Function and evolution of vertebrate globins.

Acta Physiol 2014, 211, 501–514

INVITED REVIEW

Function and evolution of vertebrate globins T. Burmester1 and T. Hankeln2 1 Institute of Zoology and Zoological Museum, University of Hamburg, Hamburg, Germany 2 Institute of Molecular Genetics, Johannes Gutenberg-University Mainz, Mainz, Germany

Received 17 March 2014, revision requested 17 April 2014, revision received 25 April 2014, accepted 30 April 2014 Correspondence: T. Burmester, Institute of Zoology and Zoological Museum, Biocenter Grindel, University of Hamburg, MartinLuther-King-Platz 3, D-20146 Hamburg, Germany. E-mail: thorsten.burmester @uni-hamburg.de

Abstract Globins are haem-proteins that bind O2 and thus play an important role in the animal’s respiration and oxidative energy production. However, globins may also have other functions such as the decomposition or production of NO, the detoxification of reactive oxygen species or intracellular signalling. In addition to the well-investigated haemoglobins and myoglobins, genome sequence analyses have led to the identification of six further globin types in vertebrates: androglobin, cytoglobin, globin E, globin X, globin Y and neuroglobin. Here, we review the present state of knowledge on the functions, the taxonomic distribution and evolution of vertebrate globins, drawing conclusions about the functional changes underlying present-day globin diversity. Keywords androglobin, cytoglobin, haemoglobin, myoglobin, neuroglobin, oxygen.

The oxygen-dependent (aerobic) metabolism allows for a highly efficient extraction of energy from food sources and thus the evolution of complex life forms. During the Cambrian explosion large animals emerged, making oxygen supply to the inner organs by simple diffusion inefficient. In addition to a circulatory system and specialized respiratory organs, proteins evolved that enhanced oxygen supply by transporting and/or storing oxygen. Three types of such respiratory proteins exist. Hemocyanins, which occur in the haemolymph of many molluscan and arthropod species, are phenoloxidase-related proteins and use copper to bind oxygen (Burmester 2002, Markl 2013). Hemerythrins are small non-haem diiron proteins that are restricted to some protostome lineages (annelids, sipunculids, branchiopods, priapulids) and bacteria, but absent in deuterostomes (Kurtz 1999, Bailly et al. 2008). (Haemo-)globins are the most widespread respiratory proteins, occurring in vertebrate and invertebrate animals, but also in prokaryotes, fungi, protozoa and plants (Hardison 1998, Weber & Vinogradov 2001, Vazquez-Limon et al. 2012, Vinogradov et al. 2013a).

Globins are small metalloproteins that typically comprise around 150 amino acids, but may also have N- and/or C-terminal extensions. Most globins cover eight a-helical segments (named A through H) with a characteristic 3-over-3 a-helical sandwich structure (referred to as globin fold) and include a haem prosthetic group (Fe2+-protoporphyrin IX), by which they reversibly bind oxygen, and in some cases, also other ligands (Perutz 1979, Bolognesi et al. 1997). While their overall structures are conserved, globin primary sequences often are not. In fact, only the proximal histidine (position F8; i.e. 8th amino acid in helix F) adjacent to the Fe2+ is present in all globins, and most of them also show a phenylalanine in inter-helical region CD1, which stabilizes the haem. There is little doubt that globins are among the bestinvestigated proteins in biological and medical sciences. Globins also represent a prime tool for the studies of the function and evolution of genes and proteins (Kendrew 1963, Goodman et al. 1975, 1987, 1989, Dickerson & Geis 1983, Ohta & Dover 1983, Perutz 1983, Hardison 1996, 1998, Wajcman et al. 2009). For about a century, haemoglobin (Hb) and myoglobin

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12312

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Vertebrate globin evolution

· T Burmester and T Hankeln

(Mb) had been considered the only globin types of vertebrates. However, recent findings suggest an unprecedented diversity of the vertebrate globin family. Here we review the current state of knowledge of the occurrence, function and evolution of vertebrate globins. The genomic perspective of vertebrate globin evolution has recently been reviewed by Storz et al. (2013).

The vertebrate globin family has (at least) eight members with distinct features In addition to Hb and Mb, six other globin types have been identified in vertebrates: Neuroglobin (Ngb) (Burmester et al. 2000), cytoglobin (Cygb) (Kawada et al. 2001, Burmester et al. 2002, Trent & Hargrove 2002), globin X (GbX) (Roesner et al. 2005), globin Y (GbY) (Fuchs et al. 2006), eye-globin or globin E (GbE) (Kugelstadt et al. 2004) and androglobin (Adgb) (Hoogewijs et al. 2012). While Hb, Mb, Ngb, Cygb and Adgb are widespread and may be present in most gnathostome (jawed) vertebrates (Burmester et al. 2004, Hoogewijs et al. 2012, Schwarze & Burmester 2013), the GbE, GbX and GbY types are

