A novel and ancient group of type I keratins with members in bichir, sturgeon and gar.

BACKGROUND: Vertebrate epithelial cells typically express a specific set of keratins. In teleosts, keratins are also present in a variety of mesenchymal cells, which usually express vimentin. Significantly, our previous studies revealed that virtually all known teleost keratins evolved independently from those present in terrestrial vertebrates. To further elucidate the evolutionary scenario that led to the large variety of keratins and their complex expression patterns in present day teleosts, we have investigated their presence in bichir, sturgeon and gar. RESULTS: We have discovered a novel group of type I keratins with members in all three of these ancient ray-finned fish, but apparently no counterparts are present in any other vertebrate class so far investigated, including the modern teleost fish. From sturgeon and gar we sequenced one and from bichir two members of this novel keratin group. By complementary keratin blot-binding assays and peptide mass fingerprinting using MALDI-TOF mass spectrometry, in sturgeon we were able to assign the sequence to a prominent protein spot, present exclusively in a two-dimensionally separated cytoskeletal preparation of skin, thus identifying it as an epidermally expressed type I keratin. In contrast to the other keratins we have so far sequenced from bichir, sturgeon and gar, these new sequences occupy a rather basal position within the phylogenetic tree of type I keratins, in a close vicinity to the keratins we previously cloned from river lamprey. CONCLUSION: Thus, this new K14 group seem to belong to a very ancient keratin branch, whose functional role has still to be further elucidated. Furthermore, the exclusive presence of this keratin group in bichir, sturgeon and gar points to the close phylogenetic relationship of these ray- finned fish, an issue still under debate among taxonomists.

Neuroglobin mRNA expression after transient global brain ischemia and prolonged hypoxia in cell culture.

Neuroglobin is a nerve-specific respiratory protein that has been proposed to play an important role in the protection of brain neurons from ischemic and hypoxic injuries. Here, we investigated the regulation of neuroglobin expression after transient global ischemia in the rat brain using mRNA in situ hybridization and under hypoxic stress in cultured neuronal cell lines (PC12, HN33) by quantitative RT-PCR. While neuroglobin mRNA expression was significantly enhanced in cell culture after severe prolonged hypoxia (0-1% O2 for 24 h), we did not find any significant increases in neuroglobin mRNA levels in the rat brain after transient global ischemia. Vegf and Glut1 mRNAs showed increases in the hippocampus as expected. Therefore, it is unlikely that neuroglobin is instrumental in the acute response of neurons to hypoxic or ischemic insults, for which the mammalian brain is not adapted.

Neuroglobin and cytoglobin in search of their role in the vertebrate globin family.

Neuroglobin and cytoglobin are two recent additions to the family of heme-containing respiratory proteins of man and other vertebrates. Here, we review the present state of knowledge of the structures, ligand binding kinetics, evolution and expression patterns of these two proteins. These data provide a first glimpse into the possible physiological roles of these globins in the animal's metabolism. Both, neuroglobin and cytoglobin are structurally similar to myoglobin, although they contain distinct cavities that may be instrumental in ligand binding. Kinetic and structural studies show that neuroglobin and cytoglobin belong to the class of hexa-coordinated globins with a biphasic ligand-binding kinetics. Nevertheless, their oxygen affinities resemble that of myoglobin. While neuroglobin is evolutionarily related to the invertebrate nerve-globins, cytoglobin shares a more recent common ancestry with myoglobin. Neuroglobin expression is confined mainly to brain and a few other tissues, with the highest expression observed in the retina. Present evidence points to an important role of neuroglobin in neuronal oxygen homeostasis and hypoxia protection, though other functions are still conceivable. Cytoglobin is predominantly expressed in fibroblasts and related cell types, but also in distinct nerve cell populations. Much less is known about its function, although in fibroblasts it might be involved in collagen synthesis.

Neuroglobin, cytoglobin, and a novel, eye-specific globin from chicken.

