CuZn superoxide dismutase (SOD-1):tetanus toxin fragment C hybrid protein for targeted delivery of SOD-1 to neuronal cells.
Increased levels of CuZn superoxide dismutase (SOD-1) are cytoprotective in experimental models of neurological disorders associated with free radical toxicity (e.g. stroke, trauma). Targeted delivery of SOD-1 to central nervous system neurons may therefore be therapeutic in such diseases. The nontoxic C-fragment of tetanus toxin (TTC) possesses the nerve cell binding/transport properties of tetanus holotoxin and has been used as a vector to enhance the neuronal uptake of proteins including enzymes. We have now produced a recombinant, hybrid protein in Escherichia coli tandemly joining human SOD-1 to TTC. The expressed hybrid protein (SOD:Tet450) has a subunit molecular mass of 68 kDa and is recognized by both anti-SOD-1 and anti-TTC antibodies. Calculated per mol, SOD:Tet450 has approximately 60% of the expected SOD-1 enzymatic activity. Analysis of the hybrid protein's interaction with the neuron-like cell line, N18-RE-105, and cultured hippocampal neurons by enzyme immunoassay for human SOD-1 revealed that SOD:Tet451 association with cells was neuron-specific and dose-dependent. The hybrid protein was also internalized, but there was substantial loss of internalized hybrid protein over the first 24 h. Hybrid protein associated with cells remained enzymatically active. These results suggest that human SOD-1 and TTC retain their respective functional properties when expressed together as a single peptide. SOD:Tet451 may prove to be a useful agent for the targeted delivery of SOD-1 to neurons.
Francis
J W
JW
Department of Anatomy, University of Maryland School of Medicine, Baltimore 21201, USA.
Hosler
B A
BA
Brown
R H
RH
Fishman
P S
PS
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006624:Hippocampus / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D000922:Immunotoxins / Q000008:administration & dosage / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D010446:Peptide Fragments / Q000008:administration & dosage / Q000096:biosynthesis / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization; D051381:Rats; D015183:Restriction Mapping; D013482:Superoxide Dismutase / Q000008:administration & dosage / Q000096:biosynthesis / Q000378:metabolism*; D013744:Tetanus Toxin / Q000008:administration & dosage / Q000096:biosynthesis / Q000378:metabolism*
D017931:DNA Primers; D000922:Immunotoxins; D010446:Peptide Fragments; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; D013482:Superoxide Dismutase