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restricted to certain taxa (Fig. 1). With the exception of Adgb, which is quite distinct in several aspects (see below), the globin fold and its general features are conserved among vertebrate globins. The most conspicuous difference found relates to the binding scheme of the iron atom of the haem. In the deoxygenated state, Hb, Mb and GbE are penta-coordinated with the sixth binding site of the Fe2+ being empty, while Ngb, GbX, Adgb and Cygb are hexa-coordinated, with the distal amino acid of the protein chain (histidine or glutamine in E7) bound to the Fe2+. No data exist on the coordination status of GbY. The physiological consequences of hexa- vs. penta-coordination are largely unknown, but hexa-coordination renders the binding of gaseous ligands less sensitive to external factors such as temperature (Uzan et al. 2004). Moreover, in hexa-coordinated globins, the binding ligand must displace the distal histidine, thereby triggering slight conformational changes of the protein, which may be instrumental in sensing processes (De Sanctis et al. 2004). While Hb and Mb are certainly best known for their respiratory functions, thus supplying adequate amounts

Figure 1 Distribution of globins in vertebrates. Data were compiled from data bases of genome and transcriptome sequences. The N- and C-terminal extensions of some globins are indicated by bars. Hexagons indicate hexa-coordination, pentagons symbolize penta-coordination. The N-terminal acylation and membrane-association of GbX are indicated by a membrane sketch. Note that haem iron-coordination of GbY is yet undetermined. Note also that some icefishes lack Hb, Mb or both, while other ray-finned fishes have duplicated Cygb and/or Mb genes.

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of O2 for aerobic energy production (Dickerson & Geis 1983, Weber & Vinogradov 2001, Wittenberg & Wittenberg 2003), in recent years various other, additional globin functions have been proposed (Fig. 2). These include roles in nitric oxide (NO) metabolism, the detoxification of reactive oxygen species (ROS), protection from apoptosis, signal transduction, lipid metabolism and, possibly, hydrogen sulphide (H2S) turnover (Hankeln et al. 2005, Burmester & Hankeln 2009, Rios-Gonzalez et al. 2014). Many of these potential new functions have only been demonstrated in vitro and still lack rigorous proof in vivo.

Haemoglobin transports oxygen and is involved in nitric oxide metabolism The red colour of the vertebrate blood results from the high amounts of Hb in the erythrocytes. Hb

Figure 2 Postulated functions of vertebrate globins. (a) Hb transports O2 in the blood, (b) Mb and GbE (and possibly Ngb) enhance intracellular O2 supply to the mitochondria, (c) Hb, Mb, Cygb and Ngb may convert NO to NO3 at high PO2 and NO2 to NO at low PO2, (d) Ngb, Cygb and Mb may detoxify reactive oxygen or nitrogen species, (e) GbX may be a component of a membrane-mediated signal transduction chain, (f) Cygb may trigger a lipid-signalling cascade, (g) Ngb may prevent hypoxia-induced apoptosis via reduction of cytochrome c or (h) may act as signal protein by inhibiting the dissociation of GDP from Ga. Note that no exact stoichiometry is given in the schemes.

· Vertebrate globin evolution

transports O2 from the respiratory surfaces (usually lungs, gills or skin) to the inner organs via the circulatory system (Fig. 2a; Dickerson & Geis 1983). In the venous blood, deoxygenated Hb also carries CO2, which is bound the protein chain, for release at the respiratory surfaces. The Hbs of Gnathostomata (jawed vertebrates) are hetero-tetramers composed of two a- and two b-type chains. The interaction between the subunits decreases the O2-affinity of Hb, which is significantly lower than that of globin monomers, and enables cooperative O2-binding (Berg et al. 2006). Different subtypes of a- and b-chains have emerged independently by gene duplication in the gnathostome classes. Subtypes usually carry out different functions, e.g. increasing O2 affinity of Hb in embryos (Brittain 2002). O2-binding properties of native Hb may be additionally modulated by temperature, pH (Bohr effect), CO2 and organic phosphate compounds

