Mechanism of action of Cholera toxin and Pertussis toxin (Action on G protein alpha subunit)

Mechanism of action of Cholera toxin and Pertussis toxin (Action on G protein alpha subunit)

  • Bacteria that are pathogenic to humans can release poisons that interrupt specific G protein linked receptor function, leading to illnesses such as pertussis, botulism, and cholera.

Cholera toxin (CT): Produced by Gram negative water-borne bacterium Vibrio cholerae.

Structure of Cholera toxin:

  • Multimeric enterotoxin that consists of a single A subunit surrounded by five B subunits.
  • Cholera toxin (also known as choleragen) is an 87kD protein composed of two functional units:

1) Enzymatic A subunit (240 residues)

  • It has 2 important segments (CTA1 and CTA2).
  • A1 domain containing the enzymatic active site, and an A2 domain that has an a-helical tail that interact with B subunit.
  • The CTA1 fragment catalyses ADP-ribosylation of the Gs alpha subunit (Gαs) proteins using NAD.

2) Intestinal receptor binding B subunit (103 residues each)

  • B subunit contains five identical peptides that assemble into a pentameric ring surrounding a central pore.
Figure 1: Structure of cholera toxin and enzymatic activation

Mechanism of action:

Cholera Toxin Stimulates Adenylate Cyclase by Permanently Activating Gsα 

  • When cholera toxin is released from the bacteria in the infected intestine, it binds to the small intestinal cells.
  • Following binding to the membrane of enteric cells, the toxin is endocytosed and travels to the endoplasmic reticulum.
  • It is then cleaved at a single site by a bacterial protease to yield two fragments, A1 (the N-terminal ~195 residues) and A2 (the C-terminal ~45 residues), that remain joined by a disulfide bond.
  • Enzymatically active A subunit (CTA1) is translocated to the cytosol.
  • In the cytoplasm, A1 catalyzes the irreversible transfer of the ADP–ribose unit from NAD to a specific Arg side chain of Gs (ADP-ribosylation of the Gsα protein).
  • The ADP-ribosylation causes the Gαs subunit to lose its catalytic activity of inactivating GTP by hydrolyzing it to GDP + Pi
  • ADP- ribosylated Gsα GTP can activate AC but is incapable of hydrolyzing its bound GTP. As a consequence, the AC remains “locked” in its active state.
  • As a result there occurs ~100-fold rise in intracellular cAMP concentration.
  • The high cAMP levels causes the phosphorylation and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein causing a dramatic efflux of ions (sodium and potassium) and water from infected enterocytes into the intestinal lumen leading to the severe diarrhea.

Figure 2: ADP – ribosylation at an arginine residue of the stimulatory Gsα subunit of heterotrimeric G protein

ADP-ribosylation is a general mechanism by which activity of many proteins is regulated, in eukaryotes (including mammals) as well as in prokaryotes.

Anti – diarrhoea treatment:

  • Stimulation of enkephalins (regulate intestinal secretion by acting directly on enterocytes).
  • Enkephalins bind to the opioid receptors on enterocytes (which act through G proteins) to inhibit the stimulation of cAMP synthesis induced by cholera toxin, thereby directly controlling ion transport.

Pertussis toxin (PT)

  • Exotoxin produced by the bacterium Bordetella pertussis ( bacterium that commonly infects the respiratory tract) which causes whooping cough.

Structure: AB5 type exotoxin (formed of 2 subunits). The “A” subunit possesses enzyme activity.

Mechanism of action:

  • PT is released from bacteria in an inactive form.
  • When PT binds to a cell membrane receptor, it is taken up in an endosome.
  • Then it is transported to trans-Golgi network and endoplasmic reticulum. During this transport, the A subunit becomes activated.
  • Pertussis toxin catalyzes ADP-ribosylation at a cysteine residue of the inhibitory Giα, making it incapable of exchanging GDP for GTP. The inhibitory pathway is blocked.
  • This prevents the G proteins from interacting with G protein-coupled receptors on the cell membrane.
  • The Gi subunits remain locked in their GDP-bound state (inactive state).
  • As consequence they are unable to inhibit adenylate cyclase activity, leading to increased cellular concentrations of cAMP.
  • Increase in cAMP in epithelial cells of the airways promotes loss of fluids and electrolytes and mucus secretion.

Figure 3: ADP – -ribosylation at a cysteine residue of the inhibitory Giα subunit of heterotrimeric G protein

Figure 4: The site of action of cholera toxin and pertussis toxin

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