Globin Genes Are Present in Ciona intestinalis

Bettina Ebner*, Thorsten Burmester{dagger} and Thomas Hankeln*,

* Institute of Molecular Genetics
{dagger} Institute of Zoology, Johannes Gutenberg University, Mainz, Germany

Correspondence: E-mail: hankeln{at}uni-mainz.de.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
The key position of the Ciona intestinalis basal to the vertebrate phylogenetic tree brings up the question of which respiratory proteins are used by the tunicate to facilitate oxygen transport and storage. The publication of the Ciona draft genome sequence suggests that globin genes are completely missing and that—like some molluscs and arthropods—the sea squirt uses hemocyanin instead of hemoglobin for respiration. However, we report here the presence and expression of at least four distinct globin gene/protein sequences in Ciona. This finding is in agreement with the ancestral phylogeny of the vertebrate globins. Moreover, it seems likely that the Ciona hemocyanin-like sequences have enzymatic instead of respiratory functions.

Key Words: Ciona intestinalis • hemoglobin • neuroglobin • cytoglobin • hemocyanin • intron


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Oxygen transport and storage in animals is mediated by either one of three distinct, unrelated types of respiratory proteins: the hemoglobins (more generally referred to as globins), hemocyanins, and hemerythrin (Kurtz 1999). Globins are small porphyrin-containing proteins that are the most widely used oxygen-binding proteins in the animal world, but they have also been found in bacteria, fungi, protists, and plants (Hardison 1996). The copper-containing hemocyanins found in many molluscan and arthropod taxa have evolved independently from unrelated tyrosinase enzymes (Markl and Decker 1992; Burmester 2001; van Holde, Miller, and Decker 2001), whereas the occurrence of iron-containing hemerythrins is limited to some annelids, brachiopods, and sipunculids (Mangum 1992).

In vertebrates, oxygen transport to the tissues is mediated by several different classes of hemoglobins (Dickerson and Geis 1983), whereas myoglobin functions as an oxygen store in the muscle (Wittenberg 1992), besides being involved as an enzyme in nitric oxide metabolism (Flögel et al. 2001). Recently, two novel classes of vertebrate globins have been described. Neuroglobin (Burmester et al. 2000) is primarily expressed in neuronal cells of the central nervous system and in the retina, where it may sustain aerobic metabolism under conditions of high oxygen demand (Sun et al. 2001; Reuss et al. 2002; Schmidt et al. 2003). Cytoglobin (Burmester et al. 2002; Trent and Hargrove 2002) is expressed in virtually all human tissues, but its physiological role is still unclear.

The sea squirt Ciona intestinalis is of special interest for the understanding of vertebrate evolution because the tunicates (Urochordata) are phylogenetically positioned at the base of the vertebrate tree (Wada and Satoh 1994). Recently, Dehal et al. (2002) reported the draft genome sequence of Ciona. In their discussion of the sea squirt gene repertoire, the authors suggested that the Ciona genome lacks globin genes and proposed that the sea squirt uses hemocyanins for oxygen transport. However, here we report the identification of four different globins in Ciona.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Database searching was performed using TBlastN (Altschul et al. 1997) with the Blosum 45 matrix on the C. intestinalis genomic DNA sequence draft at JGI (http://genome.jgi-psf.org/ciona4/ciona4.home.html) and on additional genomic data and a corresponding EST/cDNA database at Kyoto University (http://ghost.zool.kyoto-u.ac.jp/indexr1.htm). For further searches, BlastP was employed in combination with the NCBI nonredundant protein database. Phylogenetic analyses were performed using an established data set that includes the Ciona globins and 47 other selected metazoan and plant globins (Burmester et al. 2000, 2002 [alignment available from the authors upon request]). Distances between pairs of sequences were calculated using the JTT matrix (Jones, Taylor, and Thornton 1992) implemented in the PHYLIP 3.6a2 package (Felsenstein 2001). Tree reconstructions were performed by the neighbor-joining method (Saitou and Nei 1987). The reliability of the trees was tested by the bootstrap procedure with 100 replications (Felsenstein 1985). Bayesian phylogenetic analyses were performed with MrBayes 3.0 (Huelsenbeck and Ronquist 2001), assuming the WAG substitution matrix (Whelan and Goldman 2001) model of sequence evolution with gamma distribution of rates. Metropolis-coupled Markov chain Monte Carlo sampling was performed with one cold and three heated chains that were run for 150,000 generations. Prior probabilities for all trees were equal, starting trees were random, tree sampling was done every 10th generation. Posterior probabilities were estimated on 10,000 trees (burn-in = 5,000).

