Probing the surface of eukaryotic cells using combinatorial toxin libraries

The success of proteomics hinges in part on the development of approaches a ble 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-li ke toxin 1 (SLT-1), a ribosome-inactivating protein that kills eukaryotic c ells [1, 2], SLT-1 binds to the glycolipid globotriaosylceramide [3, 4], wh ich acts as a shuttle, allowing the toxin to be imported and routed near ri bosomes, We investigated the use of SLT-1 as a structural template to creat e combinatorial libraries of toxin variants with altered receptor specifici ty. Since all SLT-1 variants retain their toxic function, this property ser ved as a search engine enabling us to identify mutants from these libraries able to kill target cells expressing internalizable receptors, Random muta tions were introduced in two discontinuous loop regions of the SLT-1 recept or 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 sho wn to bind to a new receptor on these cell lines by flow cytometry. Toxin l ibraries provide a strategy to delineate the spectrum of receptors on eukar yotic cells.