Bettina Ebner

Interspecies comparison of neuroglobin, cytoglobin and myoglobin: Sequence evolution and candidate regulatory elements

Neuroglobin and cytoglobin are two novel members of the vertebrate globin family. Their physiological role is poorly understood, although both proteins bind oxygen reversibly and may be involved in cellular oxygen homeostasis. Here we investigate the selective constraints on coding and non-coding sequences of the neuroglobin and cytoglobin genes in human, mouse, rat and fish. Neuroglobin and cytoglobin are highly conserved, displaying very low levels of non-synonymous nucleotide substitutions. An oxygen supply function predicts distinct modes of gene regulation, involving hypoxia-responsive transcription factors. To detect conserved candidate regulatory elements, we compared the neuroglobin and cytoglobin genes in mammals and fish. The myoglobin gene was included to test if it also contains hypoxia-responsive regulatory elements. Long conserved non-coding sequences, indicative of gene-regulatory elements, were found in the cytoglobin and myoglobin, but not in the neuroglobin gene. Sequence comparison and experimental data allowed us to delimit upstream regions of the neuroglobin and cytoglobin genes that contain the putative promoters, defining candidate regulatory regions for functional tests. The neuroglobin and the myoglobin genes both lack conserved hypoxia-responsive elements (HREs) for transcriptional activation, but contain conserved hypoxia-inducible mRNA stabilization signals in their 3′ untranslated regions. The cytoglobin gene, in contrast, harbors both conserved HREs and mRNA stabilization sites, strongly suggestive of an oxygen-dependent regulation.

Evolution of the Globin Gene Family in Deuterostomes: Lineage-Specific Patterns of Diversification and Attrition

In the Metazoa, globin proteins display an underlying unity in tertiary structure that belies an extraordinary diversity in primary structures, biochemical properties, and physiological functions. Phylogenetic reconstructions can reveal which of these functions represent novel, lineage-specific innovations, and which represent ancestral functions that are shared with homologous globin proteins in other eukaryotes and even prokaryotes. To date, our understanding of globin diversity in deuterostomes has been hindered by a dearth of genomic sequence data from the Ambulacraria (echinoderms + hemichordates), the sister group of chordates, and the phylum Xenacoelomorpha, which includes xenoturbellids, acoelomorphs, and nemertodermatids. Here, we report the results of a phylogenetic and comparative genomic analysis of the globin gene repertoire of deuterostomes. We first characterized the globin genes of the acorn worm, Saccoglossus kowalevskii, a representative of the phylum Hemichordata. We then integrated genomic sequence data from the acorn worm into a comprehensive analysis of conserved synteny and phylogenetic relationships among globin genes from representatives of the eight lineages that comprise the superphylum Deuterostomia. The primary aims were 1) to unravel the evolutionary history of the globin gene superfamily in deuterostomes and 2) to use the estimated phylogeny to gain insights into the functional evolution of deuterostome globins. Results of our analyses indicate that the deuterostome common ancestor possessed a repertoire of at least four distinct globin paralogs and that different subsets of these ancestral genes have been retained in each of the descendant organismal lineages. In each major deuterostome group, a different subset of ancestral precursor genes underwent lineage-specific expansions of functional diversity through repeated rounds of gene duplication and divergence. By integrating results of the phylogenetic analysis with available functional data, we discovered that circulating oxygen-transport hemoglobins evolved independently in several deuterostome lineages and that intracellular nerve globins evolved independently in chordates and acoelomorph worms.

Assessing divergence time of Spirulida and Sepiida (Cephalopoda) based on hemocyanin sequences

The phylogenetic position of the mesopelagic decabrachian cephalopod Spirula is still a matter of debate. Since hemocyanin has successfully been used to calibrate a molecular clock for many molluscan species, a molecular clock was calculated based on this gene with special attention to the cephalopod genera Spirula and Sepia. The obtained partial sequence comprising ca., one third (3567 bp) of the complete gene is similar to that of Sepia officinalis. The molecular clock was calibrated using the splits of Gastropoda-Cephalopoda (ca. 550 ± 50 mya) and Heterobranchia-Vetigastropoda (ca. 380 ± 10 mya). The resulting hemocyanin-based molecular clock is stable, and the estimated divergence time of Spirulida and Sepiida, some 150 ± 30 million years ago, can be deemed reliable.

The Nerve Hemoglobin of the Bivalve Mollusc Spisula solidissima MOLECULAR CLONING, LIGAND BINDING STUDIES, AND PHYLOGENETIC ANALYSIS

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (μm). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mm), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P50 ∼0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons.

cDNA Sequences of Two Arylphorin Subunits of an Insect Biliprotein: Phylogenetic Differences and Gene Duplications during Evolution of Hexamerins—Implications for Hexamer Formation

Arylphorins represent a conserved class of hexameric ∼500 kDa insect hemolymph glycoproteins, rich in aromatic amino acids, which are produced in large quantities at the larval stage as reserves for metamorphosis and egg development. The recently isolated arylphorin from the moth Cerura vinula is unique in being complexed to a novel farnesylated bilin. Protein sequencing suggested the presence of two different ∼85 kDa subunits. Here, we report the complete coding sequences of two cDNAs encoding two arylphorins subunits with 67% identity and calculated physicochemical characteristics in agreement with the isolated holoprotein. Our phylogenetic analyses of the hexamerins revealed monophyletic origins not only for each of the arylphorins and methionine-rich proteins (H-type and M-type), the two major classes of hexamerins, but also for the minor groups of arylphorin-like and riboflavin-binding hexamerins. We named the latter proteins X-type (mixed type) hexamerins because they share sequence features with both major groups, and they show unique deletions and insertions at conserved sites located on the protein surface. We present a phylogenetic tree of lepidopteran hexamerins, which is in agreement with actual systematics. Overall, duplications of hexamerin genes occurred independently in several lepidopteran lineages. We also analyzed the hexamerin sequences for key parameters, which characterize each type of hexamerins. Based on the crystal structure of the homomeric arylphorin from Antheraea pernyi, we present a model for the heteromeric Cerura protein focusing on the role of N-glycan structures in stabilizing the hexamer structure.