Module M1 of Zebrafish Neuroglobin Acts as a Structural and Functional Protein Building Block for a Cell-Membrane-Penetrating Activity

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

Differential Loss and Retention of Cytoglobin, Myoglobin, and Globin-E during the Radiation of Vertebrates

If rates of postduplication gene retention are positively correlated with levels of functional constraint, then gene duplicates that have been retained in a restricted number of taxonomic lineages would be expected to exhibit relatively low levels of sequence conservation. Paradoxical patterns are presented by gene duplicates that have been retained in a small number of taxa but which are nonetheless subject to strong purifying selection relative to paralogous members of the same multigene family. This pattern suggests that such genes may have been co-opted for novel, lineage-specific functions. One possible example involves the enigmatic globin-E gene (GbE), which appears to be exclusively restricted to birds. Available data indicate that this gene is expressed exclusively in the avian eye, but its physiological function remains a mystery. In contrast to the highly restricted phyletic distribution of GbE, the overwhelming majority of jawed vertebrates (gnathostomes) possess copies of the related cytoglobin (Cygb) and myoglobin (Mb) genes. The purpose of the present study was 1) to assess the phyletic distribution of the Cygb, Mb, and GbE genes among vertebrates, 2) to elucidate the duplicative origins and evolutionary histories of these three genes, and 3) to evaluate the relative levels of functional constraint of these genes based on comparative sequence analysis. To accomplish these objectives, we conducted a combined phylogenetic and comparative genomic analysis involving taxa that represent each of the major lineages of gnathostome vertebrates. Results of synteny comparisons and phylogenetic topology tests revealed that GbE is clearly not the product of a recent, bird-specific duplication event. Instead, GbE originated via duplication of a proto-Mb gene in the stem lineage of gnathostomes. Unlike the Mb gene, which has been retained in all major gnathostome lineages other than amphibians, the GbE gene has been retained only in the lineage leading to modern birds and has been independently lost in at least four major lineages: teleost fish, amphibians, mammals, and nonavian reptiles. Despite the restricted phyletic distribution of this gene, our results indicate that GbE is one of the most highly conserved globins in the avian genome

NO Dioxygenase Activity in Hemoglobins Is Ubiquitous In Vitro, but Limited by Reduction In Vivo

Genomics has produced hundreds of new hemoglobin sequences with examples in nearly every living organism. Structural and biochemical characterizations of many recombinant proteins reveal reactions, like oxygen binding and NO dioxygenation, that appear general to the hemoglobin superfamily regardless of whether they are related to physiological function. Despite considerable attention to “hexacoordinate” hemoglobins, which are found in nearly every plant and animal, no clear physiological role(s) has been assigned to them in any species. One popular and relevant hypothesis for their function is protection against NO. Here we have tested a comprehensive representation of hexacoordinate hemoglobins from plants (rice hemoglobin), animals (neuroglobin and cytoglobin), and bacteria (Synechocystis hemoglobin) for their abilities to scavenge NO compared to myoglobin. Our experiments include in vitro comparisons of NO dioxygenation, ferric NO binding, NO-induced reduction, NO scavenging with an artificial reduction system, and the ability to substitute for a known NO scavenger (flavohemoglobin) in E. coli. We conclude that none of these tests reveal any distinguishing predisposition toward a role in NO scavenging for the hxHbs, but that any hemoglobin could likely serve this role in the presence of a mechanism for heme iron re-reduction. Hence, future research to test the role of Hbs in NO scavenging would benefit more from the identification of cognate reductases than from in vitro analysis of NO and O2 binding