title: Improved intracellular delivery of glucocerebrosidase mediated by the HIV-1 TAT protein transduction domain.
abstract: Enzyme replacement therapy (ERT) for Gaucher disease designed to target glucocerebrosidase (GC) to macrophages via mannose-specific endocytosis is very effective in reversing hepatosplenomegaly, and normalizing hematologic parameters but is less effective in improving bone and lung involvement and ineffective in brain. Recombinant GCs containing an in-frame fusion to the HIV-1 trans-activator protein transduction domain (TAT) were expressed in eukaryotic cells in order to obtain active, normally glycosylated GC fusion proteins for enzyme uptake studies. Despite the absence of mannose-specific endocytic receptors on the plasma membranes of various fibroblasts, the recombinant GCs with C-terminal TAT fusions were readily internalized by these cells. Immunofluorescent confocal microscopy demonstrated the recombinant TAT-fusion proteins with a mixed endosomal and lysosomal localization. Thus, TAT-modified GCs represent a novel strategy for a new generation of therapeutic enzymes for ERT for Gaucher disease.
authors.affiliation: Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 10, Room 3D04, Bethesda, MD 20892, USA.
authors.forename: Kyun Oh
authors.initials: KO
authors.lastname: Lee
authors.forename: Nga
authors.initials: N
authors.lastname: Luu
authors.forename: Christine R
authors.initials: CR
authors.lastname: Kaneski
authors.forename: Raphael
authors.initials: R
authors.lastname: Schiffmann
authors.forename: Roscoe O
authors.initials: RO
authors.lastname: Brady
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Murray
mesh_terms: D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects* / Q000502:physiology; D005057:Eukaryotic Cells / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015696:Gene Products, tat / Q000494:pharmacology*; D005962:Glucosylceramidase / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D017434:Protein Structure, Tertiary / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D005962:Glucosylceramidase