Molecular Biology and Evolution

MBE Advance Access originally published online on August 31, 2005
Molecular Biology and Evolution 2006 23(1):46-55; doi:10.1093/molbev/msj004

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Published by Oxford University Press 2005.

Research Article

Molecular Evolution of Ankyrin: Gain of Function in Vertebrates by Acquisition of an Obscurin/Titin-Binding–Related Domain

Alexander A. Hopitzan^*, Anthony J. Baines and Ekaterini Kordeli^*

^* Institut Jacques Monod/CNRS/Universités Paris VI et VII, Paris, France; and Department of Biosciences, University of Kent, Canterbury, United Kingdom

E-mail: kordeli{at}ijm.jussieu.fr.

	Abstract

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
Ankyrins form a family of modular adaptor proteins that link between integral membrane proteins and the cytoskeleton. They evolved within the Metazoa as an adaptation for organizing membrane microstructure and directing membrane traffic. Molecular cloning has identified one Caenorhabditis elegans (unc-44), two Drosophila (Dank1, Dank2), and three mammalian (Ank1, Ank2, Ank3) genes. We have previously identified a 76–amino acid (aa) alternatively spliced sequence that is present in muscle polypeptides encoded by the rat Ank3 gene. A closely related sequence in a muscle Ank1 product binds the cytoskeletal muscle proteins obscurin and titin. This obscurin/titin-binding–related domain (OTBD) contains repeated modules of 18 aa: three are encoded by Ank1 and Ank2, two by Ank3; this pattern is conserved throughout vertebrate ankyrin genes. The C. elegans ankyrin, UNC-44, contains one 18-aa module as does the ankyrin gene in the urochordate Ciona intestinalis, but the insect ankyrins contain none. Our data indicate that an ancestral ankyrin acquired an 18-aa module which was preserved in the Ecdysozoa/deuterostome divide, but it was subsequently lost from arthropods. Successive duplications of the module led to a gain of function in vertebrates as it acquired obscurin/titin-binding activity. We suggest that the OTBD represents an adaptation of the cytoskeleton that confers muscle cells with resilience to the forces associated with vertebrate life.

Key Words: ankyrin • Ank_G107 • striated muscle • module • titin • obscurin

	Introduction

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
Ankyrins mediate linkage of integral proteins to the spectrin-based cytoskeleton. Gene knockout experiments, small interfering RNA depletion, and natural mutations point to a crucial role of ankyrins in organizing membrane domains and in delivering ion channels and cell adhesion proteins to requisite membrane sites (Bennett and Baines 2001; Mohler et al. 2003, 2004; Kizhatil and Bennett 2004).

Three ankyrin genes in mammals (Ank1, Ank2, and Ank3), two in Drosophila melanogaster (Dank1, Dank2), and one in Caenorhabditis elegans (unc-44) are known to date (Lambert et al. 1990; Lux, John, and Bennett 1990; Otto et al. 1991; Dubreuil and Yu 1994; Kordeli, Lambert, and Bennett 1995; Otsuka et al. 1995; Peters et al. 1995; Bouley et al. 2000). So far, no ankyrins have been found in plants, fungi, yeast, and bacteria. Thus, ankyrins are likely to have evolved early in the evolution of metazoans to meet the requirement of animal cells for generation and maintenance of complex membrane structures (Mohler, Gramolini, and Bennett 2002b).

Ankyrins are modular proteins that contain a series of domains that are highly conserved between gene products (the N-terminal membrane-binding Ank repeats, the spectrin-binding ZU5 domain, and death domain). Additionally, the different genes encode unique regions in the C-terminal regions. Mammals generate functionally specialized isoforms during development and in different tissues by complex patterns of mRNA splicing (Bennett and Baines 2001) including by splicing the C-terminal regions (Hall and Bennett 1987; Davis, Davis, and Bennett 1992; Mohler, Gramolini, and Bennett 2002a).

