Engineering of chimeric catalase-Angiopep-2 for intracellular protection of brain endothelial cells against oxidative stress.
Blood-brain barrier (BBB) disruption and brain microvascular endothelial cells (BMVECs) death caused by excessive production of hydrogen peroxide (H2O2) have been implicated in several neurological conditions. To overcome this problem, H2O2-degrading enzyme with ability to enter the BMVECs is required. In the present study, genetic fusion of gene encoding human catalase and gene encoding Angiopep-2 (AP2), a brain targeting peptide, was performed. The fusion protein was successfully expressed in Escherichia coli and purified to homogeneity. The protein retained heme content and specific enzymatic activity in the same order of magnitude as that of native enzyme. Study of the BMVECs internalization showed that 0.1μM of the fusion protein can enter the cell within 15min, while internalization of the native protein was not observed at this condition. In addition, treatment of the BMVECs with 20 units of the fusion protein for 30min showed protection against H2O2 up to 5.0mM, whereas this protective effect was not observed from treatment with the native protein. Therefore, construction of chimeric human catalase and AP2 provides an insight into the development of potential therapeutic antioxidant with ability to penetrate the BBB for protection against neurodegenerative disorders.
Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand; Center for Innovation Development and Technology Transfer, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
Sakda
S
Yainoy
Center for Innovation Development and Technology Transfer, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
Patcharaporn
P
Houbloyfa
Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand; Center for Innovation Development and Technology Transfer, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
Warawan
W
Eiamphungporn
Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
Chartchalerm
C
Isarankura-Na-Ayudhya
Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand. Electronic address: virapong.pra@mahidol.ac.th.
Virapong
V
Prachayasittikul
D000818:Animals; D001921:Brain / Q000473:pathology*; D002374:Catalase / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D019610:Cytoprotection / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D042541:Intracellular Space / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects*; D010455:Peptides / Q000378:metabolism*; D020011:Protective Agents / Q000494:pharmacology; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000494:pharmacology*; D013056:Spectrophotometry, Ultraviolet; D054322:tat Gene Products, Human Immunodeficiency Virus
C531860:Angiopep-2; D010455:Peptides; D020011:Protective Agents; D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D006861:Hydrogen Peroxide; D002374:Catalase
Angiopep-2; Blood–brain barrier; Brain microvascular endothelial cells; Catalase; Fusion protein; Oxidative stress