C. intestinalis adult specimens were obtained from the Meeresbiologische Forschungsanstalt Helgoland, Germany. Total RNA was prepared from adult tissue (Sambrook and Russell 2001, pp. 7.4), and 5 µg of RNA were converted into cDNA by Superscript II reverse transcriptase following the supplier's protocol (Invitrogen). One-tenth of a cDNA reaction was used for standard PCR, employing globin gene–specific oligonucleotide primers that had been derived from the in silico–predicted coding regions of the Ciona globin genes. Alternatively, PCR was performed on a Ciona cDNA clone pool obtained from the German resource center RZPD (Ciona larval library 684; www.rzpd.de) and on a cDNA library made from Helgoland adult Ciona specimen provided by K. Weber (Max Planck Institut für biophysikalische Chemie, Göttingen, Germany). PCR amplificates were either sequenced directly or after cloning into plasmid vectors (pGEM T-easy, Promega) on both strands by the dye terminator cycle sequencing chemistry (Applied Biosystems). Sequencing reactions were loaded onto an Applied Biosystems Prism 3730 capillary sequencer by GENterprise GmbH, Mainz. Sequences were further manipulated using Lasergene programs (DNAstar Inc.).


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Although globin sequences in general show considerable evolutionary variability with only two residues (Phe in the CD1 corner and the proximal histidine at F8) being strictly conserved, the estimated evolution rates of vertebrate neuroglobin and cytoglobin are significantly lower (Burmester et al. 2000, 2002; Awenius et al. 2001). Thus, these sequences are particularly useful to identify globins in broad range of animal taxa. We performed TBlastN searches on the C. intestinalis genome draft and cDNA/EST data sets employing various neuroglobin and cytoglobin amino acid sequences. In contrast to the suggestions of Dehal et al. (2002), four distinct globin genes were identified in the Ciona genome (fig. 1). Two of the globin genes reside on genomic scaffold 174 (CinHb1 and CinHb2), located in head-to-tail orientation with their coding regions (CDS) only 751 bp apart from each other. The third gene (CinHb3) is included in scaffold 118, and a fourth (CinHb4) is found on overlapping scaffolds 30 and 3143. The C-terminal exon of CinHb4 was not present on the JGI genomic scaffolds and thus had to be derived from additional genomic data from Kyoto University. Transcriptional activity of CinHb1, CinHb2, and CinHb 4 was suggested by the presence of corresponding cDNA/EST database entries, but no EST clone was found for CinHb3. We therefore validated the coding sequences of all four Ciona globin gene variants by RT-PCR and sequencing (GenBank accession numbers AJ548500, AJ548501, AJ548502, and AJ557135). The numerous substitutional differences between our Ciona globin sequences and the database entries are further evidence for the known very high degree of allelic polymorphism in the sea squirt (Dehal et al. 2002). Two of the globin genes (CinHb1 and CinHb4) have in fact been annotated as gene models encoding potential globins within the Ciona genomic draft database, but this annotation was probably performed automatically and was not mentioned in the Dehal et al. (2002) paper.



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FIG. 1. Genomic organization of the four globin gene variants in Ciona. Exons encoding globin domains are depicted as black boxes and are drawn to scale. The nonglobin part of CinHb4 is hatched. The transcriptional orientation of the genes is 5' to 3' from left to right in all cases. The positions of introns are indicated by such designations as "B12.2" (i.e., the intron starts after codon position 2 within codon 12 of globin helix B)