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(ATP, GTP, inositol pentaphosphate or 2,3-diphosphoglycerate) (Berg et al. 2006). According to the current knowledge, Hb occurs in essentially all vertebrates, with the exception of some icefish species (Sidell & O’Brien 2006). It appears that the cold habitat and the sluggish lifestyle render Hb unnecessary in icefishes. On the other hand, well-elaborated examples demonstrate the potential of Hb evolution in adapting animals to conquer physiologically challenging habitats, e.g. at high altitude (Weber 2007, Storz et al. 2009, Projecto-Garcia et al. 2013), in underground burrows (Avivi et al. 2010, Campbell et al. 2010b) or in cold climates (Campbell et al. 2010a). Another important role of vertebrate Hb relates to the nitric oxide (NO) metabolism. Hb has enzymatic activity and, as oxy-Hb, can scavenge excess toxic NO and convert it to nitrate or, as deoxy-Hb, can produce NO from a circulating reservoir of nitrite (Fig. 2c). NO release, in turn, triggers blood vessel dilatation under temporary hypoxia, having profound biomedical implications (Cosby et al. 2003, Shiva et al. 2011). An additional twist to Hb’s scientific story are the still rather anecdotal reports of the protein’s expression in non-erythroid cell types like neurons (Richter et al. 2009, Russo et al. 2013), macrophages (Liu et al. 1999), lens tissue (Wride et al. 2003), alveolar epithelial cells (Newton et al. 2006), endothelial cells (Straub et al. 2012), kidney cells (Nishi et al. 2008) and cancer cells (Li et al. 2013). The functional implications of ectopic Hb expression are unclear but have been related to ROS defence (Nishi et al. 2008) or regulation of NO signalling (Straub et al. 2012). Notably, the Hbs of Gnathostomata (jawed vertebrates) and Agnatha (jawless vertebrates; i.e. hagfishes and lampreys) are functionally similar, but structurally distinct. Agnathan Hbs are monomers in the oxygenated state and polymerize to dimers or tetramers when deoxygenated (Fago et al. 2001). Moreover, ligand binding cooperativity and allosteric regulation differ mechanistically between agnathan and vertebrate Hb (Hoffmann et al. 2010a). This clearly indicates a convergent evolution of oxygen transport function from distinct globin ancestors (see below).

Myoglobin supplies oxygen to muscle cells, but is more versatile than previously thought Mb is a monomer that was initially thought to be restricted in the striated muscle (skeletal muscle and heart) (Wittenberg & Wittenberg 2003). More recent evidence shows a more widespread expression of Mb and its presence, e.g. in smooth muscle, endothelial and tumour cells, albeit at much lower concentrations (Qiu et al. 1998, Cossins et al. 2009, Gorr et al. 504

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2011) (see also below). The O2 affinity of a typical Mb (~1 Torr = 0.14 kPa) is higher than that of Hb, allowing the extraction of O2 from the blood. Within myocytes, Mb enhances O2 supply by facilitating the diffusion to the mitochondria or by storing O2 for short-term or long-term hypoxic periods. In the muscles of diving mammals, Mb may accumulate to extraordinarily high concentrations, sustaining extended dives (Davis 2014). Lineages of diving mammals have convergently evolved Mb sequences with elevated net surface charges, which increase electrostatic repulsion and thereby could have facilitated the accumulation of high Mb amounts without deleterious self-aggregation (Mirceta et al. 2013). Oxygenated Mb is also instrumental in the decomposition of toxic nitric oxide (NO), present in cells of high metabolic activity, by its dioxygenase enzyme activity (Fl€ ogel et al. 2001). On the other hand, deoxy-Mb may act as a nitrite reductase producing NO from NO2 in response to cellular hypoxia (Hendgen-Cotta et al. 2008). In smooth muscles of the vascular system, Mb thus mediates hypoxic vasodilatation independent of nitric oxide synthase (NOS) action, having profound biomedical implications for the treatment of microvascular dysfunction (Totzeck et al. 2012, Hendgen-Cotta et al. 2014). In vitro data suggest that yet another additional function of oxy-Mb may reside in its interaction with (and possible intracellular transport of) fatty acids, which could be metabolically relevant under oxygenated conditions at high energy demand (Shih et al. 2014). Most vertebrate species possess only a single Mb gene in the genome. More recently, however, two Mb isoforms with different tissue expression spectra have been detected in cyprinid fishes (Fraser et al. 2006, Roesner et al. 2008). While one Mb copy (#2) is specific to brain neurons, the Mb gene #1 displays a rather widespread expression in muscles, but also various tissues of cyprinids (Fraser et al. 2006, Cossins et al. 2009) and other ray-finned fishes (Tiedke et al. 2014). Recent in vitro data suggest that Mb #1 may exert its standard roles in O2 supply and NO production, while Mb #2 is specifically active in eliminating H2O2 and could thus function in protecting neurons from ROS in the hypoxia-adapted cyprinids (Helbo et al. 2012). Surprisingly, Mb-knockout mice show no immediate physiological defects (Garry et al. 1998), but display several physiological compensatory mechanisms ensuring survival (G€ odecke et al. 1999). Some vertebrate species in fact survive without functional Mb: While frogs entirely lack the Mb gene (Maeda & Fitch 1982, Fuchs et al. 2006), some icefish species and the stickleback have non-functional Mb copies (Hoffmann et al. 2011). It has been speculated that in the frog heart the Mb loss is functionally compensated


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for by an Hb chain (Maeda & Fitch 1982) or even by Cygb (Xi et al. 2007). A biomedically important finding is the detection of Mb mRNA and protein in human epithelial cells and the pronounced up-regulation of Mb expression in tumour cells, e.g. of breast, colon and prostate (Flonta et al. 2009, Gorr et al. 2011, Kristiansen et al. 2011). In the tumour context, Mb shows a specific, highly complex pattern of gene regulation different from the one in muscle cells (Bicker et al. 2014). Interestingly, high Mb expression levels correlate with a beneficial prognosis for breast cancer patients (Kristiansen et al. 2011). The biological function of Mb in normal epithelia and its potential suppressive effect in tumour cells is unknown, requiring further research.

