title: Identification of host cell factors required for intoxication through use of modified cholera toxin.
abstract: We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ~12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx.
authors: Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
authors: Carla P
authors: CP
authors: Guimaraes
authors: Jan E
authors: JE
authors: Carette
authors: Malini
authors: M
authors: Varadarajan
authors: John
authors: J
authors: Antos
authors: Maximilian W
authors: MW
authors: Popp
authors: Eric
authors: E
authors: Spooner
authors: Thijn R
authors: TR
authors: Brummelkamp
authors: Hidde L
authors: HL
authors: Ploegh
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D002478:Cells, Cultured; D002771:Cholera / Q000503:physiopathology; D002772:Cholera Toxin / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics; D004167:Diphtheria Toxin / Q000737:chemistry*; D005732:Gangliosides / Q000378:metabolism / Q000502:physiology; D005818:Genetic Engineering; D006238:Haploidy; D006801:Humans; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D021381:Protein Transport / Q000235:genetics*; D020539:Sequence Analysis, Protein; D012799:Sialyltransferases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D000099237:UDP-Galactose Translocators
chemical_list: D019881:Aminoacyltransferases; D001426:Bacterial Proteins; D002772:Cholera Toxin; D003546:Cysteine Endopeptidases; D004167:Diphtheria Toxin; D005732:Gangliosides; D009004:Monosaccharide Transport Proteins; D021122:Protein Subunits; D012799:Sialyltransferases; D000099237:UDP-Galactose Translocators; C120542:sortase A; C021343:haematoside synthetase