title: Membrane interactions of diphtheria toxin analyzed using in vitro synthesized mutants.
abstract: We have developed a system to study the interactions of diphtheria toxin with the cell surface using non-toxic mutant proteins synthesized in vitro. Proteins obtained by N-terminal deletions containing the whole B fragment bound strongly to cells. Deletions extending into the B fragment did not yield an autonomous binding domain. Loss of only the N-terminal 3 kd of the B fragment significantly impaired the ability to recognize the receptor. This, together with previous reports that the C-terminal end of the B fragment is required for binding, suggests that both ends of the B fragment are necessary for receptor recognition. Receptor bound diphtheria toxin undergoes a conformational change at pH less than 5.3 that results in translocation of the A fragment to the cytosol and the appearance of a B fragment-derived 25 kd polypeptide (P25) resistant to externally applied protease. Only the B fragment was required for generation of P25. N-terminal deletions of 130 amino acids or more resulted in proteins that gave rise to P25 at higher pH than full length toxin. Furthermore, a second protease-inaccessible polypeptide of 18 kd (P18) was observed.
authors.affiliation: Institute for Cancer Research Norwegian Radium Hospital, Montebello, Oslo.
authors.forename: S
authors.initials: S
authors.lastname: McGill
authors.forename: H
authors.initials: H
authors.lastname: Stenmark
authors.forename: K
authors.initials: K
authors.lastname: Sandvig
authors.forename: S
authors.initials: S
authors.lastname: Olsnes
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D007739:L Cells; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D014709:Vero Cells
chemical_list: D004167:Diphtheria Toxin; D010446:Peptide Fragments; D010447:Peptide Hydrolases