We previously identified a unique 76-residue insertion (the "76–amino acid [aa]" insert) in the C-terminal region of Ank_G107. Ank_G107 is a muscle isoform of ankyrin-G (as products of the Ank3 gene are known) which is targeted to the sarcolemma (Gagelin et al. 2002). Elucidation of the expression pattern of Ank3 in muscle (Hopitzan et al. 2005) strongly suggested that 76 aa is present in all muscle ankyrins-G but is not expressed outside muscle.

The C-terminal regions of Ank1 and Ank2 gene products (ankyrins-R and ankyrins-B, respectively) contain a sequence closely similar to 76 aa, with highest conservation in sAnk1, an Ank1 gene product. sAnk1 is a muscle-specific, integral, truncated (25 kDa) ankyrin which localizes to M and Z lines of sarcomeres and is thought to link the sarcoplasmic reticulum to myofibrils (Zhou et al. 1997; Birkenmeier et al. 1998; Gallagher and Forget 1998). Two giant myofibrillar proteins, titin and obscurin, interact with the 76-aa regions of sAnk1; obscurin also binds the 76-aa region of an Ank2 gene product (Bagnato et al. 2003; Kontrogianni-Konstantopoulos and Bloch 2003; Kontrogianni-Konstantopoulos et al. 2003). Titin and obscurin belong to the same family of modular proteins of vertebrate striated muscle. Both proteins have been suggested to play crucial roles in myofibrillogenesis (Young, Ehler, and Gautel 2001; Granzier and Labeit 2002; Russell et al. 2002; Tskhovrebova and Trinick 2003, 2004; Kontrogianni-Konstantopoulos et al. 2004). Vertebrate titin forms flexible filaments of more than 1 µm in length that span half a sarcomere from the Z line to the M line in vertebrate striated muscle. The two most N-terminal domains of titin (two Ig domains, ZIg1 and ZIg2) are involved in binding sAnk1 at the Z line (Kontrogianni-Konstantopoulos and Bloch 2003). Obscurin colocalizes with sAnk1 at the level of both, Z and M lines. The last C-terminal 400 residues were shown to bind sAnk1 (Bagnato et al. 2003; Kontrogianni-Konstantopoulos et al. 2003).

Titin and obscurin homologs (Tskhovrebova and Trinick 2003; Hooper and Thuma 2005), as well as ankyrin (C. elegans body wall muscles; Chen, Ong, and Bennett 2001), have also been reported in invertebrate muscle. Interestingly, an ankyrin-like protein has been localized in the myoplasm and muscle cells of ascidian eggs and embryos and seems to play a role in muscle development, possibly coordinating the linkage between membrane cytoskeleton, sarcoplasmic reticulum, and sarcomeres (Jeffery and Swalla 1993).

These findings raise a number of intriguing questions about the origin of the 76-aa–related sequences, as well as the functional consequences of their presence in ankyrin molecules. Here we report that 76-aa homologous sequences are present in all paralogous vertebrate ankyrin genes and show a modular architecture. Intriguingly, one of these 18-aa modules is present in both the ecdysozoan and deuterostome invertebrate lineages. We conclude that an early metazoan acquired such a module, which was adapted in vertebrate evolution by successive duplication to interact with muscle proteins, including obscurin/titin. These data indicate a gain of function in vertebrate ankyrin evolution.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
Sequence Retrieval
Protein sequences homologous to the Ank3 76-aa insert were retrieved by Psi-Blast search (Altschul et al. 1997) querying the National Center for Biotechnology Information Blast server (http://www.ncbi.nlm.nih.gov/BLAST/) against the nonredundant and RefSeq databases. Conserved sequences in the C. elegans ankyrin UNC-44 AO13 were identified using the Wise2 server (http://www.ebi.ac.uk/Wise2/). Textual Wise2 outputs were graphically displayed and processed with the genome annotation tool Artemis release 6.0 (http://www.sanger.ac.uk/Software/Artemis/v6/).

