Role of N-Terminal Amino Acids in the Potency of Anthrax Lethal Factor

Anthrax lethal factor (LF) is a Zn+2-dependent metalloprotease that cleaves several MAPK kinases and is responsible for the lethality of anthrax lethal toxin (LT). We observed that a recombinant LF (LF-HMA) which differs from wild type LF (LF-A) by the addition of two residues (His-Met) to the native Ala (A) terminus as a result of cloning manipulations has 3-fold lower potency toward cultured cells and experimental animals. We hypothesized that the “N-end rule”, which relates the half-life of proteins in cells to the identity of their N-terminal residue, might be operative in the case of LF, so that the N-terminal residue of LF would determine the cytosolic stability and thereby the potency of LF. Mutational studies that replaced the native N-terminal residue of LF with known N-end rule stabilizing or destabilizing residues confirmed that the N-terminal residue plays a significant role in determining the potency of LT for cultured cells and experimental animals. The fact that a commercially-available LF preparation (LF-HMA) that is widely used in basic research studies and for evaluation of vaccines and therapeutics is 3-fold less potent than native LF (LF-A) should be considered when comparing published studies and in the design of future experiments

Anthrax edema factor toxicity is strongly mediated by the N-end rule.

Anthrax edema factor (EF) is a calmodulin-dependent adenylate cyclase that converts adenosine triphosphate (ATP) into 3'-5'-cyclic adenosine monophosphate (cAMP), contributing to the establishment of Bacillus anthracis infections and the resulting pathophysiology. We show that EF adenylate cyclase toxin activity is strongly mediated by the N-end rule, and thus is dependent on the identity of the N-terminal amino acid. EF variants having different N-terminal residues varied by more than 100-fold in potency in cultured cells and mice. EF variants having unfavorable, destabilizing N-terminal residues showed much greater activity in cells when the E1 ubiquitin ligase was inactivated or when proteasome inhibitors were present. Taken together, these results show that EF is uniquely affected by ubiquitination and/or proteasomal degradation

Evaluation of the apparent binding affinities of LF-HMA and LF-A for PA using Schild Plot analyses.

<p>CHO WTP4 cells were incubated with various concentrations of FP59 plus a set concentration of PA (12 nM) and different concentrations of LF-HMA (A) or LF-A (B) for 3 h. After toxin removal, cells were incubated with the toxin-free medium containing 10 mM NH₄Cl for 48 h before assessment of cell viability. Schild Plot analyses were performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003130#s2" target="_blank">Methods</a>” to assess <i>K</i>_ds of each LF protein for PA. Inserts shown in panels A and B are non-linear regression curves obtained from these analyses using GraphPad Prism.</p

Toxicity of LF proteins in rats.

*<p>Fisher 344 Rats were injected with 10 µg PA or 100 µg PA plus equivalent amount of each LF preparation (IV) and monitored for minutes to death.</p

Cytotoxicity of LF proteins to RAW264.7 cells.

<p>RAW264.7 cells were incubated with various concentrations of LF-HMA, LF-A, and LF-A/X proteins (A) or LF proteins with mutated N-termini (B) and a fixed concentration of PA (250 ng/ml). Cell viability was assessed at 3 h. Percent viability was calculated relative to cells treated with medium (no toxin).</p

Schematic representation of various LF proteins.

<p>All proteins (except LF-A/St which is wild type LF produced from Sterne strain) were generated as secreted proteins from <i>B. anthracis</i> BH450. All proteins contained a signal peptide that is cleaved by signal peptidases during secretion. Constructs with a factor Xa recognition sequence alone or preceded by the Myc epitope tag are labeled “/X” or “/MyX”, respectively. These proteins were produced as precursor proteins followed by cleavage with factor Xa protease to generate the indicated N-termini. Residue (Z) circled in red indicates the residue mutated for each protein, i.e., Z = A, H, M, R, F, G in different mutated LF constructs.</p