Improved intracellular delivery of glucocerebrosidase mediated by the HIV-1 TAT protein transduction domain q Kyun Oh Lee, Nga Luu, Christine R. Kaneski, Raphael Schiffmann, Roscoe O. Brady, Gary J. Murray * Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 10, Room 3D04, 10 Center Drive, Bethesda, MD 20892, USA Received 3 May 2005 Available online 26 September 2005 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-ac- tivator 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-mod- ified GCs represent a novel strategy for a new generation of therapeutic enzymes for ERT for Gaucher disease. Published by Elsevier Inc. Keywords: Glucocerebrosidase; Gaucher disease; Enzyme replacement therapy; TAT; Protein transduction Lysosomal storage diseases (LSD) are a group of hered- itary metabolic disorders, caused by defects in catabolic en- zymes that result in pathological accumulation of unmetabolized substrates within the lysosomes of affected individuals and clinical diseases with a broad range of severity and organ system involvement [1,2]. Gaucher dis- ease (MIM 230800) is the most common LSD with a prev- alence of 1 in 50,000 [3,4]. This autosomal recessive LSD is caused by defects in the enzyme glucocerebrosidase (GC; EC 3.2.1.45) that is essential for the catabolism of glucocer- ebroside [5]. Deficiency of this enzyme leads to abnormally high concentrations of glucocerebroside in the lysosomes of mononuclear phagocytes. Over 200 different mutations have been identified in the gene encoding GC [4,6]. The broad spectrum of manifestations in Gaucher disease in- cludes hepatosplenomegaly, anemia, thrombocytopenia, bone lesions, and sometimes involvement of brain, lungs or kidneys. Patients with Gaucher disease are categorized as type 1: non-neuronopathic, type 2: acute neuronopathic or type 3: chronic neuronopathic [3,7]. Elucidation of the lectin-specific uptake of circulating glycoproteins [8,9] led to a strategy of partial enzymatic deglycosylation of native GC to direct the enzyme to mac- rophages via mannose-specific endocytosis [10]. In patients with type 1 Gaucher disease ERT reverses visceral storage of glucocerebroside resulting in dramatic reduction in hepatosplenomegaly and normalization of hematological parameters [11]. Despite this success, in one study only two-thirds of patients on high dose ERT show bone 0006-291X/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.bbrc.2005.05.207 q Abbreviations: LSD, lysosomal storage diseases; ERT, enzyme replace- ment therapy; GC, glucocerebrosidase; PTD, protein transduction domain; TAT, trans-activator; HIV-1, human immunodeficiency virus type 1; HSV, herpes simplex virus; PVDF, polyvinylidene difluoride; MOI, multiplicity of infection; 4-MUG, 4-methylumbelliferylglucopyranoside; HRP, horseradish peroxidase; FITC, fluorescein isothyocyanate; BBB, blood–brain barrier. * Corresponding author. Fax: +1 301 496 9480. E-mail address: murrayg@ninds.nih.gov (G.J. Murray). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 337 (2005) 701–707 BBRC