Phylogenomic analysis of vertebrate thrombospondins reveals fish-specific paralogues, ancestral gene relationships and a tetrapod innovation
- Patrick McKenzie†1,
- Seetharam C Chadalavada†1,
- Justin Bohrer†1 and
- Josephine C Adams1, 2Email author
© McKenzie et al; licensee BioMed Central Ltd. 2006
Received: 13 January 2006
Accepted: 18 April 2006
Published: 18 April 2006
Thrombospondins (TSPs) are evolutionarily-conserved, extracellular, calcium-binding glycoproteins with important roles in cell-extracellular matrix interactions, angiogenesis, synaptogenesis and connective tissue organisation. Five TSPs, designated TSP-1 through TSP-5, are encoded in the human genome. All but one have known roles in acquired or inherited human diseases. To further understand the roles of TSPs in human physiology and pathology, it would be advantageous to extend the repertoire of relevant vertebrate models. In general the zebrafish is proving an excellent model organism for vertebrate biology, therefore we set out to evaluate the status of TSPs in zebrafish and two species of pufferfish.
We identified by bioinformatics that three fish species encode larger numbers of TSPs than vertebrates, yet all these sequences group as homologues of TSP-1 to -4. By phylogenomic analysis of neighboring genes, we uncovered that, in fish, a TSP-4-like sequence is encoded from the gene corresponding to the tetrapod TSP-5 gene. Thus, all TSP genes show conservation of synteny between fish and tetrapods. In the human genome, the TSP-1, TSP-3, TSP-4 and TSP-5 genes lie within paralogous regions that provide insight into the ancestral genomic context of vertebrate TSPs.
A new model for TSP evolution in vertebrates is presented. The TSP-5 protein sequence has evolved rapidly from a TSP-4-like sequence as an innovation in the tetrapod lineage. TSP biology in fish is complicated by the presence of additional lineage- and species-specific TSP paralogues. These novel results give deeper insight into the evolution of TSPs in vertebrates and open new directions for understanding the physiological and pathological roles of TSP-4 and TSP-5 in humans.
The thrombospondins (TSPs) are extracellular, calcium-binding glycoproteins with roles in cell-extracellular matrix interactions, angiogenesis and tumor growth, synaptogenesis, and the organization of connective extracellular matrix (ECM) [1–4]. TSPs have been well-conserved in animal evolution as ECM components. The Drosophila melanogaster genome encodes a single TSP which is dynamically expressed during embryogenesis at sites of tissue remodeling including imaginal discs, precursor myoblasts, and muscle/tendon attachment sites . A TSP of the kuruma prawn, Marsupenaeus japonicus, is a major component of oocyte cortical rods, specialized storage structures for ECM components that are released to cover the egg upon fertilization . Five TSPs, designated TSP-1 to TSP-5, are encoded in the human and mouse genomes, all of which have dynamic and specific patterns of expression during embryogenesis and in adult life (reviewed in ). Mouse gene knockouts prepared for TSP-1, TSP-2, TSP-3, and TSP-5 have demonstrated distinct roles for these family members in normal tissue development and/or adult physiology and pathology [7–10].
All TSPs have the same domain architecture in their C-terminal regions, consisting of EGF domains, a series of calcium-binding, TSP type 3 repeats and a globular C-terminus that is related in structure to L-type lectins [11, 12]. The entire C-terminal region forms a structural unit in which calcium-binding has a critical role in the physical conformation and functional properties [13–15]. Many TSPs also contain a globular amino-terminal domain that folds as a laminin G-like domain . Vertebrate TSPs can be grouped into two structural subgroups, A and B, according to their molecular architecture and oligomerization status . TSP-1 and TSP-2, in subgroup A, are distinguished by the presence of a von Willebrand factor type_C (vWF_C) domain and three thrombospondin type 1 repeats adjacent to their N-terminal domains and oligomerize as trimers. TSP-3, TSP-4 and TSP-5, (TSP-5 is also known as cartilage oligomeric matrix protein, COMP ), in subgroup B lack these domains, contain an additional EGF domain and assemble as pentamers [19–21]. TSP-5/COMP also lacks a distinct N-terminal domain. The multidomain and multimeric organization of TSPs mediate their complex and tissue-specific physiological functions that are known in mammals.
