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.
Highlights
Globins are iron porphyrin complex-containing proteins that bind reversibly to oxygen (O2) and, as such, play an important role in respiratory function
We previously found that human Ngb binds exclusively to the GDP-bound form of the a-subunit of heterotrimeric G protein (Gai) and acts as a guanine nucleotide dissociation inhibitor (GDI) by inhibiting the rate of exchange of GDP for GTP on Gai [13,14,15]
We previously showed that zebrafish Ngb lacks GDI activity and that zebrafish Ngb cannot rescue cell death significantly under oxidative stress conditions as compared with human Ngb [17,20,21]
Summary
Globins are iron porphyrin complex (heme)-containing proteins that bind reversibly to oxygen (O2) and, as such, play an important role in respiratory function. It was recently suggested that mammalian Ngb might be involved in the neuronal response to hypoxia and ischemia [7,8,9,10,11,12]. Mammalian Ngb expression has been reported to increase in response to neuronal hypoxia in vitro and to ischemia in vivo [7,8,12]. Mammalian Ngb has been reported to protect the brain from experimentally induced stroke in vivo [8,10]. We used a protein delivery reagent, Chariot [16], to investigate whether the GDI activity of human Ngb plays an important role in its neuroprotective activity under oxidative stress conditions. We demonstrated that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity [17]
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