Nucleotide sequences homologous to the 76-aa insert were searched by BLAT (http://genome.ucsc.edu) and by the TBlastN procedure (Altschul et al. 1990) against nonredundant, expressed sequence tag (EST), RefSeq, and whole-genome databases on following sites: http://www.ncbi.nlm.nih.gov; http://www.ensembl.org/index.html (H. sapiens v25.34e.1, M. musculus v25.33a.1, R. norvegicus v25.3c.1, D. rerio v25.4.1, G. gallus v25.1b.1, A. gambiae v25.2b.1, F. rubripes v25.2c.1, D. melanogaster v25.3b.1, P. troglodytes v25.1.1, A. mellifera v25.1.1, T. nigroviridis v25.1.1, C. familiaris vBROADD1, C. elegans v25.116a.1, B. taurus vBtau_1.0); http://genome.jgi-psf.org/Xentr3/Xentr3.home.html; http://www.sanger.ac.uk/cgi-bin/blast/submitblast/x_tropicalis (X. tropicalis assembly3); http://genome.jgi-psf.org/ciona4/ciona4.home.html (Ciona intestinalis assembly1); and http://atlas.cnio.es/Caenorhabditis_briggsae/(C. briggsae v25.25.1). Predicted matches (e.g., exon-intron boarders) were confirmed by eye and in the case of less conserved fish sequences refined by annotating the 76-aa insert to genomic sequence using Wise2. For hidden Markov model analysis, the program HMMer was used (Eddy 1998). Protein sequence was deduced from extracted exonic sequences using the Lasergene (DNASTAR, Madison, Wisc.) sequence analysis software package. Nucleotide sequences corresponding to the ZU5 domain (smart entry: http://smart.embl-heidelberg.de/smart/do_annotation.pl?DOMAIN=ZU5&BLAST=DUMMY, pfam entry: http://www.sanger.ac.uk/cgi-bin/Pfam/getacc?PF00791) were retrieved as described above. All database searches were performed before December 2004; nucleotide sequence queries against nonredundant, EST, and RefSeq databases have been repeated before March 2005. Results are available online as Supplementary Material.

Sequence Alignments
All multiple nucleotide and protein sequence alignments were performed using ClustalX (Thompson et al. 1997) and were manually optimized using the BioEdit software (Hall 1999).

Sequence repeats within the obscurin/titin-binding–related domain (OTBD) were initially identified using the programs REPRO (http://ibivu.cs.vu.nl/) and RADAR (http://www.ebi.ac.uk/Radar/). The "sequence logo" representing aligned module sequences was created using WebLogo (Crooks et al. 2004). Multiple nucleotide sequence alignments are available online as Supplementary Material.

Structure Predictions
To predict the structure of the OTBD, we used the metaserver at Genesilico (http://genesilico.pl; Kurowski and Bujnicki 2003). This server is a gateway to multiple high-quality methods of secondary structure prediction and fold recognition. Additionally, we used servers for PONDR (http://www.pondr.com) and GLOBPLOT (http://globplot.embl.de; Linding et al. 2003) to analyze the potential for disordered structure.

Phylogenetic Analysis
Phylogenetic analysis was conducted on 48 nucleic acid sequences corresponding to the ZU5 domain (for sequence alignment, see Supplementary Material online). The construction of the maximum likelihood (ML) tree was performed with the program Treefinder (www.treefinder.de) using the general time reversible substitution model for nucleotides with four rate categories and default parameters (Jobb, von Haeseler, and Strimmer 2004). Robustness of the topology was assessed using 1,000 bootstrap replicates. The tree was rerooted using C. elegans and C. briggsae as outgroups.

	Results and Discussion

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
The Muscle-Specific 76-aa Insert of Ankyrins-G Defines a Novel Domain Conserved Among Vertebrate Ankyrins
Figure 1 shows a schematic diagram of the muscle ankyrin Ank_G107, encoded by the rat Ank3 gene. In the C-terminal region is a 76-aa insert encoded by three consecutive exons (Gagelin et al. 2002; Hopitzan et al. 2005). We previously reported that sequences homologous to the 76 aa are encoded by all three mammalian ankyrin genes (Gagelin et al. 2002). To gain further insight into the nature of the 76 aa, we performed a multiple sequence alignment of C-terminal domains of ankyrins from humans, mice, and rats (fig. 2A).