Importantly, TSP family members have multiple roles in inherited and acquired human disease. TSP-5/COMP is most highly expressed in cartilage and point mutations in its type 3 repeats and L-lectin domain are causal in pseudoanchrondroplastic dysplasia (PSACH) and some forms of multiple epiphyseal dysplasia (MED) (OMIM 117170 and 132400). These mutations cause functional perturbation through effects on calcium-binding and intra- or intermolecular interactions that impair both the post-translational processing and secretion of TSP-5/COMP and its interactions with other ECM molecules in cartilage ECM (reviewed in ). Single nucleotide polymorphisms (SNPs) in the coding sequences of TSP-1 and TSP-4 are associated with increased risk of familial premature heart disease [23, 24]. These coding SNPs also alter the calcium-binding and physical properties of TSP C-terminal regions, correlating with altered interactions with and signaling effects on vascular cells [25–27]. In contrast, a SNP in the 3' untranslated region of TSP-2 has protective effects against myocardial infarction . Also indicative of a protective role in the myocardium, TSP-2 gene knockout mice have increased susceptibility to angiotensin II-induced cardiac failure . TSP-1 and TSP-2 are also known as natural inhibitors of angiogenesis that can suppress the vascularization of tumors by triggering microvascular endothelial cell apoptosis by binding CD36 (reviewed in ). Down-regulation of TSP-1 has been documented in certain human tumors and the expression level of TSP-1 impacts on tumor growth [29–31]. A TSP-1 peptide mimetic is in clinical trial as a novel anti-cancer therapy .
To date, the functions of TSPs in vivo have been examined experimentally only in mice, yet in general the zebrafish is proving an excellent model for analysis of the musculoskeletal and cardiovascular systems and has the definite advantages of a faster lifecycle, large numbers of progeny, and accessibility of all embryonic stages for experimental analysis and imaging [33, 34]. However, despite an intense research focus on mammalian TSPs, the phylogeny of TSPs in other vertebrates is not well understood. With these considerations in mind, we have combined molecular phylogenetic and phylogenomic approaches to address whether fish would be appropriate model organisms for future experimental study of TSPs in relation to their roles in human disease.
An overview of TSPs in vertebrate subphyla
Dataset of vertebrate TSPs compiled from fully-sequenced genomes and genome-predicted proteins for this study.
a: SINFRUP00000078556 (scaffold_376)
b: SINFRUP00000091179 (scaffold_260)
a :CAG03524 (chro 14)
b :CAG10667 (Chro 10)
CAG09456 (chro 17)
a:CAG07859 (chro 12)
b:CAG00605 (chro 1)
c:CAG06350 (chro 4)
CAI20599 (chro 20)
a:ENSDARP00000057230 (chro 13)
b:ENSDARP00000030477 (chro 12)
a:NP_775332 (chro 16)
b:XP_690679 (unmap, Zv5 NA2846)
c:ENSDARP00000022442 (partial sequence, chro 5)
NP_001011401 (scaffolds_3931 and _3512)
XP_421205 (chro 5)
XP_419599 (chro 3)
XP_424763 (chro Z)
XP_418238 (chro 28)
A40558 (chro2 band F)
Q03350 (chro17 band F)
NP_038719 (chro 3 band E3)
In contrast, our searches of the three fish genomes identified 6 to 8 TSPs encoded in each genome (Table 1). The T. rubripes and T. nigroviridis genomes each encoded six TSP sequences. By BLASTP searches, these sequences grouped as homologues of TSP-1, TSP-2, TSP-3 or TSP-4 (Table 1). In the case of T. rubripes, two sequences were most similar to TSP-1, two sequences were most similar to TSP-4, and the remaining two sequences were most similar to TSP-2 or TSP-3, respectively. Each TSP-encoding sequence was located on a different genomic scaffold (Table 1). In T. nigroviridis, two sequences were most closely-related to TSP-1, one to TSP-2, and three were most similar to TSP-4. The T. nigroviridis genome has been mapped physically  and each of the six TSPs were located on a different chromosome (Table 1).
From the zebrafish genome, assembly Zv5 of August 2005, we identified 8 TSP-like sequences. As in the pufferfish, the D.rerio TSP sequences appeared homologous to either TSP-1, -2, -3 or -4 (Table 1). Two of the TSPs corresponded exactly to published sequences for D. rerio TSP-3 and TSP-4 predicted from cDNA  (Table 1). The other six sequences encoded a predicted TSP-1, two TSP-2s, another TSP-3, and two other predicted TSP-4-like polypeptides. The six mapped genes are encoded at separate loci (Table 1). We took advantage of the large number of ESTs available from zebrafish in dbEST (634, 605 as of August 1, 2005) to establish whether all eight TSPs are transcribed : ESTs of 100 % identity were identified for six of the TSPs, but not for the TSP-2 on chromosome 12 or the partial TSP-4c sequence on chromosome 5. Our further analysis therefore focused on the six TSPs that are definitely transcribed.
