Anthrax Antigen

Anthrax Protective Antigen

Ansley Scott '02 and
Dawn Stancik '02
Biology 363
December 2001

I. Introduction

Recently anthrax has caused much concern as a public health threat because of its use as a bioterrorism weapon. However, anthrax has been a concern for farmers since biblical times, especially in agricultural regions like South America and Africa. Although it is rare, anthrax has been found sporadically in cattle, sheep, and goats throughout the midwest and the western United States after injestion or inhalation of the Bacillus anthracis spores found in the soil. Farmers rarely contract the disease and are usually only exposed to Cutaneous anthrax through open wounds, whereby the bacteria can enter the skin. Cutaneous anthrax is the form most encountered in naturally occurring cases and is treatable; it is only deadly in about 20% of untreated cases (Anthrax on the Farm ). Inhalational anthrax is very rare and usually fatal; after inhalation of the spores, cold symptoms set in early and are abruptly followed by respiratory distress and death. This form is the one used as a bioterrorist weapon. Anthrax can be treated with antibiotics (ciprofloxacin) if diagnosed early enough, and it is not a communicable disease. In bioterrorism, anthrax works well because it is easy to prepare and disperse and can inflict sufficiently severe disease to paralyze a city and perhaps a nation (Nature's Anthrax Page ).

The Anthrax bacterium produces a toxin which consists of the three proteins, the protective antigen (PA), the lethal factor (LF), and the oedema (OF) factor. These factors along with aa poly-D-glutamic acid capsule are the major factors imparting virulence in B. anthracis. The PA protein works by forming a heptamer which binds to the surface of a host cell and serves as a channel by which the oedema factor and the lethal factor enter the cell (Heptamer). Once in the cell, the PA protein is the common binding moiety of two toxins, lethal and oedema, which are also composed of the lethal factor and the edema factor proteins (Petosa, 1997).

Bacillus anthracis colonies

II. General Structure

The PA monomer is predominantly made up of antiparallel b-sheets and it consists of four functional domains (Petosa, 1997). Domain I (residues 1-258) , the amino terminal domain, contains two calcium ions and the cleavage site for proteases to activate the protein. The product of the cleavage results in the creation of the amino terminal fragment a20 (Brossier, 1999). Domain II (residues 259-487) is involved in the formation of the hexamer and has a flexible loop that aids in membrane insertion. As of now, Domain III (residues 488-595) does not have a known function. Domain IV (residues 596-735) is needed for receptor binding ( Petosa, 1997).

III. Domain I and II

Domain I
PA is activated by proteolysis on the N-terminal loop in Domain I. This causes the release of a 20 K fragment of Domain I (PA20) . Loss of PA20 triggers the heptamer formation of PA. This heptamer is soluble in water and inserts itself into the host membrane (Heptamer) (Petosa, 1997).

Domain II
The large loop in Domain II (302-325) is involved with the toxicity of anthrax and may be involved in the PA channel interactions with the host. Residues 304, 306, and 308 are located within the channel lumen (Petosa, 1997).

IV. Receptor Binding Region of Domain IV

Domain IV was thought to contain two receptor binding domains: the slarge loop(amino acid 704-723) and the small loop (amino acids 679-693) . It turns out that only the small loop is involved in receptor binding (Varughese et al., 1999). In addition to the small loop, residues at the end of the C-terminus aid in receptor binding. Deletions in both the C-terminus and in the small loop decrease anthrax toxicity by inhibiting binding. It has been shown that two antibodies, 3B6 and 14B7, bind to the region between amino acids 671 and 721 and decrease toxicity by inhibiting the binding of PA to the receptor (Varughese et al., 1999) .

V. Interactions With the Lethal Factor

The lethal factor (LF) is the major factor causing death in anthrax patients, but the mechanism is not clearly understood. Internalization and translocation of the lethal factor into the cytosol of Bacillus anthracis occurs when the PA protein binds to a specific, yet undefined, cell surface receptor (Pannifer, 2001). The highly specific LF enzyme has four domains (I, II, III, IV) that have evolved through the gene duplication process, fusion and mutation . After the LF has bound to the PA, the lethal toxin leads to an increase in the permeability of Na⁺ and K⁺ ions after its internalization and is followed by the hydrolysis of ATP. This inhibits macromolecular synthesis involved with the immune response, and results in cell death (Gupta et al., 2001). Interestingly, when the PA and LF are injected in animals intravenously, they work in concert to induce rapid death.

Domain III has a hydrophobic core (282-382). It also contains a five-tandem repeat 101 amino acid sequence that seem to have been created through a duplication event (282-382) .

VI. References

1. Brossier, Fabien, Martine Weber-Levy, Michele Mock, and Jean-Claude Sirard. 2000. Role of Toxin Functional Domains in Anthrax Pathogenesis. Infection and Immunity. 68:1781-1786.

2. Gupta, Pankaj, Samer Singh, Ashutosh Tiwari, Rajiv Bhat, and Rakesh Bhatnagar. 2001. Effect of pH on Stability of Anthrax Lethal Factor: Correlation Between Denaturation and Activity. Biochemical and Biophysical Research Communications. 284: 568-573.

3. Pannifer, Andrew. 2001. Crystal Structure of the Anthrax Lethal Factor. Nature. 414:229-233.

4. Petosa, Carlo, R. John Collier, Kurt R. Klimpel, Stephan H. leppla, and Rover C. Liddington. 1997. Crystal Structure of the Anthrax Toxin Protective Antigen. Nature. 385: 833-838.