View larger version (11K): [in this window] [in a new window]   FIG. 1.— Schematic representation of AnkG107 containing the muscle-specific 76-residue insert (76 aa). Ankyrin domains are designated as follows: SpBd, spectrin binding; DD, death domain; and C-ter, C-terminal. Amino acid sequence of 76 aa and encoding Ank3 exons are shown on top and bottom, respectively.

 

View larger version (63K): [in this window] [in a new window]   FIG. 2.— Multiple alignment of C-terminal ankyrin sequences corresponding to the rat ankyrin-G 76-aa insert. (A) Shown are the protein sequences encoded by Ank1 (Homo sapiens AnkR, NP_065211 [GenBank] ; Mus musculus AnkR, AAC24156 [GenBank] ), Ank2 (H. sapiens AnkB, CAD98033 [GenBank] ; Rattus norvegicus AnkB, XP_227735), Ank3 (R. norvegicus AnkG, AJ428573 [GenBank] ), and unc-44 (Caenorhabditis elegans UNC-44, AAB41827 [GenBank] ). Amino acid residues of human Ank1 gene product (AnkR, NP_065211 [GenBank] ) implicated in binding titin and obscurin are indicated on top of the alignment (double underlined and in bold). Underneath the sequences are structure predictions, generated as described in the text. Predicted -helices are shown as cylinders; ß-strand as an arrow; and disorder as open rectangles. Note that the ß-strand predicted by JUFO is suggested by PONDR to be disordered, so both predictions are indicated in the figure. (B) Identification of three internal sequence repetitions (modules I–III) within ankyrin sequences corresponding to the 76-aa insert by optimized ClustalX alignment. Amino acid sequence of the Ciona intestinalis module was deduced from EST clone BW435037 [GenBank] (nt 556–621). Modules are underlined in (B) and indicated by open boxes in (A). Conserved amino acids in more than 80% (A) or 60% (B) of the sequences are highlighted in black (identical residues) or gray (similar residues according to Blosum62 residue weight table). (C) Graphical representation of the aligned module sequences shown in (B) as sequence logo.

 
In previous alignments using available ankyrin-B isoforms, the Ank2 gene products displayed conservation over only about half of the 76-aa sequence (Gagelin et al. 2002). However, in database searches, we found sequences in which homology extends further: these sequences include human partial protein product CAD98033 [GenBank] (derived from the uterus EST DKFZp686M09125) and rat RefSeq entry XP_227735. Figure 2A includes alignment of these sequences and indicates that homology extends to the whole region corresponding to the 76 aa. Ank2 mRNAs can evidently be spliced to include or exclude the first exon of the 76-aa homologous sequence.

Psi-Blast searches revealed that no proteins other than ankyrins contain the 76-aa sequence. Surprisingly, in the C. elegans ankyrin UNC-44, a short stretch of about 20 residues shows homology with mammalian 76-aa sequence. The alignment is with the central region of the 76 aa. By contrast, no homology was found with either of the D. melanogaster ankyrins (Dank1 and Dank2).

Further analysis of the 76-aa region using the programs REPRO and RADAR revealed two internal sequence repetitions which we termed modules I and II (fig. 2A). Both modules are present within all three vertebrate gene products as confirmed by a multiple sequence alignment (fig. 2B). A less conserved third module (module III) was identified only in the C-terminal domains of Ank1 and Ank2 transcripts. The pattern of sequence conservation among modules is shown in figure 2C. It is noteworthy that the sequence conserved in C. elegans corresponds to module II. All modules, except module III of Ank2, are made of 18 residues. The only known protein-protein interactions occurring within this region are between the Ank1-encoded sAnk1 and the two muscle proteins titin and obscurin (Bagnato et al. 2003; Kontrogianni-Konstantopoulos and Bloch 2003; Kontrogianni-Konstantopoulos et al. 2003). The sites of interaction reside, at least partially, within modules I and II, respectively (fig. 2A).

