RRC ID 10755
Author Watanabe S, Wakasugi K.
Title Module M1 of zebrafish neuroglobin acts as a structural and functional protein building block for a cell-membrane-penetrating activity.
Journal PLoS One
Abstract Neuroglobin (Ngb) is a recently discovered vertebrate globin that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection during oxidative stress that occurs, for example, during ischemia and reperfusion. Recently, we found that zebrafish, but not human, Ngb can translocate into cells. Moreover, we demonstrated that a chimeric ZHHH Ngb protein, in which the module M1 of human Ngb is replaced by the corresponding region of zebrafish Ngb, can penetrate cell membranes and protect cells against oxidative stress-induced cell death, suggesting that module M1 of zebrafish Ngb is important for protein transduction. Furthermore, we recently showed that Lys7, Lys9, Lys21, and Lys23 in module M1 of zebrafish Ngb are crucial for protein transduction activity. In the present study, we have investigated whether module M1 of zebrafish Ngb can be used as a building block to create novel cell-membrane-penetrating folded proteins. First, we engineered a chimeric myoglobin (Mb), in which module M1 of zebrafish Ngb was fused to the N-terminus of full-length human Mb, and investigated its functional and structural properties. Our results showed that this chimeric Mb protein is stable and forms almost the same heme environment and α-helical structure as human wild-type Mb. In addition, we demonstrated that chimeric Mb has a cell-membrane-penetrating activity similar to zebrafish Ngb. Moreover, we found that glycosaminoglycan is crucial for the cell-membrane-penetrating activity of chimeric Mb as well as that of zebrafish Ngb. These results enable us to conclude that such module substitutions will facilitate the design and production of novel functional proteins.
Volume 6(2)
Pages e16808
Published 2011-2-3
DOI 10.1371/journal.pone.0016808
PMID 21304818
PMC PMC3033418
MeSH Animals Animals, Genetically Modified CHO Cells Cell Membrane / metabolism* Cells, Cultured Cricetinae Cricetulus Globins / chemistry* Globins / genetics Globins / metabolism* Globins / physiology* HeLa Cells Humans Membrane Proteins / metabolism Models, Biological Mutagenesis, Site-Directed Myoglobin / chemistry Myoglobin / genetics Myoglobin / metabolism Nerve Tissue Proteins / chemistry* Nerve Tissue Proteins / genetics Nerve Tissue Proteins / metabolism* Nerve Tissue Proteins / physiology* Neuroglobin Protein Engineering / methods Protein Structure, Tertiary / genetics Protein Structure, Tertiary / physiology Protein Transport / genetics Recombinant Fusion Proteins / chemistry Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / physiology Signal Transduction / genetics Signal Transduction / physiology Zebrafish / genetics* Zebrafish / metabolism
IF 2.74
Times Cited 8
Human and Animal Cells HeLa(RCB0007)