reconstitution with the H chain, the Tyr227 mutant L chain failed to show any neuroparalytic activity in either of these assays. This methodology allows, for the first time, routine preparation of recombinant forms of the L chain that are needed to decipher the molecular details of its interaction with substrate and, thereby, assist the design of effective inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of Release of Neurotransmitters from Rat Dorsal Root Ganglia by a Novel Conjugate of a Clostridium botulinumToxin A Endopeptidase Fragment and Erythrina cristagalliLectin

“…It was not possible to estimate an EC 50 for recLH N /A(H227Y) and recLH N /A(H227Y)-ECL due to insufficient cleavage of SNAP-25; however, data indicated that the endopeptidase activity of the recLH N /A(H227Y) species was reduced ϳ300-fold compared with recLH N /A. This is in reasonable agreement with the data previously reported for this H227Y mutation in the light chain of BoNT/A (32).…”

Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A

“…The zinc coordinating residues identify a primary sphere of residues essential to the catalytic function. Site-directed mutagenesis of TeNT and BoNT/A residues of the zinc-binding motif (11)(12)(13) and of the conserved glutamic acid 271 of TeNT (14,15) confirmed the essential role of these residues in catalysis. From the analysis of the available structures, it appears that a secondary layer of residues is present at the active site of BoNT/A and BoNT/B.…”

Clostridial neurotoxins: structure-function led design of new therapeutics

“…The largest body of work on the expression and purification of recombinant fragments containing the enzyme activity has been carried out on the A serotype, and a wide range of LC/A fragments have been purified from the soluble fraction and using refolding techniques [33][34][35][36][37][38][39][40][41][42][43]. From these various reports, it is apparent that the resulting LC/A proteins differ considerably in stability and retained protease activity.…”