title: Defining the pathway for Tat-mediated delivery of beta-glucuronidase in cultured cells and MPS VII mice.
abstract: We used recombinant forms of human beta-glucuronidase (GUS) purified from secretions from stably transfected CHO cells to compare the native enzyme to a GUS-Tat C-terminal fusion protein containing the 11-amino-acid HIV Tat protein transduction domain for: (1) susceptibility to endocytosis by cultured cells, (2) rate of clearance following intravenous infusion, and (3) tissue distribution and effectiveness in clearing lysosomal storage following infusion in the MPS VII mouse. We found: (1) Native GUS was more efficiently taken up by cultured human fibroblasts and its endocytosis was exclusively mediated by the M6P receptor. The GUS-Tat fusion protein showed only 30-50% as much M6P-receptor-mediated uptake, but also was taken up by adsorptive endocytosis through binding of the positively charged Tat peptide to cell surface proteoglycans. (2) GUS-Tat was less rapidly cleared from the circulation in the rat (t(1/2) = 13 min vs 7 min). (3) Delivery to most tissues of the MPS VII mouse was similar, but GUS-Tat was more efficiently delivered to kidney. Histology showed that GUS-Tat more efficiently reduced storage in renal tubules, retina, and bone. These studies demonstrate that Tat modification can extend the range of tissues corrected by infused enzyme.
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104, USA.
authors.forename: Koji O
authors.initials: KO
authors.lastname: Orii
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Grubb
authors.forename: Carole
authors.initials: C
authors.lastname: Vogler
authors.forename: Beth
authors.initials: B
authors.lastname: Levy
authors.forename: Yun
authors.initials: Y
authors.lastname: Tan
authors.forename: Kamelia
authors.initials: K
authors.lastname: Markova
authors.forename: Beverly L
authors.initials: BL
authors.lastname: Davidson
authors.forename: Q
authors.initials: Q
authors.lastname: Mao
authors.forename: Tadao
authors.initials: T
authors.lastname: Orii
authors.forename: Naomi
authors.initials: N
authors.lastname: Kondo
authors.forename: William S
authors.initials: WS
authors.lastname: Sly
mesh_terms: D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism*; D015316:Genetic Therapy; D005966:Glucuronidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008360:Mannosephosphates / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D016538:Mucopolysaccharidosis VII / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D015696:Gene Products, tat; D008360:Mannosephosphates; D011994:Recombinant Proteins; C047217:mannose 1-phosphate; D005966:Glucuronidase