Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells.
Antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) have been considered to have a beneficial effect against various diseases mediated by reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against the oxidative stresses, the lack of their transduction ability into cells resulted in limited ability to detoxify intracellular ROS. To render the catalase enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant catalase proteins were generated. A human liver catalase gene was cloned and fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) and arginine-rich peptides (RRRRRRRRR) in a bacterial expression vector to produce genetic in-frame Tat-CAT and 9Arg-CAT fusion proteins, respectively. The expressed and purified fusion proteins can be transduced into mammalian cells (HeLa and PC12 cells) in a time- and dose-dependent manner when added exogenously in culture medium, and transduced fusion proteins were enzymatically active and stable for 60 h. When exposed to H(2)O(2), the viability of HeLa cells transduced with Tat-CAT or 9Arg-CAT fusion proteins was significantly increased. In combination with transduced SOD, transduced catalase also resulted in a cooperative increase in cell viability when the cells were treated with paraquat, an intracellular antioxide anion generator. We then evaluated the ability of the catalase fusion proteins to transduce into animal skin. This analysis showed that Tat-CAT and 9Arg-CAT fusion proteins efficiently penetrated the epidermis as well as the dermis of the subcutaneous layer when sprayed on animal skin, as judged by immunohistochemistry and specific enzyme activities. These results suggest that Tat-CAT and 9Arg-CAT fusion proteins can be used in protein therapy for various disorders related to this antioxidant enzyme.
Jin
L H
LH
Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon, Kangwondo, South Korea.
Bahn
J H
JH
Eum
W S
WS
Kwon
H Y
HY
Jang
S H
SH
Han
K H
KH
Kang
T C
TC
Won
M H
MH
Kang
J H
JH
Cho
S W
SW
Park
J
J
Choi
S Y
SY
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics*; D001483:Base Sequence; D002374:Catalase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D015870:Gene Expression; D015696:Gene Products, tat / Q000235:genetics*; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D016716:PC12 Cells; D010269:Paraquat / Q000494:pharmacology; D010455:Peptides / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000493:pharmacokinetics; D012867:Skin / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D010455:Peptides; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D001120:Arginine; D006861:Hydrogen Peroxide; D002374:Catalase; D013482:Superoxide Dismutase; D010269:Paraquat