Reductive Methylation and Mutation of an Anthrax Toxin Fusion Protein Modulates its Stability and Cytotoxicity

Abstract

We characterized an anti-cancer fusion protein consisting of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas exotoxin A by (i) mutating the N-terminal amino acids and by (ii) reductive methylation to dimethylate all lysines. Dimethylation of lysines was achieved quantitatively and specifically without affecting binding of the fusion protein to PA or decreasing the enzymatic activity of the catalytic moiety. Ubiquitination in vitro was drastically decreased for both the N-terminally mutated and dimethylated variants, and both appeared to be slightly more stable in the cytosol of treated cells. The dimethylated variant showed greatly reduced neutralization by antibodies to LF. The two described modifications offer unique advantages such as increased cytotoxic activity and diminished antibody recognition, and thus may be applicable to other therapeutic proteins that act in the cytosol of cells.

Frontline Science: Anthrax lethal toxin-induced, NLRP1-mediated IL-1β release is a neutrophil and PAD4-dependent event

“…Endotoxin‐free LF, active‐site nonproteolytic mutant LF (E687C), PA, and FP59 (AGG variant) were purified from B. anthracis as previously described 16,17 . FP59 is a fusion of the receptor‐binding N‐terminus of LF to the enzymatic domain of Pseudomonas exotoxin A and inhibits protein synthesis upon entry into the mammalian cytosol 17 .…”

“…Endotoxin‐free LF, active‐site nonproteolytic mutant LF (E687C), PA, and FP59 (AGG variant) were purified from B. anthracis as previously described 16,17 . FP59 is a fusion of the receptor‐binding N‐terminus of LF to the enzymatic domain of Pseudomonas exotoxin A and inhibits protein synthesis upon entry into the mammalian cytosol 17 . LF used in all studies has an amino acid sequence of “HMAGG” at its N‐terminus, except where indicated as LF‐OS or LT‐OS, where the sequence is “AGG.” These LF preparations have different potencies in vivo, 18 which are reflected by different doses used in some experiments, as the final HMAGG preparations in this laboratory have been depleted.…”

Heme Oxygenase-1 Regulation of Matrix Metalloproteinase-1 Expression Underlies Distinct Disease Profiles in Tuberculosis

“…A DNA fragment encoding ESAT6 followed by a 6-residue linker and then a C-terminal hexa-histidine tag was synthesized by GeneArt (Life Technologies). The fragment was subcloned into the expression plasmid FP59AGGpYS (24) using MluI and XmaI restriction sites in frame with the first 255 N-terminal amino acids of anthrax lethal factor. A B. anthracis strain deficient in extracellular proteases (25) was transformed with the resulting plasmid, and LFn-ESAT6-His6 fusion protein was purified from culture supernatants by nickel-affinity chromatography.…”

Anthrax Toxin as a Molecular Platform to Target Nociceptive Neurons and Modulate Pain

“…Although the exact intracellular half-life of EF is unknown, these observations suggest that the functional persistence of EF in the cytoplasm is fairly short. Methods that have successfully prolonged the intracellular half-life of LFN-DTA may potentially be applied to EF, such as attaching a D-amino acid at the N-terminus (91) or dimethylating all lysines (92) to avoid ubiquitination and proteosomal degradation. Other potential strategies may involve combination treatment with PDE inhibitors to prolong the duration of elevated cAMP.…”