 
BlastP searches (Altschul et al. 1997 [Blosum 45 matrix]) in the nonredundant NCBI protein database using the derived amino acid sequences of the Ciona globins show that all four proteins consistently identify the vertebrate neuroglobins and cytoglobins at the highest ranking positions (28% to 31% amino acid identities; E-values of 10-7 to 10-10). In all four putative globins, the functional determinants of true globin oxygen carriers (Dickerson and Geis 1983), such as the proximal and distal histidines at amino acid positions F8 and E7 and a phenylalanine residue at CD1, are conserved (fig. 2). CinHb1, CinHb2, and CinHb3 encode proteins that conform to the standard length of globins (approximately 150 amino acids). CinHb4, however, is substantially elongated and shows an unusual extension of 135 amino acids at its N-terminus. A search for known protein domains (www.ebi.ac.uk/interpro/scan.html) within this N-terminal extension reveals the presence of a 77-amino-acid region that matches INTERPRO domain IPR000472, characteristic of the TGF-ß receptor type I/II protein family involved in signal transduction (Massague and Weis-Garcia 1996). Additionally, the SMART analysis tool (http://smart.embl-heidelberg.de) predicts the presence of a signal peptide comprising N-terminal amino acids 1 to 24 of CinHb4. The functional relevance of these matches is currently unknown.



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FIG. 2. Comparison of Ciona globin amino acid sequences (CinHb1 to CinHb4) with human neuroglobin (HsaNGB [GenBank accession number AF422797]), myglobin (HsaMB, [GenBank accession number M14603]), and hemoglobins (HsaHBA [GenBank accession number J00153] and HsaHBB [GenBank accession number M36640]). The globin consensus numbering is given below the sequences. The secondary structure of the human hemoglobin ß is superimposed in the upper row with the {alpha}-helices designated A through H. Strictly conserved amino acids are shown with a black background, and residues conserved in more than 50% of the sequences are shaded in gray

 
The four putative Ciona globins are rather diverged in sequence compared with one another. Pairwise comparisons of the globin domains reveal that the two clustered variants CinHb1 and CinHb2 have the highest level of amino acid identity (59%), suggesting they have indeed been derived from a common ancestor by a more recent duplication and subsequent divergence. This notion is supported by the vicinity of the genes on scaffold 174 and by phylogenetic analyses (fig. 1). All other CinHb variants are approximately equally distant to one another (36% to 40% identity).

The phylogeny of Ciona globins relative to vertebrate and nonvertebrate globins was investigated using established amino acid sequence alignments (Burmester et al. 2000, 2002) and various tree reconstruction methods. The plant globins were considered as outgroup. In all types of analyses, the globins of Ciona form a well-supported monophyletic clade (fig. 3). However, due to a lack of sufficient phylogenetic signal in the globin sequences, the exact branching orders in the tree of the metazoan globins could not be resolved with sufficient confidence, giving rise to low bootstrap support values and posterior probabilities. Nevertheless, the vertebrate globins form a common clade distinct from both Ciona and the other invertebrate globins (fig. 3).



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FIG. 3. A simplified globin amino acid phylogenetic tree based on Bayesian evolutionary interference. The values on the tree represent posterior probabilities of clade support, given the data and the model of sequence evolution. The tree root was set by taking plant globins as outgroup. The Ciona globins are monophyletic and stand basal to the vertebrate hemoglobin, myoglobin, and cytoglobin

 
The monophyletic relationship of the Ciona globin variants with respect to the globin tree is further substantiated by the distribution of introns within the genes (fig. 4). All four CinHb genes contain an intron at position B12.2, and all but CinHb2 contain introns at position G7.0. These B12.2 and G7.0 intron positions have been found in all vertebrate globins known and, because of their presence in many invertebrate and even plant globin genes, are thought to have already existed in an ancestral globin gene (Hardison 1996; Burmester et al. 2000, 2002). We therefore conclude that CinHb2 has lost its G7.0 intron after the CinHb1/CinHb2 duplication event. Such intron loss has previously been observed in globin genes (e.g., of insect taxa [Hankeln et al. 1997; Burmester and Hankeln 1999]) and was possibly generated by recombination of the intron-containing gene with an intronless cDNA gene copy (Derr and Strathern 1993). All four Ciona globin genes have an additional intron at position E10.2. Such "central" E helix introns are present in many globin genes of vertebrate, invertebrate, and even plant origin, albeit being located at slightly different positions within the E helix (fig. 4). There has been debate concerning the homology and evolutionary antiquity of these central introns (Stoltzfus and Doolittle 1993) because their presumed positional conservation was used as evidence for the "exon theory of genes" (Go 1981). In vertebrates, only the presumably very ancient neuroglobin gene, but not hemoglobins, myoglobin, or cytoglobin, contains a central E helix intron at position E11.0 (Burmester et al. 2000). It was not clear, however, if this neuroglobin E11.0 intron was indeed old or a more recent acquisition during vertebrate evolution. The finding of E10.2 helix introns (only a 1-bp shift relative to E11.0) in all four CinHbs suggests that the neuroglobin E11.0 intron position may predate vertebrate evolution. If the central introns in Ciona and in vertebrate neuroglobin are indeed homologous (and ancient), their 1-bp positional difference must be explained by an "intron sliding" event (Rogers 1986). Such a shift of two intron borders is not easily explained in functionally important single-copy genes, as it probably involves transient nonfunctional states of the gene. This may not so strongly be the case for a gene family such as the one in Ciona, in which intron sliding in one copy could be temporarily compensated for by the function of the other gene variants. The presence of an E10.2 intron in all four Ciona globin genes either could be the result of gene conversion (as hypothesized for Chironomus globin gene introns [Hankeln et al. 1997]) or could be interpreted as evidence that these genes duplicated from one copy within the ancestral Ciona line.