Neuroglobin is a nerve globin, whose function is still uncertain Ngb was initially identified as a neuron-expressed globin (Burmester et al. 2000). It is phylogenetically ancient (see below) and apparently occurs in most vertebrates, although genome data suggest absence in Agnatha and Chondrichthyes. Ngb is a monomer with about 150 amino acids that binds reversibly to O2 with an affinity in the range of a typical Mb (half saturation pressure P50 = 0.9 to 2.2 Torr (0.12–0.29 kPa) (Burmester et al. 2000, Dewilde et al. 2001). Despite ~14 years of research, the true function of Ngb is still a matter of intense debate (Hankeln et al. 2005, Burmester & Hankeln 2009). Ngb is predominantly expressed in neurons of the central and peripheral nervous systems and in endocrine tissues (Reuss et al. 2002, Laufs et al. 2004). The relative intensity of expression in different regions of the mammalian brain is controversially discussed (Hankeln et al. 2004, Hundahl et al. 2012b) and additional sites of ectopic Ngb expression, e.g. in hematopoietic stem cells (D’Aprile et al. 2014) and tumour tissue (Emara et al. 2014) have been proposed. We like to point out that rigorous quantitative studies of regional Ngb gene expression, including intracellular protein levels as a pivotal clue to understand its physiological function(s), are still missing. On the subcellular level, there is conclusive evidence that Ngb is associated with the mitochondria and therefore the oxidative metabolism (Bentmann et al. 2005, Mitz et al. 2009). It has also been proposed that – at least in the retina – a proportion of Ngb protein resides within the mitochondria (Lechauve et al. 2012, Yu et al. 2012), thereby possibly interacting with the respiratory chain (Lechauve et al. 2012, 2013). An astonishingly wide collection of possible molecular functions of Ngb has been proposed (Fig. 2). Conceivable roles of Ngb include local or temporal O2 supply or protection from ROS (Burmester &



Hankeln 2009). In fact, levels of Ngb are positively correlated with the hypoxia-tolerance of particular animal species (Roesner et al. 2008, Avivi et al. 2010, Schneuer et al. 2012). In this context, it has been suggested that Ngb serves as a general neuroprotective protein (Sun et al. 2001, Khan et al. 2006, Greenberg et al. 2008, Raida et al. 2013), a viewpoint that also has received criticism (Schmidt-Kastner et al. 2006, Kelsen et al. 2008, Di Pietro et al. 2014). Ngb may also be involved in the detoxification of harmful NO (Brunori et al. 2005) or act as a nitrite reductase in the production on NO to temporarily inhibit and protect mitochondrial respiration (Tiso et al. 2011). It has been suggested that Ngb functions as a Ga protein GDP dissociation inhibitor in mouse, but not zebrafish (Wakasugi et al. 2003, 2011), and that it inhibits apoptosis by reduction of cytochrome c released from the mitochondria (Fago et al. 2006, Raychaudhuri et al. 2010). Recently, even a function of Ngb in neurite development via interference with the PTEN/Akt pathway was proposed (Li et al. 2014). While many of such studies reflect in vitro results, Ngb knock-out mice have provided surprisingly little clues so far: Upon hypoxia, the expression of c-FOS and HIF1A was enhanced (Hundahl et al. 2011), the behavioural response to light was altered (Hundahl et al. 2012a) and, rather surprisingly, the infarct size was reduced in the cerebral cortex of knock-out animals after experimental stroke (Raida et al. 2012), clearly contradicting a protective function. The lack of a pronounced hypoxic up-regulation – at least in mammals – may also argue against a general function of Ngb in acute stress response (Burmester et al. 2007). To elucidate the role(s) of Ngb, it will be necessary to unify detailed quantitative expression information (if possible at single-cell levels, and distinguishing between highly and lowly expressing cell types) and phenotypes preferentially obtained from in vivo models.

Cytoglobin is a tissue globin with roles in NO metabolism, fibrosis and tumourigenesis Cygb was independently discovered by three groups (Kawada et al. 2001, Burmester et al. 2002, Trent & Hargrove 2002) and occurs in all vertebrates studied so far, including lampreys (K. Schwarze and T. Burmester, unpublished data). Recombinant Cygb is a dimer (Sugimoto et al. 2004) with an O2 affinity in the Mb-like range of 1 Torr (0.14 kPa) (Trent & Hargrove 2002). Most studies agree that Cygb is present in fibroblast-related cell lineages as well as in distinct neurons (Nakatani et al. 2004, Schmidt et al. 2004, Motoyama et al. 2014). With regard to the additional presence of Cygb in many other cell types including epithelia, macrophages, muscles and diverse tumour


