Interactions of diphtheria toxin B-fragment with cells. Role of amino- and carboxyl-terminal regions.
The B-fragment of diphtheria toxin binds to cell surface receptors and facilitates entry of the enzymatically active A-fragment into the cytosol. The roles of the amino- and carboxyl-terminal regions of the B-fragment in interactions with the cell membrane were studied by measuring specific binding, insertion into membranes at low pH, and formation of cation-selective channels, as well as by toxicity measurements after association with active A-fragment. Deletion of the amino-terminal 12 amino acids of the B-fragment did not affect its ability to bind to receptors and to form ion channels at low pH, whereas both abilities were strongly impaired when one more amino acid (Trp206) was removed. Replacement of the amino-terminal 31 residues with an amphipathic sequence from human apolipoprotein A1 restored receptor binding but not ion channel formation. The binding to cells was virtually abolished when 9 residues were deleted from the carboxyl terminus. Deletion of only 4 residues or extension by 12 residues did not prevent specific binding, but reduced insertion, channel formation, and toxicity. Those deletions that reduced receptor binding ability increased the trypsin sensitivity of the B-fragment. The results indicate that the amino- and carboxyl-terminal regions of diphtheria toxin B-fragment are important for receptor binding, possibly because they contribute to keep the B-fragment in a binding-competent conformation. Small alterations in the carboxyl-terminal end reduced insertion, channel formation, and toxicity more than the ability of the B-fragment to bind to cells.
Stenmark
H
H
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
Ariansen
S
S
Afanasiev
B N
BN
Olsnes
S
S
D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004167:Diphtheria Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D006863:Hydrogen-Ion Concentration; D036341:Intercellular Signaling Peptides and Proteins; D007473:Ion Channels / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D014176:Protein Biosynthesis; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000378:metabolism; D014158:Transcription, Genetic; D014709:Vero Cells
D016632:Apolipoprotein A-I; D004167:Diphtheria Toxin; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007473:Ion Channels; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D004247:DNA