Enhancement of diphtheria toxin potency by replacement of the receptor binding domain with tetanus toxin C-fragment: a potential vector for delivering heterologous proteins to neurons.
This study describes the expression, purification, and characterization of a recombinant fusion toxin, DAB(389)TTC, composed of the catalytic and membrane translocation domains of diphtheria toxin (DAB(389)) linked to the receptor binding fragment of tetanus toxin (C-fragment). As determined by its ability to inhibit cellular protein synthesis in primary neuron cultures, DAB(389)TTC was approximately 1,000-fold more cytotoxic than native diphtheria toxin or the previously described fusion toxin, DAB(389)MSH. The cytotoxic effect of DAB(389)TTC on cultured cells was specific toward neuronal-type cells and was blocked by coincubation of the chimeric toxin with tetanus antitoxin. The toxicity of DAB(389)TTC, like that of diphtheria toxin, was dependent on passage through an acidic compartment and ADP-ribosyltransferase activity of the DAB(389) catalytic fragment. These results suggest that a catalytically inactive form of DAB(389)TTC may be useful as a nonviral vehicle to deliver exogenous proteins to the cytosolic compartment of neurons.
Francis
J W
JW
Cecil B. Day Center for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA. francis@helix.mgh.harvard.edu
Brown
R H
RH
Figueiredo
D
D
Remington
M P
MP
Castillo
O
O
Schwarzschild
M A
MA
Fishman
P S
PS
Murphy
J R
JR
vanderSpek
J C
JC
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002250:Carbon Radioisotopes; D003001:Cloning, Molecular; D003342:Corpus Striatum / Q000166:cytology; D003600:Cytosol; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics; D004167:Diphtheria Toxin / Q000737:chemistry / Q000235:genetics*; D004705:Endocytosis; D015870:Gene Expression; D005822:Genetic Vectors; D006822:Hybrid Cells; D007930:Leucine / Q000493:pharmacokinetics; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013744:Tetanus Toxin / Q000737:chemistry / Q000235:genetics*
D002250:Carbon Radioisotopes; D003603:Cytotoxins; D004167:Diphtheria Toxin; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; D007930:Leucine