Based on these observations, we propose that the conserved sequences corresponding to the ankyrin-G 76 aa should be designated "obscurin/titin-binding–related domain." Having noted the presence of a module II–like sequence in C. elegans UNC-44, we wondered if vertebrate and worm sequences have common origins. If this is the case, related sequences might be in simple chordates too. The genome of the urochordate C. intestinalis is now available (Dehal et al. 2002). We searched the genome scaffolds initially by TBlastN, using rat ankyrin-G OTBD as a query sequence. This yielded no significant hits. As a more sensitive alternative, we constructed a hidden Markov model of the aligned 18-aa modules from humans, mice, rats, and C. elegans. This was used to search all possible translations of the C. intestinalis ankyrin gene. A single hit was obtained. This sequence is shown in figure 2B aligned with other 18-aa modules. To establish if the C. intestinalis sequence is likely to be expressed as part of an ankyrin protein, we used the predicted amino acid sequence to query the EST database by TBlastN. This revealed 16 ESTs with 100% identity; thus, this single module represents part of an expressed protein. The ESTs are part of Unigene cluster Cin.5740: assembly of this cluster using CAP3 reveals a sequence-encoding part of a conventional ankyrin with ZU5 and death domains, as well as the module. The HMM detects the 18-aa module in this peptide with an E value of 0.005.

Because the HMM for the 18-aa module was a sensitive means for detecting related sequences in the urochordate, we wondered if our initial Blast searches had failed to detect such a module in insect ankyrins. Reanalysis of the D. melanogaster ankyrins revealed no significant hits. We also used this HMM to search the Ensembl databases of Anopheles gambiae and Apis mellifera peptides: again, no significant hits were found. We conclude that the 18-aa module is represented in both ecdysozoan and deuterostomal organisms but that it is not preserved in insect ankyrins.

We further investigated the possibility that the OTBD represents a known protein structure. We used the 18-aa HMM to probe sequences represented in the Protein Data Bank (PDB). This search revealed no significant hits. Direct BlastP analysis of sequences in the PDB using the OTBD sequence of human ankyrin-R gave no hits with expectation values better than 0.3, so we cannot assign a fold to the OTBD with confidence. We also submitted the OTBD sequence of human ankyrin-R to the Genesilico metaserver for structure prediction. The algorithms INGBU, 3DPSSM, MGENTHREADER, and FFAS failed to give a confident or consistent prediction of fold. However, the secondary structure prediction methods PSIPRED, JNET, SABLE, PROF, JUFO, and PROFSEC indicated possible secondary structure elements in the OTBD sequence. The output of the JUFO program is typical of the predictions obtained; its -helix and ß-strand predictions are annotated in figure 2A. To probe the sequences for disordered structure, the programs DISEMBL, DISOPRED, GLOBPLOT, and PONDR were used. All these programs predicted elements of disordered structure between the modules. To illustrate this, figure 2A also shows the PONDR predictions for disordered sequence. Note that the second half of each 18-aa module is predicted to be -helical, and the modules are connected by disordered polypeptide. Kyte-Doolittle (Kyte and Doolittle 1982) hydropathy analysis indicates that the sequence is relatively hydrophilic, and JNET (Cuff and Barton 2000) solvation analysis indicates that most of the OTBD is likely to be accessible to water. It seems possible that the OTBD is flexible, with structured regions contributed by 18-aa modules joined by flexible linkers.

Exons Encoding the OTBD: Expression and Organization
To investigate the conservation of the OTBD among species, we extended the multiple protein sequence alignment to all so far available ankyrin data, including genomic sequences (fig. 3). Protein sequences shown in figure 2A were annotated to genomes to localize OTBD-encoding exons. Nucleotide sequences were subsequently extracted, translated, and aligned (fig. 3A).

