Tetanus toxin is a protein neurotoxin of 1315 amino acids. The mechanisms by which tetanus toxin binds to and enters cells are not well understood. Hc is about 50,000 M.W. peptide corresponding to the carboxyl terminus of tetanus toxin that retains the ability of holotoxin to bind to receptors and undergo retrograde axonal transport. We have previously prepared a number of deletion mutants of Hc and identified the carboxyl terminal 5 amino acids as a region critical for binding to receptors. Current studies are designed to determine 1) if Hc and truncated variants that retain receptor-binding activity can undergo retrograde axonal transport and 2) if Hc can undergo retrograde axonal transport fused to a second protein. Genes coding for full-length Hc and cell-binding deletion mutants of Hc were inserted into the pMAL(TM)c2 vector downstream from the malE gene. Fusion proteins consisting of maltose-binding protein and different Hc sequences were expressed in E. coli, purified, radioiodinated and assayed for retrograde axonal transport in the rat sciatic nerve. Fusion proteins containing full length Hc or Hc lacking 5 amino acids from the carboxyl terminal were found to undergo retrograde axonal transport. Fusion proteins containing Hc with 163 or 263 amino acids deleted from the amino terminal, which have been demonstrated to bind to target cells, did not undergo retrograde axonal transport. These data suggest that domains from the amino terminal of Hc may be important for retrograde axonal transport either by increasing the stability of Hc or by maintaining Hc in a product conformation.