Neuroglobin and cytoglobin are two recently discovered respiratory proteins of vertebrates. Here we report the first identification and expression analyses of these proteins in bird species. Neuroglobin from the domestic chicken Gallus gallus differs in approximately 30% from the mammalian proteins, but its genome structure shows the conservation of the B12.2, E11.0, and G7.0 intron positions. The chicken cytoglobin protein is shorter than the mammalian orthologs, from which it differs overall by approximately 25%, due to the absence of the C-terminal exon in the gene. Comparison of chicken and mammalian gene order shows that neuroglobin and cytoglobin are located on conserved syntenic chromosomal segments. While neuroglobin is expressed in the chicken's brain and eye, cytoglobin RNA was detected in all investigated tissues. In addition, a novel globin-type has been identified that is only expressed in the chicken's eye. The gene of this eye-globin contains the typical globin introns at B12.2 and G7.0. Phylogenetic analyses suggest that this globin is most closely related to the cytoglobin lineage. Although the function of this eye-globin remains presently uncertain, it adds an additional diversity to the vertebrate globin family.

Zebrafish reveals different and conserved features of vertebrate neuroglobin gene structure, expression pattern, and ligand binding.

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.

Neuroglobin and cytoglobin: genes, proteins and evolution.

Hemoglobin and myoglobin are oxygen transport and storage proteins of most vertebrates. Neuroglobin (Ngb) and cytoglobin (Cygb)--two recent additions to the vertebrate globin superfamily--have still disputed functions. Combining the data from all available resources, we investigate the evolution of these novel globins. Both Ngb and Cygb show little sequence variation in vertebrate evolution, suggesting conserved structures and functions, and an important role in the animal's metabolism. Exon-intron patterns remained unchanged in Ngb and Cygb, with the exception of the addition of a 3' exon to Cygb early in mammalian evolution. In phylogenetic analyses, Ngb forms a common branch with globin X, another recently identified globin with undefined function in lower vertebrates, and with some invertebrate nerve globins. This shows an early divergence of this branch in animal evolution. Cygb is related to myoglobin, and associated with an eye-specific globin from birds. The pattern of globin evolution shows that proteins with clear respiratory roles evolved independently from intracellular globins with uncertain functions. This result suggests either multiple independent functional changes or a yet undefined respiratory role of tissue globins like Ngb and Cygb.

Type I keratin cDNAs from the rainbow trout: independent radiation of keratins in fish.

Five different type I keratins from a teleost fish, the rainbow trout Oncorhynchus mykiss, have been sequenced by cDNA cloning and identified at the protein level by peptide mass mapping using MALDI-MS. This showed that the entire range of type I keratins detected biochemically in this fish has now been sequenced. Three of the keratins are expressed in the epidermis (subtype Ie), whereas the other two occur in simple epithelia and mesenchymal cells (subtype Is). Among the Is keratins is an ortholog of human K18; the second Is polypeptide is clearly distinct from K18. We raised a new monoclonal antibody (F1F2, subclass IgG1) that specifically recognizes trout Is keratins, with negative reactions on zebrafish. A phylogenetic tree has been constructed from a multiple alignment of the rod domains of the new sequences together with type I sequences from other vertebrates such as shark, zebrafish, and human; a recently sequenced lamprey Is keratin was applied as outgroup. This tree shows one branch defining the K18 orthologs and a second branch containing all other type I keratins (mostly subtype Ie). Within this second branch, the teleost keratins form a separate, highly bootstrap-supported twig. This tree leaves little doubt that the teleost Ie keratins diversified independently from the mammalian Ie keratins.

Type II keratin cDNAs from the rainbow trout: implications for keratin evolution.

From a teleost fish, the rainbow trout Oncorhynchus mykiss, we have cloned and sequenced cDNAs encoding five different type II keratins. The corresponding protein spots, as separated by 2D-PAGE of trout cytoskeletal preparations, have been identified by peptide mass mapping using MALDI mass spectrometry. Three of the sequenced keratins are expressed in the epidermis (subtype IIe), and two in simple epithelia and mesenchymal cells (subtype IIs). The IIs keratins are both orthologs of human K8. This leaves unsequenced only the trace component S3 of the biochemically established trout keratin catalog. A phylogenetic tree has been constructed from a multiple alignment of the rod domains of the new keratin sequences together with type II sequences from other vertebrates such as shark, zebrafish, and human; lamprey K8 (recently sequenced in our laboratory) has been used as outgroup. This tree suggests, in a highly bootstrap-supported manner, that the teleost IIe keratins diversified independently from the mammalian IIe keratins. In contrast, all the species investigated express K8-like keratins, suggesting that the different IIe branches evolved from K8-like progenitors. The tree also indicates that the published zebrafish sequences represent IIe keratins and that the biochemically identified K8 ortholog in zebrafish has not yet been sequenced.