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.
Highlights
Transport and storage of oxygen in vertebrate animals are typically mediated by globins, small proteins that bind O2 by the means of a porphyrin-coordinated Fe2ϩ ion [1,2,3]
Whereas Ngb was originally identified in mammalian species [6], it is present in fishes [24], suggesting a widespread occurrence in vertebrates
We designed multiple primers that allowed the amplification of overlapping fragments from the D. rerio Ngb gene
Summary
Cloning and Sequencing of D. rerio Ngb cDNA and Gene—D. rerio were kept at 28 °C in a freshwater aquarium. Genomic DNA of D. rerio fication of cDNA ends; HRE, hypoxia-responsive sequence elements; ISH, in situ hybridization; NRSE, neuron-restrictive silencer element; PBS, phosphate-buffered saline; TBST, Tris-buffered saline with Tween 20. After washing 3 ϫ 5 min with 2ϫ SSC and 5 min in 1ϫ PBS, 0.1% Tween 20, and 0.2% bovine serum albumin, nonspecific antibody binding sites were blocked with 1% blocking reagent (Roche Applied Science), 150 mM NaCl, and 100 mM Tris-HCl, pH 7.4. Specimens were incubated for 30 min at room temperature with an anti-digoxigenin antibody (Roche Applied Science) coupled with alkaline phosphatase (diluted 1:100 in PBS) and washed 2 ϫ 5 min in PBS, 0.1% Tween 20, and 0.2% bovine serum albumin and 5 min in 100 mM Tris/HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2. The sections were washed 3 ϫ 8 min in PBS, mounted in Elvanol polyvinyl alcohol (Mowiol, Calbiochem), and examined with a Leitz DM RD microscope
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
