Probing the surface of eukaryotic cells using combinatorial toxin libraries

The success of proteomics hinges in part on the development of approaches able to map receptors on the surface of cells. One strategy to probe a cell surface for the presence of internalized markers is to make use of Shiga-like toxin 1 (SLT-1), a ribosome-inactivating protein that kills eukaryotic cells [1, 2]. SLT-1 binds to the glycolipid globotriaosylceramide [3, 4], which acts as a shuttle, allowing the toxin to be imported and routed near ribosomes. We investigated the use of SLT-1 as a structural template to create combinatorial libraries of toxin variants with altered receptor specificity. Since all SLT-1 variants retain their toxic function, this property served as a search engine enabling us to identify mutants from these libraries able to kill target cells expressing internalizable receptors. Random mutations were introduced in two discontinuous loop regions of the SLT-1 receptor binding subunit. Minimal searches from screening 600 bacterial colonies randomly picked from an SLT-1 library identified toxin mutants able to kill cell lines resistant to the wild-type toxin. One such mutant toxin was shown to bind to a new receptor on these cell lines by flow cytometry. Toxin libraries provide a strategy to delineate the spectrum of receptors on eukaryotic cells.