title: Tat-mediated protein transduction of human brain pyridoxine-5-P oxidase into PC12 cells.
abstract: Pyridoxine-5-P oxidase catalyses the terminal step in the biosynthesis of pyridoxal-5-P, the biologically active form of vitamin B6 which acts as an essential cofactor. Here, a human brain pyridoxine-5-P oxidase gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat-pyridoxine-5-P oxidase fusion protein. Expressed and purified Tat-pyridoxine-5-P oxidase fusion protein transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-pyridoxine-5-P oxidase protein showed catalytic activity and was stable for 48 h. Moreover, the formation of pyridoxal-5-P was increased by adding exogenous Tat-pyridoxine-5-P oxidase to media pre-treated with the vitamin B6 precursor pyridoxine. In addition, the intracellular concentration of pyridoxal-5-P was markedly increased when Tat-pyridoxal kinase was transduced together with Tat-pyridoxine-5-P oxidase into cells.These results suggest that the transduction of Tat-pyridoxine-5-P oxidase fusion protein presents a means of regulating the level of pyridoxal-5-P and of replenishing this enzyme in various neurological disorders related to vitamin B6.
authors.affiliation: Department of Biomedical Sciences and Research, Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea.
authors.forename: So Young
authors.initials: SY
authors.lastname: Kim
authors.forename: Jae Jin
authors.initials: JJ
authors.lastname: An
authors.forename: Dae Won
authors.initials: DW
authors.lastname: Kim
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Choi
authors.forename: Sun Hwa
authors.initials: SH
authors.lastname: Lee
authors.forename: Seok-Il
authors.initials: SI
authors.lastname: Hwang
authors.forename: Oh-Shin
authors.initials: OS
authors.lastname: Kwon
authors.forename: Tae-Cheon
authors.initials: TC
authors.lastname: Kang
authors.forename: Moo Ho
authors.initials: MH
authors.lastname: Won
authors.forename: Sung-Woo
authors.initials: SW
authors.lastname: Cho
authors.forename: Jinseu
authors.initials: J
authors.lastname: Park
authors.forename: Won Sik
authors.initials: WS
authors.lastname: Eum
authors.forename: Kil Soo
authors.initials: KS
authors.lastname: Lee
authors.forename: Soo Young
authors.initials: SY
authors.lastname: Choi
mesh_terms: D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000201:enzymology*; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016716:PC12 Cells; D011734:Pyridoxaminephosphate Oxidase / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D011734:Pyridoxaminephosphate Oxidase