Abstract
Botulinum neurotoxins are most potent of all toxins. Their N-terminal light chain domain (Lc) translocates into peripheral cholinergic neurons to exert its endoproteolytic action leading to muscle paralysis. Therapeutic development against these toxins is a major challenge due to their in vitro and in vivo structural differences. Although three-dimensional structures and reaction mechanisms are very similar, the seven serotypes designated A through G vastly vary in their intracellular catalytic stability. To investigate if protein phosphorylation could account for this difference, we employed Src-catalyzed tyrosine phosphorylation of the Lc of six serotypes namely LcA, LcB, LcC1, LcD, LcE, and LcG. Very little phosphorylation was observed with LcD and LcE but LcA, LcB, and LcG were maximally phosphorylated by Src. Phosphorylation of LcA, LcB, and LcG did not affect their secondary and tertiary structures and thermostability significantly. Phosphorylation of Y250 and Y251 made LcA resistant to autocatalysis and drastically reduced its kcat/Km for catalysis. A tyrosine residue present near the essential cysteine at the C-terminal tail of LcA, LcB, and LcG was readily phosphorylated in vitro. Inclusion of a competitive inhibitor protected Y426 of LcA from phosphorylation, shedding light on the role of the C-terminus in the enzyme’s substrate or product binding.
Highlights
Botulinum neurotoxins (BoNTs)1 are among the most lethal of all toxins and are potential biowarfare agents (Cochrane, 1947; Gill, 1982; Montecucco and Schiavo, 1995; Arnon et al, 2001)
Our results showed while Lc of BoNT/A (LcA), light chain domain (Lc) of BoNT/B (LcB), light chain of serotype C1 (LcC1), and light chain of serotype G (LcG) were readily phosphorylated, light chain of serotype D (LcD) and light chain of serotype E (LcE) were poorly phosphorylated
TYROSINE PHOSPHORYLATION OF botulinum neurotoxin (BoNT) LCS To understand structural and environmental factors affecting the stability of BoNT Lcs (Ahmed et al, 2001, 2003, 2004; Toth et al, 2009), we investigated in vitro tyrosine phosphorylation of LcA, LcB, LcC, LcD, LcE, and LcG by Src
Summary
Botulinum neurotoxins (BoNTs) are among the most lethal of all toxins and are potential biowarfare agents (Cochrane, 1947; Gill, 1982; Montecucco and Schiavo, 1995; Arnon et al, 2001). A thorough understanding of their structure under cellular environment is essential These 150 kDa exotoxins are produced by strains of Clostridium botulinum as seven distinct serotypes, designated BoNT/A-G. After finding their way through oral, respiratory, or wound routes to the animal body, BoNTs travel to peripheral cholinergic neuronal cells and are internalized by endocytosis followed by translocational delivery of its 50 kDa light chain (Lc) from endosome into the cytosol (Simpson, 2004). The free Lc, a zinc-endopeptidase (Schiavo et al, 1992a), exerts its proteolytic activity at specific sites on one of the three synaptosomal proteins, SNAP-25, VAMP, or syntaxin. The resultant effect is muscle paralysis and eventual death if the intoxication is severe and not treated
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