Replacement of negative by positive charges in the presumed membrane-inserted part of diphtheria toxin B fragment. Effect on membrane translocation and on formation of cation channels.
Diphtheria toxin B fragment is capable of forming cation-selective channels in the plasma membrane. Such channels may be involved in the translocation of the toxin A fragment to the cytosol. Seven negatively charged amino acids in the B fragment were replaced one by one by lysines, followed by studies of cytotoxicity and channel-forming ability of the different mutants. The mutant D392K showed a strong reduction in binding to cell surface receptors. Of the six mutants that showed wild-type binding affinity, the two mutants D295K and D318K were very inefficient in forming channels. These two mutants had the lowest ability to mediate A fragment translocation. The mutant E362K was able both to induce cation channel formation and to mediate A fragment translocation at a higher pH value than the wild-type B fragment. The results support the notion that formation of cation channels is of importance for the translocation of the A fragment across the plasma membrane, and they indicate that the pH requirement for translocation of the A fragment to the cytosol is partly determined by the B fragment.
Falnes
P O
PO
Institute for Cancer Research, Norwegian Radium Hospital, Oslo, Norway.
Madshus
I H
IH
Sandvig
K
K
Olsnes
S
S
D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000378:metabolism; D004563:Electrochemistry; D019286:Endopeptidase K; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D014709:Vero Cells
D002412:Cations; D004167:Diphtheria Toxin; D007473:Ion Channels; D010446:Peptide Fragments; C039171:diphtheria toxin fragment B; D000643:Ammonium Chloride; D012964:Sodium; D012697:Serine Endopeptidases; D019286:Endopeptidase K