Relationship of fish and tetrapod TSPs : assessment by molecular phylogeny
We examined the domain architecture of the fish TSPs through the CDD, SMART and InterPro databases. All the fish-encoded sequences identified as homologous to mammalian TSP-1 and TSP-2 contained vWF_C and TSP type 1 domains and were thus confirmed as belonging to TSP subgroup A. Those identified as subgroup B homologues on the basis of the oligomerization domain lacked these domains and included an additional EGF domain. All known TSPs contain at least one EGF domain with a consensus sequence for beta-hydroxylation of an asparagine residue, indicative of a capacity for calcium-binding, , and this trait was conserved in all the newly-identified fish TSPs (data not shown). Human and mouse TSP-3 and TSP-4 are distinguished from TSP-5 by the presence of a 4-amino acid insert motif, PPGP, at the end of the sixth type 3 repeat that may alter calcium-binding activity . Examination of the sixth type 3 repeat of the subgroup B TSPs in our dataset revealed that PPGP motifs were present in the fish TSP-3, TSP-4a and TSP-4b sequences and also, unexpectedly, in each of TSP-3, TSP-4 and TSP-5 from X. tropicalis and chicken. D. rerio TSP-3a has an unusually long repeat that contains a variant motif, GIGP (Fig. 1B). These results reveal that the absence of the PPGP motif from mammalian TSP-5 is a secondary trait that is not inherent to all forms of TSP-5.
Relationships of fish TSP-4-like sequences to human TSP-3, TSP-4, or TSP-4.
% Identity to Human :
TSP C terminal region of:
Syntenic relationships of tetrapod and fish TSP genes : TSP-5/COMP is encoded at an ancient locus
The species-specific encoding of paralogous pairs of TSP -1-, TSP-3-, or TSP-4 in fish raised the possibility that these TSP genes exist as a result of the additional genome duplication that took place early in the Actinopterygii (ray-finned fish) lineage [36, 45, 46]. In addition, the intriguing possible relationship between fish TSP-4-like sequences and TSP-5 suggested that tetrapod TSP-5/COMP might have arisen through a relatively recent gene duplication of TSP-4 with subsequent loss of the exons encoding the amino-terminal domain. If TSP-5/COMP did arise from a recent TSP-4 gene duplication then, according to the molecular clock hypothesis, the encoded protein would be expected to have closer sequence identity to TSP-4 than to other members of subgroup B . Our molecular phylogenies (Fig. 2) and other phylogenetic studies have not convincingly resolved the relationships of tetrapod TSP-3, TSP-4 and TSP-5 [48, 49]. The overall pairwise sequence identities of TSP-3, TSP-4, and TSP-5 are very similar in any given tetrapod species. For example, in pairwise comparisons of the region from the coiled-coil domain to the C-terminus of human subgroup B TSPs, the identity between TSP-3 and TSP-4 is 60 %, between TSP-3 and TSP-5 is 58 %, and between TSP-4 and TSP-5 is 63 %. Similar results are obtained if the comparison is made in other tetrapod species (data not shown). Furthermore, the exon organization of the TSP-3, TSP-4 and TSP-5 genes in human and mouse are near-identical, with the TSP-5/COMP gene lacking the four exons that encode the amino-terminal domain [50–52]. Therefore, as an independent approach to understand the evolutionary relationships between fish and tetrapod TSPs, in particular the relationship of TSP-4 and TSP-5, we undertook a phylogenomic analysis of the conservation of neighboring genes around each TSP gene locus in the available mapped fish and tetrapod genomes. Conservation of synteny is a powerful approach to reconstruct evolutionary processes when multiple physically-mapped genome sequences are available. The criterion for conservation of synteny is that orthologous gene loci are linked in different species, irrespective of the exact gene order or the presence of non-conserved intervening genes .
Interestingly, the TSP-4 genes of human, mouse, and chicken are all immediately adjacent to the gene encoding another member of the metaxin family, metaxin-3 (MTX3). Three other flanking genes are conserved between human, mouse and chicken, CMYA5, PAPD4 and ZFYVE16. The gene encoding Riken A130038L21 is also conserved adjacent to the mouse and chicken TSP-4 genes (Fig. 5B). Of the TSP-4-like genes of fish, TSP-4a in T. nigriviridis is encoded adjacent to MTX3, CMYA5 and A130038L21 and was thus established as syntenic with tetrapod the TSP-4 gene (Fig. 5B). Genes adjacent to T. rubripes TSP-4a did not include MTX3 but were similar to those adjacent to T. nigriviridis TSP-4c (discussed further below).
