A genetic fusion construct between the tetanus toxin C fragment and the lysosomal acid hydrolase beta-glucuronidase expresses a bifunctional protein with enhanced secretion and neuronal uptake.
The neurotropic atoxic fragment of tetanus toxin has been used as a carrier for transporting macromolecules into neurons but all studies to date have tested cytosolic proteins. In this study we investigated the effect of a genetic addition of the tetanus toxin C fragment sequence to a lysosomal enzyme which contains a signal sequence for insertion into the membrane-bound compartment and must be extensively modified in the endoplasmic reticulum (ER) and Golgi to attain functionality. In-frame fusion constructs between the atoxic C fragment and beta-glucuronidase were compared with the wild-type enzyme for: (i) enzymatic activity; (ii) heat stability; (iii) pH dependence; (iv) specific activity; (v) apparent molecular mass and (vi) receptor-mediated uptake by fibroblasts and neurons. The modified proteins had biochemical properties similar to wild-type enzyme but exhibited different enzyme secretion profiles. Addition of the secreted fusion enzyme to cultures of primary neurons showed significantly increased neuronal uptake of the modified protein compared with the wild-type, demonstrating the bifunctionality of the chimeric molecule.
Jiang
Kanli
K
Center for Comparative Medical Genetics, School of Veterinary Medicine, University of Pennsylvania and Division of Neurology, Stokes Research Institute, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.
Watson
Deborah J
DJ
Wolfe
John H
JH
D000818:Animals; D004355:Drug Stability; D005347:Fibroblasts / Q000378:metabolism; D005966:Glucuronidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D051379:Mice; D008809:Mice, Inbred C3H; D008970:Molecular Weight; D009474:Neurons / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics*; D010957:Plasmids; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D013744:Tetanus Toxin / Q000235:genetics*
D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; D005966:Glucuronidase