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Α genetic fusion construct between the tetanus toxin C fragment and the lysosomal acid hydrolase β‐glucuronidase expresses a bifunctional protein with enhanced secretion and neuronal uptake
Author(s) -
Jiang Kanli,
Watson Deborah J.,
Wolfe John H.
Publication year - 2005
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2005.03133.x
Subject(s) - endoplasmic reticulum , enzyme , biochemistry , secretion , fusion protein , chemistry , toxin , hydrolase , signal peptide , golgi apparatus , cytosol , biology , peptide sequence , microbiology and biotechnology , recombinant dna , gene
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 β‐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.