In T. nigroviridis, the TSP-4c gene has gene neighbors unrelated to those of TSP-4 or TSP-5. The same gene neighbors were conserved adjacent to T. rubripes TSP-4a (Fig. 5B). We infer that the fish-specific duplication of the TSP-4 gene was accompanied in the puffer-fish lineage by transposition of one of the duplicated genes. Both paralogues have been retained in T. nigriviridis whereas the TSP-4 gene at the ancestral locus has been lost in T. rubripes.
Evidence for paralogous relationships between four TSP-encoding loci in the human genome
The above results clarified the identities of fish TSP genes in relation to tetrapod TSP genes, yet still did not resolve certain ambiguities with regard to the relationships of the TSP-3, TSP-4 and TSP-5 genes. At the level of genome organization, the conserved synteny of both the TSP-3 and TSP-4 genes with genes encoding members of the metaxin family suggests that the TSP-3 and TSP-4 genes lie within paralogous genomic regions that arose from the same ancestral DNA duplication event . On this basis, the TSP-3 and TSP-4 genes can be considered closely related. Because no metaxin gene is found adjacent to the TSP-5/COMP locus, or indeed on the same chromosome in any of the organisms studied, and other local conserved gene neighbors of the TSP-5/COMP gene are distinct from those conserved adjacent to the TSP-4 gene (Fig. 5B and Fig. 6), the TSP-5 gene appears more remote from TSP-3 and TSP-4. Yet, by criteria of protein sequence relationships, the new data from fish demonstrate a very close relationship between TSP-4-like coding sequences and TSP-5 (Fig. 2). To integrate these separate and apparently paradoxical pieces of data, we took advantage of the extensive analysis of human genome sequence organization that has identified large paralogous chromosomal regions within the human genome itself. The existence of such regions provides evidence for the rapid evolution of vertebrate genomes through large-scale block or genome-wide DNA duplication in an ancestral chordate [57–59]. We tested whether any of the five TSP-encoding loci are located in paralogous region of the human genome by searching the "dataset of paralogons in the human genome", version 5.28 . The human genome is suitable for this form of analysis because the rate of DNA rearrangement has been slower than in rodents .
To substantiate these findings, additional paralogy searches were carried out for the three members of the metaxin family: the searches with metaxin-1 and metaxin-3 again identified the paralogy between the chromosomal regions of TSP-3 and TSP-4. No paralogous region was identified with regard to the metaxin-2 locus on chromosome 2 (data not shown). Of the other gene pairs identified within the paralogous regions of the TSP-3, TSP-4 and TSP-5 genes, members of the MEF (myocyte enhancer factor) and KCNN (potassium intermediate/small conductance calcium-activated channel, subfamily N) families were consistently present in all the paired blocks (Fig. 7A–C; KCNN paralogy is not shown in Fig. 7A, but KCNN2 is located at 113.73 Mb of chromosome 5 and KCNN3 is located at 151.7Mb of chromosome 1; . Thus, the ancestral chromosomal region likely included ancestral MEF and KCNN genes in the vicinity of a TSP gene. We tested this idea by examining whether MEF or KCNN family members are also syntenic with TSPs in other vertebrates. From the available mapping information, MEF-2D in mouse and zebrafish are located on the same chromosomes as the TSP-3 gene (TSP-3a on chromosome 16 in the case of zebrafish), and MEF-2C and MEF-2B in the mouse are located on the same chromosomes as TSP-4 and TSP-5, respectively. KCNN1 is also on mouse chromosome 8; KCNN2 is syntenic with TSP-4 in chicken but not in mouse, and KCNN3 is syntenic with TSP-4b (i.e. the TSP-5 locus) in T. nigriviridis. These data reinforce the intepretation that the TSP-3, TSP-4 and TSP-5 genes have evolved as a consequence of duplications of the same ancestral genomic region.
Our study, initiated with the aim of assessing the suitability of zebrafish as a model organism for future experimental study of TSPs in relation to their roles in human disease, delivers some unexpected conclusions that change current perspectives on the TSP gene family in vertebrates. Based on a combination of molecular phylogenetic and phylogenomic approaches, we propose a new model for the evolution of TSPs in vertebrates.
