Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain.

Abstract

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 3.2.2.46) 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.

• Authors: Xian-Yang Zhang, Annie Dinh, James Cronin, Su-Chen Li, Jakob Reiser
• Journal: Journal of neurochemistry (J Neurochem) Vol. 104 Issue 4 Pg. 1055-64 (Feb 2008) ISSN: 1471-4159 [Electronic] England
• PMID: 17986221 (Publication Type: Comparative Study, Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• tat Gene Products, Human Immunodeficiency Virus
• Galactosylceramidase

Topics

• Amino Acid Sequence
• Animals
• COS Cells
• Cells, Cultured
• Chlorocebus aethiops
• Fibroblasts (metabolism)
• Galactosylceramidase (genetics, metabolism)
• HeLa Cells
• Humans
• Lysosomes (enzymology, genetics)
• Mice
• Mice, Neurologic Mutants
• Molecular Sequence Data
• Protein Structure, Tertiary (genetics)
• Transduction, Genetic
• tat Gene Products, Human Immunodeficiency Virus (chemistry, genetics, metabolism)