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entities (reviewed by (Oleksiewicz et al. 2011), a general cautionary note on standards of antibody validation and on the lack of cell-type specific quantitative expression data should be made. While Cygb is a cytoplasmic protein in fibroblasts, it appears to reside in both the nucleus and the cytoplasm in neurons (Schmidt et al. 2004). This observation hints to distinct functions in neuronal versus non-neuronal cells and to an intracellular transport of the protein. The presence of two distinct Cygb gene copies in fish species is in line with the hypothesis of a dual function (Fuchs et al. 2005). The fish Cygb genes have evolved by gene duplication and display complementary, nonoverlapping expression patterns. There is no correlation of Cygb with O2 consumption, rendering a respiratory role of Cygb unlikely (Hankeln et al. 2005, Schmidt et al. 2005). It has been speculated that Cygb may supply O2 to collagen synthesis (Schmidt et al. 2004), in augmenting O2-consuming NO synthase (Hankeln et al. 2005), in a lipid-based signalling process (Reeder et al. 2011), or in more general ROS protection (Xu et al. 2006, Li et al. 2007, Hodges et al. 2008, Fang et al. 2011, Singh et al. 2014). The colocalization of Cygb and nNOS in brain neurons (Avivi et al. 2010, Hundahl et al. 2013) might in fact argue for the involvement of Cygb in NO metabolism in this cell type. Special attention has been paid to the role of Cygb in diverse human malignancies. Results so far demonstrate a tumour-suppressor activity of Cygb, e.g. in lung, oesophagus, head and neck, breast, bladder and colon (Oleksiewicz et al. 2011), but also a potential tumour-promoting role in cells under stress (Oleksiewicz et al. 2013). The mechanistic basis of this complex, possibly cell-type-specific phenotype is not understood. Cygb is augmented in fibrotic liver, pancreas and kidney, showing a damage-reducing phenotype possibly by reducing oxidative stress (Xu et al. 2006). Moreover, Cygb knockout mice were recently reported showing defects in muscle regeneration and repair (Singh et al. 2014).

Globin E is an eye-specific respiratory protein The eye-specific globin GbE was first found in chicken (Kugelstadt et al. 2004) and later discovered in other bird genomes (Blank et al. 2011a, Hoffmann et al. 2011, Storz et al. 2011). Initially, GbE has been suggested to be a bird-specific protein, while recent data identified orthologous GbE genes in the coelacanth Latimeria chalumnae (Schwarze & Burmester 2013) and in turtles (Kim Schwarze and Thorsten Burmester, unpublished data). Together with the phylogenetic analyses this proves that GbE originated early in 506

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vertebrate evolution. Immunohistochemical studies showed that GbE is highly expressed in the chicken’s eye (~10 lM GbE protein in total eye tissue) and is preferentially located in the outer segments of the photoreceptor cells (Blank et al. 2011a). GbE binds O2 with a somewhat lower affinity than Mb and displays slow autoxidation. GbE is phylogenetically related to Mb (Blank et al. 2011a, Hoffmann et al. 2011, Storz et al. 2011, 2013) and may have a similar function in O2 supply to the metabolically very active retina (Blank et al. 2011a).

Globin Y is developmentally regulated, but still an enigma The structure of GbY shows a typical globin. Initially identified in Xenopus (Fuchs et al. 2006), more recent studies identified orthologues of GbY in other species, such as the platypus (Ornithorhynchus anatinus), the lizard Anolis carolinensis, the elephant shark Callorhinchus milii, the coelacanth and turtles (Patel et al. 2008, Hoffmann et al. 2010a, 2011, Schwarze & Burmester 2013) (Fig. 1). No GbY genes were found in the genomes of ‘higher mammals’ (Placentalia and Marsupialia), birds or ray-finned fishes (Actinopterygii). In adult Xenopus, GbY shows strongest mRNA expression in ovary, kidney, eye and lung (Fuchs et al. 2006). During development, transcription peaks during organogenesis and metamorphosis (C. Fuchs, T. Burmester and T. Hankeln, unpublished data). However, there are still too few data to speculate about GbY’s function.

Globin X is a membrane-bound globin in the nervous system GbX genes have been identified in many metazoan animals (Roesner et al. 2005, Blank & Burmester 2012). An additional paralogous GbX-like gene is restricted to invertebrates, including hemichordates and cephalochordates (Blank & Burmester 2012, Hoffmann et al. 2012a). In vertebrates, GbX occurs in lampreys, sharks, ray-finned fishes, coelacanth, amphibians and reptiles, but has not been found in birds and mammals (Roesner et al. 2005, Fuchs et al. 2006, Blank & Burmester 2012). In zebrafish, GbX is expressed in parts of the CNS that may be associated with the sensory system (Blank et al. 2011b). GbX sequences are longer than those of a typical globin (~200 amino acids). It differs from other globins in carrying N-terminal acylation sites (myristoylation at Gly2 and palmitoylation at Cys3), by which GbX binds to the membrane (Blank et al. 2011b). Thus, GbX may be involved in the protection of membrane lipids or a membrane-related cellular signalling process, although the exact function is unknown (Blank


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et al. 2011b). It can further be speculated that the convergent loss of GbX in birds and mammals may be related to their endothermic lifestyle, which provides a constant inner environment to the nervous system, which might render the GbX function unnecessary.

