title: Probing the surface of eukaryotic cells using combinatorial toxin libraries.
abstract: 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.
authors.affiliation: Ontario Cancer Institute, Princess Margaret Hospital Rm. 7-117, 610 University Avenue, Ontario, M5G 2M9, Toronto, Canada.
authors.forename: M R
authors.initials: MR
authors.lastname: Bray
authors.forename: S
authors.initials: S
authors.lastname: Bisland
authors.forename: S
authors.initials: S
authors.lastname: Perampalam
authors.forename: W M
authors.initials: WM
authors.lastname: Lim
authors.forename: J
authors.initials: J
authors.lastname: Gariépy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D020650:Combinatorial Chemistry Techniques; D005057:Eukaryotic Cells; D005434:Flow Cytometry; D006801:Humans; D008958:Models, Molecular; D015335:Molecular Probes; D022622:Shiga Toxin 1 / Q000737:chemistry* / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014709:Vero Cells
chemical_list: D015335:Molecular Probes; D022622:Shiga Toxin 1