View larger version (95K): [in this window] [in a new window]   FIG. 3.— Multiple alignment of OTBD amino acid and nucleotide sequences as deduced from vertebrate genomes. (A) Compared sequences correspond to the most conserved modules I and II. Human Ank1 OTBD exons 39a–41 are indicated on top. Conserved amino acids in 80% or more of the sequences are highlighted in black (identical residues) or gray (similar residues according to Blosum62 residue weight table). (B) Multiple alignment of nucleotide sequences corresponding to human Ank1 exon 40. Coding sequence is in uppercase, splice acceptor and donor sites (ag/gt) are in lower case. Exons are represented by boxes on top of the alignment; shown below is the protein sequence of human sAnk1 (NP_065211 [GenBank] ). Residues implicated in binding of titin and obscurin are in bold and double underlined. Introns are not drawn to scale. Identical nucleotides in 80% or more of the sequences are highlighted in black.

 
In mammals, the OTBD is encoded by three consecutive exons corresponding to rat Ank3 exons 43–45 (Hopitzan et al. 2005) and human Ank1 exons 39a–41 (Gallagher et al. 1997). Exon 41 has three internal splice sites giving rise to four segments. In muscle-specific sAnk1 isoforms, alternative splicing joins segments 1 and 4 (Birkenmeier et al. 1993, 1998). It is of interest that segment 4 encodes module III. We found that Ank2 exons 44 and 45, as defined by Mohler and colleagues (Mohler et al. 2003), corresponded to the last two OTBD exons. The first exon was here identified by annotating the partial protein sequence shown in figure 2A to the Ank2 gene locus.

Several lines of evidence point to a muscle-restricted expression of the first OTBD exon. (1) Expression of exon 39a is restricted to muscle under the control of an alternative promoter (Birkenmeier et al. 1998; Gallagher and Forget 1998). (2) All three OTBD exons of Ank3 have been found so far only in muscle tissue (Gagelin et al. 2002; Hopitzan et al. 2005). (3) The first OTBD exon encodes the entire module I, which contains half of the region that binds muscle protein titin (figs. 2A and 3A). It will be of importance to determine the critical residues implicated in titin binding as has been done for obscurin. (4) Blast analysis of the EST databases reveals no expression outside muscle-rich tissues. (5) Although multiple full-length cDNAs have been obtained for ankyrin-B splice variants, none have yet been obtained from muscle; correspondingly, none contain the first exon of the OTBD.

Based on these data, we propose that module I bestows ankyrins with important muscle-specific functions, such as providing binding sites for muscle proteins. The last two OTBD exons are not restricted to muscle tissues; they have also been found in transcripts from reticulocytes, liver, bone marrow, and brain (Otto et al. 1991; Gallagher et al. 1997). It is noteworthy that both exons are needed to constitute module II. Crucial residues for binding obscurin are localized within both last OTBD exons; this requirement could apply to other yet unidentified protein interactions and could be the reason why these two exons have always been found coexpressed.

Our results strongly suggest the presence of the three OTBD exons in vertebrates in general, including birds, amphibians, and fishes (fig. 3A).

To further study the conservation of the OTBD among vertebrates, we aligned nucleotide sequences corresponding to the most conserved middle exon (fig. 3B). A striking observation was that both exon structure and nucleotide sequence are conserved among all paralogous ankyrin genes from fish to man.

Phylogenetic Relationships Among Ankyrin Genes
To understand more of the origin and evolution of the OTBD, we conducted phylogenetic analyses on available ankyrin sequences. A defining characteristic of ankyrins is the ZU5 domain. This domain binds spectrin and is highly conserved in all known ankyrins. An ML tree was constructed from 48 nucleotide sequences encoding ankyrin ZU5 domains, retrieved from genomic databases. The tree defines two major clusters corresponding to vertebrate and invertebrate ankyrins.

