Zebrafish Reveals Different and Conserved Features of Vertebrate Neuroglobin Gene Structure, Expression Pattern, and Ligand Binding

Abstract

Neuroglobin has been identified as a respiratory protein that is primarily expressed in the mammalian nervous system. Here we present the first detailed analysis of neuroglobin from a non-mammalian vertebrate, the zebrafish Danio rerio. The zebrafish neuroglobin gene reveals a mammalian-type exon-intron pattern in the coding region (B12.2, E11.0, and G7.0), plus an additional 5-non-coding exon. Similar to the mammalian neuroglobin, the zebrafish protein displays a hexacoordinate deoxy-binding scheme. Flash photolysis kinetics show the competitive binding on the millisecond timescale of external ligands and the distal histidine, resulting in an oxygen affinity of 1 torr. Western blotting, immune staining, and mRNA in situ hybridization demonstrate neuroglobin expression in the fish central nervous system and the retina but also in the gills. Neurons containing neuroglobin have a widespread distribution in the brain but are also present in the olfactory system. In the fish retina, neuroglobin is mainly present in the inner segments of the photoreceptor cells. In the gills, the chloride cells were identified to express neuroglobin. Neuroglobin appears to be associated with mitochondria-rich cell types and thus oxygen consumption rates, suggesting a myoglobin-like function of this protein in facilitated oxygen diffusion.Transport and storage of oxygen in vertebrate animals are typically mediated by globins, small proteins that bind O 2 by the means of a porphyrin-coordinated Fe 2ϩ ion (1-3). The heterotetrameric hemoglobin is present in red blood cells of nearly all vertebrates and transports O 2 in the circulatory system from the respiratory surfaces to the inner organs. The monomeric myoglobin, typically found in the myocytes of cardiac and striated muscles, facilitates intracellular O 2 diffusion to the mitochondria and stores O 2 (3, 4) but also functions as an nitric-oxide dioxygenase (5). Neuroglobin (Ngb) 1 and cytoglobin are two recently discovered vertebrate globins (6 -9). Whereas cytoglobin might play a role in collagen synthesis (10, 11), the leading hypotheses suggest Ngb to be involved in neuronal oxygen homeostasis (6,(12)(13)(14)(15)(16). Ngb shares only a few amino acids with vertebrate hemoglobin and myoglobin (Ͻ25% identity) but rather resembles the nerve-specific globins known in some invertebrates (6, 13). In fact, phylogenetic analyses suggest an ancient origin of Ngb, probably diverging from the other globins before the Protostomia-Deuterostomia split (6, 8). Mouse and human deoxy-Ngb display hexacordinated hemochrome-binding schemes at the Fe 2ϩ (17, 18). The proximal histidine can be replaced by an external ligand such as O 2 , resulting in an oxygen affinity (P 50 ) of ϳ1-2 torr, similar to that of myoglobin (6, 17).Initially, Ngb was found to be predominantly expressed in the brains of man and mouse (6), but recent analyses show Ngb to be also present in the peripheral nervous system and some non-neuronal endocrine tissues (19 -21). Particularly high amounts of Ngb were observed in the m...

Human Neuroglobin Functions as an Oxidative Stress-responsive Sensor for Neuroprotection

“…Although Ngb was originally identified in mammalian species, it is also present in non-mammalian vertebrates (17,18). Mammalian and fish Ngb proteins share about 50% amino acid sequence identity.…”

Structure and dynamics of Antarctic fish neuroglobin assessed by computer simulations

“…As shown in Fig. 2A, in fish Ngbs, the average Cys-Cys distance in the CD loop is several-Å shorter than in the human protein, in which Cys46-CD7 and Cys55-D5 are known to form a disulfide bridge and appear to be involved in redox-state sensing (11,21,30). Thus, although the residues are in the reduced form, they remain very close to each other during the time scale of the simulations (Fig.…”

“…Additionally, in human Ngb, two cysteyl residues in the CD region, Cys46-CD7 and Cys55-D5, may form an internal disulfide bond that modulates oxygen affinity (10). This regulation was not observed in zebrafish Ngb, where the first cysteine is shifted by two positions (11), or in murine Ngb, where Cys46-CD7 is absent (12).…”

“…Mammalian and zebrafish Ngbs share about 50% sequence identity and their oxygen-binding properties are similar (11).…”

“…Ngb was originally identified in mammals but is widespread in all nonmammalian vertebrates, e.g., the zebrafish Danio rerio and other teleost fishes (11). Mammalian and zebrafish Ngbs share about 50% sequence identity and their oxygen-binding properties are similar (11).…”