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Zhang, XY, Dinh, A, Cronin, J, Li, SC and Reiser, J (2008) Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain. J. Neurochem. 104:1055-64
A number of studies have shown that a short peptide, the protein transduction domain (PTD) derived from the HIV-1 Tat protein (Tat-PTD) improved cellular uptake in vitro and distribution in vivo of recombinant proteins bearing such PTDs when administered systemically. To investigate the effects of Tat-PTD addition on the subcellular localization of the lysosomal enzyme galactocerebrosidase (GALC, EC 184.108.40.206) and with a view towards designing improved therapeutic strategies for Krabbe disease (globoid cell leukodystrophy), mouse GALC was tagged C-terminally with the Tat-PTD. Compared with unmodified GALC, GALC bearing a Tat-PTD, a myc epitope and 6 consecutive His residues [GALC-TMH (Tat-PTD, a myc epitope and 6 consecutive His residues)] was found to be secreted more efficiently. Also, GALC-TMH was found to be taken up by cells both via mannose-6-phosphate receptor (M6PR)-mediated endocytosis as well as by M6PR-independent mechanisms. GALC-TMH displayed increased M6PR-independent uptake in fibroblasts derived from twitcher mice (a murine model of globoid cell leukodystrophy) and in neurons derived from the mouse brain cortex compared with GALC lacking a Tat-PTD. Immunocytochemical analyses revealed that Tat-modified GALC protein co-localized in part with the lysosome-associated membrane protein-1. Complete correction of galactosylceramide accumulation was achieved in twitcher mouse fibroblasts lacking GALC activity following addition of GALC-TMH. Therefore, GALC-TMH not only maintained the features of the native GALC protein including enzymatic function, intracellular transport and location, but also displayed more efficient cellular uptake.
Amino Acid Sequence; Animals; COS Cells; Cells, Cultured; Cercopithecus aethiops; Fibroblasts/metabolism; Galactosylceramidase/genetics; Galactosylceramidase/metabolism; HeLa Cells; Humans; Lysosomes/enzymology; Lysosomes/genetics; Mice; Mice, Neurologic Mutants; Molecular Sequence Data; Protein Structure, Tertiary/genetics; Transduction, Genetic; tat Gene Products, Human Immunodeficiency Virus/chemistry; tat Gene Products, Human Immunodeficiency Virus/genetics; tat Gene Products, Human Immunodeficiency Virus/metabolism