In previous analysis of ankyrins, Bouley et al. (2000) examined the phylogeny of ankyrins by comparing the sequences of ANK repeats of a small number of ankyrins. They concluded that C. elegans and D. melanogaster ankyrins do not represent direct orthologs of any vertebrate ankyrins and that duplication events that led to fruitfly ankyrins 1 and 2 occurred independently of the expansion of the vertebrate genes. In these respects, their data are consistent with the proposed divergence of bilateran Metazoa into Ecdysozoa and deuterostomes (Aguinaldo et al. 1997; Halanych 2004).

Figure 4 shows the results of our more extensive analysis of the nucleotide sequences of the spectrin-binding ZU5 domain. We have taken advantage of recent genome and EST sequencing to analyze a much wider range of organisms that was available to Bouley et al. (2000). Like Bouley et al. (2000), our data support independent gene expansion events in the arthropod and vertebrate lineages. The sequences of all insects available have two ankyrin genes, compared to single genes in available nematode sequences.

View larger version (23K): [in this window] [in a new window]   FIG. 4.— Phylogenetic tree of ankyrins. The ML tree shows the relation between ankyrins based on sequences corresponding to the ZU5 domain; Caenorhabditis elegans and Caenorhabditis briggsae served as outgroups. Bootstrap values calculated from 1,000 replicates are indicated.

 
All vertebrate sequences fall into three categories that we name Ank1, Ank2, and Ank3 in accordance with mammalian gene nomenclature. Interestingly, our data reveal six ankyrins in teleost fish, with two in each of the three ankyrin groups. This is consistent with the recently reported whole-genome duplication event in the ray-finned fish lineage (Van de Peer 2004; Volff 2005). An interesting question is whether the three paralog ankyrin gene pairs are functional or whether one copy of each has evolved as a pseudogene. To the extent that the Ensembl annotations reveal that the key functional domains are preserved in each predicted protein, any degeneration is limited.

The single ankyrin gene in the urochordate C. intestinalis appears to be the ortholog of all vertebrate ankyrins. This echoes the pattern in most other vertebrate superfamilies, which have a single C. intestinalis ortholog (Leveugle et al. 2004).

Origin and Function of the OTBD; Relation to Titin and Obscurin
Based on all the data above, we propose that the full OTBD represents a vertebrate adaptation. We looked for further arguments in favor of this hypothesis by analyzing the OTBD-binding sequences of titin and obscurin. The first two N-terminal Ig domains of human titin, which bind the sAnk1 OTBD, were used in a Psi-Blast search. Retrieved sequences revealed very high conservation among vertebrates (94%, 93%, and 82% identity when compared to Mus musculus, Gallus gallus, and Tetraodon nigroviridis, respectively). Homologous invertebrate sequences were much less conserved: C. elegans titin showed 40% identity; the most related insect titin sequence (an A. mellifera kettin isoform; Lakey et al. 1993) showed only 36% identity.

Regarding obscurin, we performed a Psi-Blast search using the C-terminal 400 residues of the human sequence. Unlike titin, overall conservation of this sequence among vertebrates is rather low: 64%, 38%, and 30% identity when compared to Rattus norvegicus, T. nigroviridis, and G. gallus, respectively. However, highly conserved, short sequence stretches observed within this region could be important for ankyrin binding. No significant hit with an invertebrate sequence was found, and no obscurin is annotated in either Wormbase or Flybase.

These results strongly suggest that protein-protein interactions between the cytoskeleton and muscle proteins titin and obscurin have evolved differently in vertebrates and invertebrates. We propose that vertebrate ankyrins gained specific functions in muscle by the acquisition of the OTBD, whereas any related functions in invertebrates are mediated by different protein interactions. This hypothesis is supported by the diverse molecular architecture of invertebrate sarcomeres and by the strong relationship between titin isoforms and sarcomere properties (Tskhovrebova and Trinick 2003).

