Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum alpha-toxin
Aerolysin 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.
Keywords: Animals ; Bacterial Toxins/chemistry/*metabolism ; Base Sequence ; Binding Sites ; CHO Cells ; Carbohydrate Conformation ; Carbohydrate Sequence ; Clostridium/chemistry ; Cricetinae ; DNA Primers ; Glycosylphosphatidylinositols/*metabolism ; Hemolysin Proteins/chemistry/*metabolism ; Molecular Sequence Data ; Polymerase Chain Reaction ; Polysaccharides/*chemistry/*metabolism ; Pore Forming Cytotoxic Proteins ; Protein Binding ; Recombinant Proteins/chemistry/metabolism ; Transfection
Departments of Immunoregulation and Bacterial Toxinology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
Record created on 2009-01-30, modified on 2016-08-08