Androglobin is an ancestral chimeric globin type in the testis Most recently, a family of chimeric proteins with an N-terminal calpain-like domain, an internal globin domain, and an IQ calmodulin-binding motif was discovered (Hoogewijs et al. 2012). In humans and mice, this protein comprises around 1600 amino acids and is predominantly expressed in testis (therefore ‘androglobin’; Adgb). There is evidence for an increased gene expression in fertile compared to infertile males, suggesting a role in reproduction (Hoogewijs et al. 2012). The globin domain satisfies the criteria of a globin-like fold, but is split into two parts and permutated, with helices C to H followed by helices A and B. Recombinant human ADGB globin (comprising helices C to H) exhibits an absorption spectrum characteristic of hexa-coordination. Adgb orthologues have been identified in the genomes of almost all Metazoan lineages and in choanoflagellates, suggesting an early origin before the radiation of animals. Notably, some model taxa (e.g. the nematode C. elegans and the fly D. melanogaster) lack Adgb.

The emergence of animal globins: three ancestral lineages The phylogenetic ancestry and their widespread occurrence in bacteria, fungi, plants, protists and animals



(Hardison 1996, 1998, Weber & Vinogradov 2001, Vazquez-Limon et al. 2012, Vinogradov et al. 2013a, b) demonstrate that globins are derived from a protein that was already present in the last common ancestor of all living organisms more than 1.5 billion years ago. The function of the first animal globin is unknown. However, metazoans and thus globins evolved in an oxygenated atmosphere and it can be assumed that they already had O2-binding properties. It is nevertheless unlikely that these proteins had a true respiratory function because the first animals were probably small and relied on O2 supply to the respiratory chain by diffusion. Thus, one may speculate that the last common ancestor of animal globins had rather a role in O2 sensing, served in the detoxification of ROS/RNS, or was another type of O2-dependent enzyme. Similar functions have actually been proposed for prokaryote globins (Freitas et al. 2005, Vinogradov et al. 2013a,b). Within animals, phylogenetic analyses agree that Ngb, GbX and Adgb represent major clades that are distinct from the other globin types, suggesting their early evolutionary origin (Fig. 3). In fact, Ngb, GbX and Adgb genes have been identified in both vertebrates and invertebrates, showing that they emerged before the separation of Protostomia and Deuterostomia (Burmester et al. 2000, Blank & Burmester 2012, Dr€ oge et al. 2012, Hoogewijs et al. 2012) (Fig. 3). The predecessors of Ngb, GbX and Adgb thus formed the minimal globin repertoire of the first animals, which can be taken as an indicator that they had distinct, non-overlapping function. Because of a lack of phylogenetic signal it must remain unclear whether Adgb, Ngb or GbX emerged first. Notably, all three globins are hexa-coordinated, which suggests that

Figure 3 Hypothesized evolution of animal globins. The tree represents a simplified version from Blank & Burmester (2012) and Hoogewijs et al. (2012). For globin symbols and abbreviations, see Fig. 1. Note that O2 transport function in the circulatory system arose convergently during globin gene evolution (arrows). © 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12312

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hexa-coordination was the original coordination state of the Fe2+ in the haem group of deoxy-globins. Penta-coordination, which is typical for most globins with respiratory roles like, e.g. Hb and Mb, is therefore the derived state that evolved later. GbX- and GbX-like proteins are widespread in Metazoa, including both Protostomia and Deuterostomia, and similar putatively N-acylated globins also occur in plants, brown algae and fungi (Blank & Burmester 2012). This may suggest an early origin of Nacylated globins, although these metazoan and nonmetazoan genes may be not orthologous. For zebrafish GbX, it has been conclusively demonstrated that Nacylation of GbX mediates membrane binding (Blank et al. 2011b), a location that rather precludes a function as a common respiratory protein. Rather GbX may protect membrane lipids from oxidation or may be involved in signalling, which is mediated by various other acylated membrane-bound proteins. Similar membrane-related functions have also been suggested for some prokaryote globins (Hou et al. 2001, Freitas et al. 2005). Thus, the respiratory globins of animals may actually derive from a membrane-bound ancestor whose function is mirrored today by GbX. Globins in the nervous system have been observed in various invertebrates (Wittenberg 1992), and it was suggested that these invertebrate nerve globins are orthologous to vertebrate Ngb (Burmester et al. 2000). In fact, phylogenetic analyses showed a close relationship of Ngb with the nerve-globin of the annelid Aphrodite aculeata. More recent analyses (Ebner et al. 2010, Blank & Burmester 2012) further identified a cephalochordate globin and a polymeric sea urchin globin as closely related and probably orthologous to Ngb, which confirms an early origin, but provides little clues to Ngb function. In contrast, the nerve-globin of the bivalve Spisula solidissima, which is thought to protect the surf clam from hypoxic damage during burrowing, is not related to Ngb, suggesting a convergent evolution of nerve-specific globins (Dewilde et al. 2006). This may also indicate that Ngb and unrelated nerve globins fulfil different physiological functions in nerve cells. Invertebrate globins display an enormous structural and functional diversity, which actually may exceed that of vertebrate globins (Weber & Vinogradov 2001). Protostome and deuterostome Hbs and Mbs are not monophyletic, but convergently evolved to respiratory function from distinct globin types (Blank & Burmester 2012). This may be interpreted in terms of a predisposition of globins to reversibly bind O2, which repeatedly allowed, after a few amino acid substitutions, the evolution of proteins that had the ability to transport or store O2 efficiently. The globins of invertebrate deuterostomes (hemichordates, tunicates 508

