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.
D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003013:Clostridium / Q000737:chemistry; D006224:Cricetinae; D017931:DNA Primers; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D052899:Pore Forming Cytotoxic Proteins; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D001427:Bacterial Toxins; D017931:DNA Primers; D017261:Glycosylphosphatidylinositols; D006460:Hemolysin Proteins; D011134:Polysaccharides; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; C064270:hemolytic toxin, Clostridium septicum; C032676:aerolysin