Hong, Y.Ohishi, K.Inoue, N.Kang, J. Y.Shime, H.Horiguchi, Y.van der Goot, F. G.Sugimoto, N.Kinoshita, T.2009-01-302009-01-302009-01-30200210.1093/emboj/cdf508https://infoscience.epfl.ch/handle/20.500.14299/34635Aerolysin of the Gram-negative bacterium Aeromonas hydrophila consists of small (SL) and large (LL) lobes. The alpha-toxin of Gram-positive Clostridium septicum has a single lobe homologous to LL. These toxins bind to glycosylphosphatidylinositol (GPI)-anchored proteins and generate pores in the cell's plasma membrane. We isolated CHO cells resistant to aerolysin, with the aim of obtaining GPI biosynthesis mutants. One mutant unexpectedly expressed GPI-anchored proteins, but nevertheless bound aerolysin poorly and was 10-fold less sensitive than wild-type cells. A cDNA of N-acetylglucosamine transferase I (GnTI) restored the binding of aerolysin to this mutant. Therefore, N-glycan is involved in the binding. Removal of mannoses by alpha-mannosidase II was important for the binding of aerolysin. In contrast, the alpha-toxin killed GnTI-deficient and wild-type CHO cells equally, indicating that its binding to GPI-anchored proteins is independent of N-glycan. Because SL bound to wild-type but not to GnTI-deficient cells, and because a hybrid toxin consisting of SL and the alpha-toxin killed wild-type cells 10-fold more efficiently than GnTI- deficient cells, SL with its binding site for N-glycan contributes to the high binding affinity of aerolysin.AnimalsBacterial Toxins/chemistry/*metabolismBase SequenceBinding SitesCHO CellsCarbohydrate ConformationCarbohydrate SequenceClostridium/chemistryCricetinaeDNA PrimersGlycosylphosphatidylinositols/*metabolismHemolysin Proteins/chemistry/*metabolismMolecular Sequence DataPolymerase Chain ReactionPolysaccharides/*chemistry/*metabolismPore Forming Cytotoxic ProteinsProtein BindingRecombinant Proteins/chemistry/metabolismTransfectiontext::journal::journal article::research article