9-polylysine protein transduction domain: enhanced penetration efficiency of superoxide dismutase into mammalian cells and skin.
Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), have been considered to have a beneficial effect against various diseases that are mediated by the reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against oxidative stresses, the lack of their transduction ability into cells resulted in a limited ability to detoxify intracellular ROS. To render the SOD enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant SOD proteins were generated. A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment that encodes the 9 amino acids Tat protein transduction domain (RKKRRQRRR) of HIV-1 and lysine rich peptide (KKKKKKKKK) in a bacterial expression vector in order to produce a genetic in-frame Tat-SOD and 9Lys-SOD fusion protein, respectively. The expressed and purified Tat-SOD and 9Lys-SOD fusion proteins can transduce into human fibroblast cells, and they were enzymatically active and stable for 24 h. The cell viability of the fibroblast cells that were treated with paraquat, an intracellular superoxide anion generator, was increased by the transduced Tat-SOD or 9Lys-SOD. The transduction efficacy of 9Lys-SOD was more efficient than that of Tat-SOD. We evaluated the ability of the SOD fusion pmteins to transduce into animal skin. This analysis showed that Tat-SOD and 9Lys-SOD fusion proteins efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin (judged by the immunohistochemistry and specific enzyme activities). The enzymatic activity of the transduced 9Lys-SOD was higher than that of Tat-SOD, indicating that the penetration of 9Lys-SOD was more efficient when put into the skin. These results suggest Tat-SOD and 9Lys-SOD fusion proteins can be used as anti-aging cosmetics, or in protein therapy, for various disorders that are related to this antioxidant enzyme and ROS.
Park
Jinseu
J
Division of Life Sciences, Hallym University, Chunchon, Korea.
Ryu
Jiyoon
J
Jin
Li Hua
LH
Bahn
Jae Hoon
JH
Kim
Ju A
JA
Yoon
Chang Sik
CS
Kim
Dae Won
DW
Han
Kyu Hyung
KH
Eum
Won Sik
WS
Kwon
Hyeok Yil
HY
Kang
Tae-Cheon
TC
Won
Moo Ho
MH
Kang
Jung Hoon
JH
Cho
Sung-Woo
SW
Choi
Soo Young
SY
D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016166:Free Radical Scavengers / Q000378:metabolism*; D015696:Gene Products, tat / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D010269:Paraquat / Q000494:pharmacology; D011107:Polylysine / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic
D000975:Antioxidants; D016166:Free Radical Scavengers; D015696:Gene Products, tat; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D011107:Polylysine; D013482:Superoxide Dismutase; D010269:Paraquat