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and cephalochordates) add another level of complexity to globin evolution. While orthologues of Ngb, GbX and Adgb are mostly present, other globins form distinct taxon-specific clades that cannot be assigned to any other know globin type (Ebner et al. 2010, Blank & Burmester 2012, Hoffmann et al. 2012a, Storz et al. 2013). This pattern reflects a substantial degree of globin diversity in early deuterostomes, which still has to be explained in functional terms.

Evolution of globins within vertebrates The last common ancestor of vertebrates had at least four globins: Adgb, Ngb, GbX and a globin that eventually gave rise to gnathostome and agnathan Hbs and Mbs, Cygb, GbE and GbY (Fig. 3). This clade is monophyletic and emerged after the divergence of Protostomia and Deuterostomia (Fuchs et al. 2006, Ebner et al. 2010, Hoffmann et al. 2010a, 2011, Storz et al. 2011, Blank & Burmester 2012). There is conclusive phylogenetic and genomic evidence for a common clade of the Hb a- and b-genes, but otherwise the support values of the other clades are poor and various relationships have been proposed (Burmester et al. 2002, Gillemans et al. 2003, Hardison 2008, Patel et al. 2008, 2010, Hoffmann et al. 2010b, 2011, Blank et al. 2011a, Storz et al. 2011, 2013). However, recent combinations of phylogenetic, genetic and functional analyses provide some clues about the evolutionary events. Hbs of lampreys (Petromyzontiformes) and hagfishes (Myxiniformes) are similar (Fago et al. 2001), supporting the hypothesis that the Agnatha are monophyletic (Kuratani & Ota 2008). However, the agnathan and gnathostome Hbs are structurally distinct and there is conclusive evidence that they are of convergent origin (Hoffmann et al. 2010a). Agnathan Hbs may either be related to Cygb (Hoffmann et al. 2010a) or may be the sister group to a clade comprising gnathostome Hb, Mb, GbE and GbY (Blank & Burmester 2012). The latter view is tentatively supported by the identification of a Cygb gene in the sea lamprey Petromyzon marinus, which further indicates that Cygb diverged from the vertebrate globin clade before Agnatha and Gnathostomata separated (K. Schwarze and T. Burmester, unpublished data) (Fig. 3). This interpretation is in line with the hexacoordinate binding scheme of deoxy-Cygb, which, as explained above, can be considered as ancestral. Cygb displays a widespread expression in various cell types and diverged first, indicating the origin of the other vertebrate-specific globins from such a more general tissue-globin. Deoxy-Hb, Mb, GbE and the agnathan globins are penta-coordinated, the coordination state of GbY is unknown. Thus, it might be speculated that


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the conversion to the penta-coordinate state occurred after separation of the globin clade from Cygb. In addition to Adgb, GbX, Ngb and Cygb, the last common ancestor of jawed vertebrates (Gnathostomata) thus had a globin ancestor that subsequently gave rise to Hb, Mb, GbE and GbY. The duplication and diversification of these globin types took place in a relatively short time frame after the separation of Agnatha and Gnathostomata in the Cambrian or Precambrian period (~550 million years ago), and the separation of Chondrichthyes (sharks and rays) and Teleostomi (Osteichtyes and ‘higher’ vertebrates) some 450 million years ago. This might also explain the lack of resolution in gene phylogenies. The evolution of respiratory globins was probably accompanied and spurred by the rise in atmospheric O2 levels and the evolution of complex life forms, which rendered O2 supply by simple diffusion inefficient. In most fish, the principal site of high Mb expression is the heart, suggesting that this protein first evolved to sustain O2 supply to this organ, which is supplied in the fish’s circulatory system by O2-poor blood. Shared paralogues in the Mb gene loci of various tetrapods and the GbE region of the coelacanth support a relationship between these genes, which is supported by the recent molecular phylogenetic analyses (Hoffmann et al. 2011, 2012b, Schwarze & Burmester 2013). This is in line with the similar O2 binding characteristics and probably similar functions of Mb and GbE, albeit in different organs (Blank et al. 2011a). The reasons why GbE has only been retained in few lineages, but was lost in other vertebrates, are unknown. However, it may be speculated that morphological adaptations that have improved O2 supply to the retina (e.g. the emergence of vascularized retinae in mammals) rendered GbE unnecessary (Blank et al. 2011a). The position of GbY can currently not be resolved by phylogenetic methods, but the inclusion of GbY in the Hb cluster in various vertebrate genomes tentatively supports a close relationship of these proteins (Patel et al. 2008, 2010, Hoffmann et al. 2010b). However, it is unlikely that GbY may be an alternative Hb chain, although its main sites of expression and its function remain to be determined. Hb is destined for O2 transport in the erythrocytes, a function so crucial that it has required conservation of Hb genes in all vertebrates except icefish. All gnathostome Hbs consist of two a- and two b-chains, which diverged before the separation of Chondrichthyes and Teleostomi. Multiple different a- and b-genes occur in gnathostomes, mainly reflecting developmentally regulated Hb chains, which fulfil the specific requirements of embryos and larvae. Phylogenetic analyses show remarkable differences in the evolution of Hb-chains that are expressed in the