	Conclusions

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
Here, we characterize a novel sequence from the C-terminal domain of ankyrins which is unique to vertebrate ankyrin genes. We describe this sequence as an OTBD because it is established that the two giant muscle proteins, obscurin and titin, bind this sequence in a product of the mammalian Ank1 gene.

The domain is encoded by three exons and is composed of up to three modules that potentially contain -helical elements; the modules are likely to be joined to each other by flexible linkers. Amino acid and nucleotide sequences as well as splice sites corresponding to modules I and II are highly conserved from fish to man. Tissue expression information derived from sequence analysis of cloned isoforms and ESTs suggests that the first OTBD exon, which encodes the entire module I, is expressed exclusively in muscle tissues.

The presence of conserved sequence corresponding to module II in C. elegans and C. intestinalis suggests that the OTBD has evolved by successive duplications of an ancestor sequence that first arose before the divergence of Ecdysozoa and deuterostomes (fig. 5).

View larger version (23K): [in this window] [in a new window]   FIG. 5.— Proposed model of evolutionary events leading to OTBD in present-day ankyrins. The figure shows a simplified summary of our data. An early metazoan acquired a sequence module (shown in black at the root of the tree), which was preserved after the Ecdysozoa/deuterostome split. Nematodes retain this in present-day UNC-44 ankyrin, but it has been lost from arthropod ankyrins. DNA closely related to this module is present in the ankyrin gene of the urochordate Ciona intestinalis. In vertebrates, the module was preserved in the coding sequence of ankyrin genes, underwent successive duplications to form modules I, II, and III, and thus gained function as an obscurin/titin-binding domain. The ankyrin gene itself also underwent successive duplications giving rise to Ank1, Ank2, and Ank3; module III has been lost from Ank3. The ray-finned fish lineage went through a round of tetraploidization/rediploidization, the legacy of which is that each of Ank1, Ank2, and Ank3 is duplicated in present-day teleosts and retains modules of the OTBD.

 
Insect ankyrin genes duplicated independently and have lost module II. Comparison of titin and obscurin sequences implicated in binding to Ank1-encoded OTBD show conservation only among vertebrates. These data support the hypothesis that the OTBD confers specialized function(s) to vertebrate ankyrins.

One of the features of vertebrate life is the increased size that vertebrate organisms have compared to their precursors. One view of ankyrins is that they are a solution to the problems of independent motility in Metazoa (Bennett and Baines 2001): for example, they contribute to membrane resilience to the forces of muscle contraction. By extension, the OTBD, by linking two giant cytoskeletal proteins to membrane structures, would consolidate the linkage of sarcomeres to the sarcoplasmic reticulum and eventually to the sarcolemma. Identification of further protein interactions of the OTBDs encoded by the other two ankyrin genes will greatly advance our understanding of the complex functions of the ankyrin family in vertebrate tissues.

	Supplementary Material

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
Supplementary Materials are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).

Table indicating the sources of nucleotide sequences used in this study (Hopitzan05-0255_ Table.pdf).

Multiple nucleotide sequence alignment corresponding to ZU5 domains from which the phylogenetic tree (fig. 4) was built (Hopitzan05-0255_ ZU5.pdf and Hopitzan05-0255_ ZU5.fasta).

Multiple nucleotide sequence alignment of conserved exonic sequences homologous to the 76-aa insert from which the multiple protein sequence alignment (fig. 3) was deduced (Hopitzan05-0255_ OTBD.pdf and Hopitzan05-0255_ OTBD.fasta).

	Acknowledgements

TOP Abstract Introduction Materials and Methods Results and Discussion Conclusions Supplementary Material Acknowledgements References

 
This work was supported by the Centre National de la Recherche Scientifique, the Universités Paris VI et VII, and by grants from the European Commission (HPRN-CT-2000-00096) and the Association Française contre les Myopathies (AFM). A.J.B. was a recipient of a Leverhulme Trust Research Fellowship and A.A.H. was supported in part by a fellowship from the AFM.

	Footnotes

 
Billie Swalla, Associate Editor

	References

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Accepted for publication August 26, 2005.

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