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FIG. 4. Distribution of introns in vertebrate and nonvertebrate globin genes, shown relative to the globin {alpha}-helices A to H. The position of the B12.2 and G7.0 introns is widely conserved, whereas the "central" globin gene introns of diverse taxa often occupy slightly different locations. The homology (and, thus, ancestry) of these "central" intron positions is debated

 
Our model of the evolution of globin function in vertebrates (Burmester et al. 2000, 2002) suggests that the earliest vertebrate already harbored two globin variants, one expressed primarily in neuronal cells (neuroglobin) and one putatively expressed in all tissues (the common ancestor of cytoglobin and myoglobin). Detailed analysis of the expression patterns and the kinetic and ligand-binding properties will have to show whether the four Ciona globin variants perform physiological roles homologous to vertebrate globins. Based on the protein structure, however, we propose that the tunicates may indeed use these oxygen-binding proteins for respiratory metabolism. CinHb1, CinHb2, and CinHb3 will most probably be intracellular, cytoplasmic globins, whereas the situation is uncertain for the "composite" CinHb4 with its signal peptide and the TGF-ß receptor–like domain. The current EST data set and our own RT-PCR experiments only give preliminary information about the possible gene expression patterns. It is noteworthy that CinHb1 and especially CinHb2 ESTs seem to be enriched in Ciona heart tissue and blood cells, which are lymphoid-like cells with cytotoxic activity towards mammalian cells (Di Bella and De Leo 2000). Although it is uncertain whether the tunicate blood cells are evolutionarily homologous to vertebrate erythrocytes, the expression of globins in these cell types—regardless of the ancient globin function—may in fact predate the separation of the chordate and urochordate lineage.

Dehal et al. (2002) put forward the idea that copper-containing hemocyanins are used for oxygen transport in Ciona. This would be surprising because hemocyanins have so far only been found in molluscan or arthropod species. The hemocyanins of these two phyla represent two separate protein families that derived independently from distinct types of tyrosinases (Burmester 2001). Although there are in fact two genes in Ciona that display significant similarities to the arthropod hemocyanin superfamily (Burmester 2002), phylogenetic analyses indicate that respiratory hemocyanins evolved only within the arthropod phylum (Immesberger and Burmester, unpublished data). At least three more Ciona genes resemble the molluscan hemocyanins and other members of the large tyrosinase superfamily. However, neither phylogenetic nor structural analyses (single domain proteins in Ciona in contrast to seven or eight domains in molluscan hemocyanins) support a role of these Ciona proteins as respiratory proteins (data not shown). Although the exact role of the Ciona globins in the oxygen metabolism of tunicates requires further studies, it should be considered extremely unlikely that Ciona uses hemocyanins for respiration as suggested (Dehal et al. 2002).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
We would like to thank M. Krüss (Biologische Forschungsanstalt Helgoland) for sending Ciona specimens and K. Weber (MPI Göttingen) for making his Ciona cDNA library available to us. T.H. and T.B. gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (Ha2103/3 and Bu956/6) and by the EU (QLG-CT-2002–01548).


    Footnotes
 
Kenneth Wolfe, Associate Editor Back


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 Introduction
 Materials and Methods
 Results and Discussion
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Accepted for publication May 5, 2003.