embryonic and larval stages, which reflect the specific O2 requirements in this phase of life (Tiedke et al. 2011, Hoffmann et al. 2012b, Opazo et al. 2013, Schwarze & Burmester 2013). At least some embryonic Hba chains of teleosts and embryonic Hba of tetrapods (amphibian larval Hba, avian p- and mammalian f-Hb) share a common origin, which suggests an early evolution of Hba in gnathostome evolution. In contrast, Hbb chains expressed in early life stages in different gnathostome subphyla are not orthologous, but emerged several times convergently from adult Hbb by gene duplications (Hoffmann & Storz 2007, Hoffmann et al. 2012b, Opazo et al. 2013, Schwarze & Burmester 2013). The adaptive values of the ‘novel’ members of the globin gene family in vertebrates, all produced by gene duplication and sequence diversification, are thus successively becoming evident through evolutionary and functional studies. However, it is equally intriguing to observe that family members as GbE, GbX and even Ngb, which appear essential components of the gene repertoire of many vertebrate lineages, can yet be entirely missing in others, and their loss obviously has been compensated for perfectly. If globins can indeed substitute for each other, this may mean that they each fulfil several functions in vivo, which will then be hard to disentangle experimentally.

Conclusions Fifteen years ago, it was widely accepted that Hb and Mb were the only globin types, and both were primarily involved in O2 supply. This view has strikingly changed. First, the advent of the genomic era led to the unprecedented discovery of multiple additional globin types with still not fully understood occurrence and expression in multiple tissues other than the blood or striated muscles. Second, several functions other than O2-transport and storage have been conclusively demonstrated for some globins, although many in vitro results have still to be confirmed by in vivo studies. Nevertheless, occurrence and evolution of globins mirror their enormous functional flexibility to meet specific, adaptive needs of diverse animal lineages.

Conflict of interest The authors declared no conflict of interest. The authors thank the Deutsche Forschungsgemeinschaft (DFG) for funding of the globin-related research topics. They also greatly acknowledge the skilful work and great input contributed by many talented graduate and undergraduate students over the past 15 years. We further thank Andrej Fabrizius and Kim Schwarze (University of Hamburg) for critical reading of the manuscript.


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Evolution and Expression of Tissue Globins in Ray-Finned Fishes.

Development and evolution of vertebrate cranial placodes.

More than hemoglobin - the unexpected diversity of globins in vertebrate red blood cells.

Homeobox genes in vertebrate evolution.

Form and function remixed: developmental physiology in the evolution of vertebrate body plans.

Chromosomal changes in vertebrate evolution.

Hormonally active phytochemicals and vertebrate evolution.

Evolution of the Vertebrate Resistin Gene Family.

Evolution of Vertebrate Phototransduction: Cascade Activation.

Evolution of mitochondrial power in vertebrate metazoans.

Evolution of the vertebrate Hox homeobox genes.

Acquisition of germ plasm accelerates vertebrate evolution.

Evolution of the vertebrate immune system.

Evolution of Epigenetic Regulation in Vertebrate Genomes.

The folding pathway for globins.

Bioinformatics and evolution of vertebrate nociceptin and opioid receptors.

Comparative studies of vertebrate Beta integrin genes and proteins: ancient genes in vertebrate evolution.

Development: Germ plasm 'fuels' vertebrate evolution.

The sarcoplasmic reticulum and the evolution of the vertebrate heart.

Evolution of the vertebrate skeleton: morphology, embryology, and development.

Evolution and Diversity of Transposable Elements in Vertebrate Genomes.

The evolution of vertebrate corticotrophin and melanocyte stimulating hormone.

Evolution, structure, and synthesis of vertebrate egg-coat proteins.