title: 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)
authors.affiliation: Department of Biochemistry, Imperial College of Science, Technology and Medicine, London.
authors.forename: L
authors.initials: L
authors.lastname: Zhou
authors.forename: A
authors.initials: A
authors.lastname: de Paiva
authors.forename: D
authors.initials: D
authors.lastname: Liu
authors.forename: R
authors.initials: R
authors.lastname: Aoki
authors.forename: J O
authors.initials: JO
authors.lastname: Dolly
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001906:Botulism / Q000139:chemically induced; D002352:Carrier Proteins / Q000235:genetics; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015870:Gene Expression; D058619:Maltose-Binding Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016296:Mutagenesis; D009466:Neuromuscular Blocking Agents / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D013329:Structure-Activity Relationship; D015032:Zinc / Q000494:pharmacology
chemical_list: D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009466:Neuromuscular Blocking Agents; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D010450:Endopeptidases; D001905:Botulinum Toxins; D015032:Zinc