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Double-site ricin B chain mutants retain galactose binding
A Frankel1, E Tagge, J Chandler
1Department of Medicine, Medical University of South Carolina, Charleston 29425, USA.
Protein Engineering
|April 1, 1996
Summary
Researchers engineered ricin B chain (RTB) mutants to understand its sugar-binding properties. Mutants showed reduced sugar binding but retained cell binding and cytotoxicity, revealing key amino acids involved in RTB function.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
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 carbohydrates.
- Understanding RTB's structure-function relationship is crucial for toxin neutralization and therapeutic applications.
Purpose of the Study:
- To investigate the role of specific amino acid residues in ricin B chain (RTB) sugar binding.
- To characterize the binding affinity and cytotoxic activity of RTB mutants.
- To identify subdomains within RTB involved in carbohydrate recognition and avidity.
Main Methods:
- Expression and purification of recombinant RTB mutants in insect cells.
- Assays for sugar binding (asialofetuin ELISA) and cell surface binding (immunofluorescence).
- Assessment of cytotoxicity by measuring inhibition of protein synthesis (ID50) after heterodimerization with RTA.
Main Results:
- Recombinant RTB mutants were soluble, immunoreactive, and had correct molecular weight.
- All mutants bound asialofetuin and mammalian cells, though sugar binding affinity varied.
- Double-site mutants showed 1-2 log reductions in sugar binding, while single-site mutants also exhibited reduced binding.
- Mutant heterodimers with RTA retained cytotoxicity, with ID50 values 1-2 logs higher than native ricin.
Conclusions:
- Amino acid residues in the 1 alpha and 2 gamma subdomains of RTB are critical for sugar binding.
- Other RTB subdomains contribute to the overall avidity of ricin for cell surface oligosaccharides.
- RTB mutants offer insights into ricin-carbohydrate interactions and potential therapeutic targets.