Interaction of tetanus toxin derived hybrid proteins with neuronal cells.
The non-toxic ganglioside binding domain of tetanus toxin (Hc fragment C or TTC) has been studied as a vector for delivering therapeutic proteins to neurons. There is little information on the cellular processing of proteins delivered by linkage to TTC. We have evaluated the cellular handling of a multi-domain hybrid protein containing TTC and both the human enzyme superoxide dismutase and the maltose binding protein from E. coli. Binding, internalization, and cleavage of this protein during prolonged incubation with fetal cortical neurons or cells of the N18-RE-105 line was evaluated by immunoblot analysis, ELISA, and immunocytochemistry. Hybrid proteins were bound and internalized in a manner very similar to TTC. Internalized proteins showed long-term stability within cells, and were degraded into predictable large protein fragments in both cell types. Fragments that were cleaved away from the TTC domain were released into extracellular fluid after internalization. Proteins coupled to TTC share its long-term stability after cellular internalization. After internalization, dissociation of proteins linked to TTC facilitates their release from the cell, but not into other cellular compartments such as the cytosol. TTC linked proteins are probably enclosed within a stable endosomal compartment throughout their cellular lifetime.
Figueiredo
D M
DM
Neurology Service and, Department of Neurology, Baltimore VAMC, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Matthews
C C
CC
Parks
D A
DA
Fairweather
N F
NF
Dougan
G
G
Wilt
S G
SG
Fishman
P S
PS
D018528:ATP-Binding Cassette Transporters; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D016503:Drug Delivery Systems; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D006801:Humans; D015151:Immunoblotting; D018796:Immunoconjugates; D007150:Immunohistochemistry; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology; D011506:Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics*; D013482:Superoxide Dismutase / Q000378:metabolism*; D013744:Tetanus Toxin / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D018796:Immunoconjugates; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011506:Proteins; D013744:Tetanus Toxin; C461599:maltose transport system, E coli; D013482:Superoxide Dismutase