The knockout of cytoglobin 1 in zebrafish (Danio rerio) alters lipid metabolism, iron homeostasis and oxidative stress response.

Cytoglobin (Cygb) is an evolutionary ancient heme protein with yet unclear physiological function(s). Mammalian Cygb is ubiquitously expressed in all tissues and is proposed to be involved in reactive oxygen species (ROS) detoxification, nitric oxide (NO) metabolism and lipid-based signaling processes. Loss-of-function studies in mouse associate Cygb with apoptosis, inflammation, fibrosis, cardiovascular dysfunction or oncogenesis. In zebrafish (Danio rerio), two cygb genes exist, cytoglobin 1 (cygb1) and cytoglobin 2 (cygb2). Both have different coordination states and distinct expression sites within zebrafish tissues. The biological roles of the cygb paralogs are largely uncharacterized. We used a CRISPR/Cas9 genome editing approach and generated a knockout of the penta-coordinated cygb1 for in vivo analysis. Adult male cygb1 knockouts develop phenotypic abnormalities, including weight loss. To identify the molecular mechanisms underlying the occurrence of these phenotypes and differentiate between function and effect of the knockout we compared the transcriptomes of cygb1 knockout at different ages to age-matched wild-type zebrafish. We found that immune regulatory and cell cycle regulatory transcripts (e.g. tp53) were up-regulated in the cygb1 knockout liver. Additionally, the expression of transcripts involved in lipid metabolism and transport, the antioxidative defense and iron homeostasis was affected in the cygb1 knockout. Cygb1 may function as an anti-inflammatory and cytoprotective factor in zebrafish liver, and may be involved in lipid-, iron-, and ROS-dependent signaling.

The More, the Merrier? Multiple Myoglobin Genes in Fish Species, Especially in Gray Bichir (Polypterus senegalus) and Reedfish (Erpetoichthys calabaricus).

The members of the globin superfamily are a classical model system to investigate gene evolution and their fates as well as the diversity of protein function. One of the best-known globins is myoglobin (Mb), which is mainly expressed in heart muscle and transports oxygen from the sarcolemma to the mitochondria. Most vertebrates harbor a single copy of the myoglobin gene, but some fish species have multiple myoglobin genes. Phylogenetic analyses indicate an independent emergence of multiple myoglobin genes, whereby the origin is mostly the last common ancestor of each order. By analyzing different transcriptome data sets, we found at least 15 multiple myoglobin genes in the polypterid gray bichir (Polypterus senegalus) and reedfish (Erpetoichthys calabaricus). In reedfish, the myoglobin genes are expressed in a broad range of tissues but show very different expression values. In contrast, the Mb genes of the gray bichir show a rather scattered expression pattern; only a few Mb genes were found expressed in the analyzed tissues. Both, gray bichir and reedfish possess lungs which enable them to inhabit shallow and swampy waters throughout tropical Africa with frequently fluctuating and low oxygen concentrations. The myoglobin repertoire probably reflects the molecular adaptation to these conditions. The sequence divergence, the substitution rate, and the different expression pattern of multiple myoglobin genes in gray bichir and reedfish imply different functions, probably through sub- and neofunctionalization during evolution.

Genetic and functional diversity of the multiple lungfish myoglobins.

It is known that the West African lungfish (Protopterus annectens) harbours multiple myoglobin (Mb) genes that are differentially expressed in various tissues and that the Mbs differ in their abilities to confer tolerance towards hypoxia. Here, we show that other lungfish species (Protopterus dolloi, Protopterus aethiopicus and Lepidosiren paradoxa) display a similar diversity of Mb genes and have orthologous Mb genes. To investigate the functional diversification of these genes, we studied the structures, O2 binding properties and nitrite reductase enzymatic activities of recombinantly expressed P. annectens Mbs (PanMbs). CD spectroscopy and small-angle X-ray scattering revealed the typical globin-fold in all investigated recombinant Mbs, indicating a conserved structure. The highest O2 affinity was measured for PanMb2 (P50  = 0.88 Torr at 20 °C), which is mainly expressed in the brain, whereas the muscle-specific PanMb1 has the lowest O2 affinity (P50  = 3.78 Torr at 20 °C), suggesting that tissue-specific O2 requirements have resulted in the emergence of distinct Mb types. Two of the mainly neuronally expressed Mbs (PanMb3 and PanMb4b) have the highest nitrite reductase rates. These data show different O2 binding and enzymatic properties of lungfish Mbs, reflecting multiple subfunctionalisation and neofunctionalisation events that occurred early in the evolution of lungfish. Some Mbs may have also taken over the functions of neuroglobin and cytoglobin, which are widely expressed in vertebrates but appear to be missing in lungfish.