Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain.
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.
Zhang
Xian-Yang
XY
Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA.
Dinh
Annie
A
Cronin
James
J
Li
Su-Chen
SC
Reiser
Jakob
J
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D005347:Fibroblasts / Q000378:metabolism; D005698:Galactosylceramidase / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000201:enzymology* / Q000235:genetics*; D051379:Mice; D008818:Mice, Neurologic Mutants; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000235:genetics; D014161:Transduction, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D054322:tat Gene Products, Human Immunodeficiency Virus; D005698:Galactosylceramidase