Cell surface and intracellular functions for ricin galactose binding.

Area of Science:

• Toxicology
• Molecular Biology
• Cell Biology

Background:

• Ricin is a potent toxin composed of ricin A chain (RTA) and ricin B chain (RTB).
• RTB mediates cell entry by binding to cell surface galactose residues.
• The specific contribution of RTB's two galactose-binding sites to ricin toxicity is not fully understood.

Purpose of the Study:

• To investigate the role of the two galactose-binding sites in RTB on ricin's overall toxicity.
• To determine if these sites are essential for cell entry and/or intracellular cytotoxic mechanisms.

Main Methods:

• Site-directed mutagenesis was used to create three ricin B chain point mutants: site 1, site 2, and a double site mutant.
• Mutant ricins were expressed in Xenopus oocytes and reassociated with recombinant ricin A chain.
• Cytotoxicity was assessed using Vero cells (galactose receptor-mediated) and macrophages (mannose receptor-mediated), with and without lactose to block galactose binding.

Main Results:

• Wild-type ricin and single-site mutants showed toxicity via galactose receptors, with lactose blocking this effect.
• Site 1 and 2 mutants were 20-40 times less potent than wild-type ricin; the double mutant was non-cytotoxic.
• All tested ricins, including mutants, inhibited protein synthesis in mannose receptor-bearing cells.
• Even with mannose receptor uptake, at least one galactose-binding site was necessary for cytotoxicity, and two sites increased potency.

Conclusions:

• Both galactose-binding sites of ricin B chain are critical for potent ricin toxicity.
• These sites contribute to both initial cell surface binding and intracellular cytotoxic functions.
• Targeting galactose-binding activity offers a potential strategy for mitigating ricin's toxic effects.