Anthrax Toxins

Bacillus anthracis is the etiological agent of anthrax. Major virulence factors produced by Bacillus anthracis are the gamma-linked, poly-D-glutamic acid capsule and an exotoxin composed of three components, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA is the cell binding and delivery component where LF and EF are enzymes which cause changes in infected cells. These proteins are not toxic, held separately; but, act as toxins in binary combinations. Combination of the cell binding component, PA, with LF is termed lethal toxin (LeTx) and PA with EF is edema toxin (EdTx). After secretion, in a cellular environment, the 83 kDa PA protein is cleaved by membrane peptidases, producing 63 kDa carboxy terminal fragments which combine, often in groups of seven forming a heptamer. Cleavage is an essential step in allowing oligomerization and in exposing the binding sites on PA oligomer for EF and LF. The assembled PA complex and associated LF or EF enters the cell through endocytosis. PA mediates the transfer of LF and EF to the cytoplasm where these enzymes recognize and act on their targets. The LF component, a zinc-dependent metalloprotease, cleaves the mitogen-activated protein kinase kinase (MAPKK), impairing its function and thereby blocking the MAPK signaling pathway. EF is a calmodulin-dependent adenylate cyclase which creates in the cell excess amounts of cyclic-AMP, disrupting AMP-mediated signaling. In the early stage of infection, LF and EF work together to block the patient’s immune response. List provides several forms of PA, LF and EF. FRET peptide substrates containing a cleavage site for anthrax LF are available as MAPKKide® and can be used to assess the enzymatic activity of LF. Antibodies directed to PA and LF are also in the List Labs catalog.

Protective Antigen

Protective antigen (PA) is the receptor binding component of anthrax toxin. The original 83 kDa protein is cleaved by cellular proteases to form an activated 63 kDa monomer, PA63. In this form, PA63 aggregates with other PA63 molecules forming a ring-shaped heptamer. PA63 has binding sites specific for the enzymes LF and EF allowing for attachment, endocytosis, and translocation. PA63 should be used in approximately seven-fold excess along with either EF or LF to intoxicate cells. List Labs provides PA in several different forms,

• Product #171 is the entire 83 kDa native protein;
• Product #174 is PA63, which has been created by enzymatic digestion and purified;
• Product #177 is PA20, the approximately 20 kDA fragment cleaved from PA.
• Product #175 is purified PA63 conjugated to FITC for visualization.
• Product #771 purified IgG from goat, is directed to PA.

Lethal Factor

Lethal Factor (LF) is the enzymatic subunit of anthrax lethal toxin. When intoxicating a cell, LF binds to PA on the cell surface and translocates through a pore made by seven PA monomers. List Labs offers several forms of LF, all made recombinantly in the native Bacillus anthracis host.

• Product #169 (LF-A), is native LF, recommended for most users and is provided in a liquid form;
• Product #172 (LF-HMA), the original LF made by List Labs, has been found to carry two additional amino acids, histidine and methionine, on the N-terminal. This product is recommended only for use in assays validated with this product; and finally
• Product #176 (LF E687C) is an enzymatic inactive form of LF, carrying a Glu to Cys substitution in the zinc-binding site.

LF Substrates and Antibodies

List Labs has designed substrates available as MAPKKide® which mimic the native targets for LF, proteins in the MAPKK family. These FRET peptides contain the cleavage site for LF and fluorophores which are quenched based on fluorescence resonance energy transfer (FRET). Cleavage of each substrate by LF releases the fluorophore creating an increase in fluorescent intensity. The following are available LF peptide substrates carrying three different fluorophores:

• Product #532 is MAPKKide® Plus Specific Substrate for Anthrax Lethal Factor. This peptide is remarkable in its resistance to non-specific cleavage by enzymes which may be present in natural samples. MAPKKide® Plus carries the fluorophore 7-amido-4-methylcoumarin (AMC).
• Product #530 is MAPKKide® utilizing the o-Abz/Dnp FRET pair. A calibration peptide, Product #539 is available for use as an unquenched calibration peptide for Product #530
• Product #531 is MAPKKide® with the FRET pair DABCYL/FITC.

List offers pairs of antibodies directed to LF, suitable for use in ELISAs.

• Product #768L affinity purified and biotinylated Anti-Lethal Factor from B. anthracis (chicken lgY) works to detect LF as described in the C of A.
• Product #769B Anti-Lethal Factor from B. anthracis (chicken lgY) may be used to capture LF. Additionally, we have demonstrated that the antibody #769B may be used to capture LF and used with the MAPKKide® Plus substrate to quantitate the amount of LF in a complex matrix.

Edema Factor

Edema Factor (EF) is the enzymatic subunit of anthrax edema toxin. When intoxicating a cell, EF binds to PA on the cell surface and translocates through a pore made by seven PA monomers. List Labs offers two forms of EF, made recombinantly in the native Bacillus anthracis host.

• Product #178 is comprised of the native sequence with an additional amino acid, histidine on the N-terminal.
• Product #173 is a mutant S414N EF mutant with reduced adenylate cyclase activity.

An antibody for EF is also available,

• Product #773L, goat anti-edema factor. Use in a dot blot and direct ELISA is described in the C of A.