The encoding of large numbers of TSPs in three species of fish, that include paralogous pairs of TSP-1, TSP-3, or TSP-4 genes, is in line with the strong evidence that ray-finned fish underwent an additional whole genome duplication after the divergence of the bony fish and tetrapod lineages around 450 million years ago [36, 45–47]. In general, after a gene duplication event, reduced selection pressure on one of the paralogous genes can have several consequences. One gene may be lost relatively rapidly, or both genes may be retained and diverge functionally, either by sub-specialization of the original function or by evolving new functions [62, 63]. For the TSP family, the three fish species provide evidence of distinct lineage-specific events involving loss or retention of different TSP paralogues. For example, T. nigriviridis encodes two TSP-1s but does not encode a TSP-3, whereas D. rerio encodes two TSP-3s and a single TSP-1 (Table 1). The retention of both members of a paralogous pair may have resulted in functional specialization. Thus, each of the fish TSP-1 or TSP-3 paralogues could have a subset of the functions of tetrapod TSP-1 or TSP-3, or may have evolved distinct and novel functions.
We could readily identify synteny of the TSP-encoding loci in fish with the chromosomal regions of tetrapod TSP genes. This finding establishes that precursors of the TSP-1 to TSP-5 genes were all present within corresponding ancestral genomic contexts in the last common ancestor of bony fish and tetrapods. This state appears to have originated within the chordate lineage. The Ciona intestinalis (an invertebrate chordate) genome encodes a smaller number of TSPs; yet, because both A and B forms of TSPs are present, it is clear that the existence of A and B forms predates the whole genome duplications that occurred in the early stages of vertebrate evolution ([5, 64]; our unpublished data). These conclusions are supported by evidence that large scale gene duplication activity increased substantially after the divergence of amphioxus (a cephalochordate) from the vertebrate lineage . Whereas Ciona intestinalis encodes a single subgroup A TSP (GenBank AAS45620; ), inspection of available ESTs from a cartilaginous fish, the little skate Leucoraja erinacea, indicates that transcripts corresponding to both TSP-1 and TSP-2 are present (GenBank CV068535 and CV067510). Thus, for subgroup A, an expansion of gene number appears common to both cartilaginous and bony fish. This observation is in agreement with a recent statistical estimate that most vertebrate-specific gene duplications occurred before the separation of cartilaginous and bony fish . For additional clarification of the phasing of expansion of the TSP gene family in the chordate and vertebrate lineages, the genome sequences of a jawless vertebrate (i.e., lamphrey or hagfish) and a cephalochordate are needed.
Our studies also lead to the novel and surprising conclusion that the TSP-5/COMP protein sequence has evolved to its current state as an innovation of tetrapods. In human, mouse, chicken and X. tropicalis, TSP-4 and TSP-5 protein sequences are readily distinguished by BLAST searches or multiple sequence alignment, even without consideration of the presence or absence of the TSP amino-terminal domain (e.g. [5, 11]; Table 2). In contrast, in fish, the proteins encoded at the TSP-5/COMP locus have sequence character most similar to TSP-4, even when the full-length sequence is used as the BLASTP query. None of the invertebrate TSPs identified to date has TSP-5 character ([5, 6, 68]; our unpublished observations). Thus, on the basis that the TSP-5 locus arose through duplication of an ancestral TSP-4-like gene, it appears that the encoded protein retained TSP-4-like character in fish and has evolved distinct and novel features in tetrapods. Given the significant role of TSP-5/COMP in mammalian cartilage, it is tempting to speculate that the polypeptide sequence evolved rapidly in tetrapods under the altered selection pressures imposed on the bony endoskeleton by the switch from aquatic swimming to terrestrial locomotion. Although it has been accepted that TSP-4 and TSP-5 have separate biological activities in mammals, there are interesting hints of over-lap. For example, both TSP-4 and TSP-5 are expressed in blood vessel walls [69, 70]. In chick embryos, TSP-4 is transiently expressed in cartilage in association with the initial stages of osteogenesis . Further consideration of similarities and differences in the characteristics, regulation, and pathologies of TSP-4 and TSP-5 may open fruitful novel directions for future research.
Combining the approaches of molecular phylogeny and phylogenomic analysis of chromosomal context is a generally applicable strategy to improve the identification of orthologous relationships between members of complex gene families across species. The identification of numerous fish TSPs and the discovery of the unexpectedly close relationship between TSP-4 and TSP-5 raise fascinating questions about the fundamental roles of TSPs in fish. New directions are identified for studies of the pathophysiological roles of TSP-4 and TSP-5 in human disease.
Dataset of known vertebrate TSPs
The following TSP protein sequences, predicted from sequencing of full-length cDNAs, were included in our studies : from Homo sapiens, TSP-1 (GenBank Accession P07996); TSP-2 (P35442); TSP-3 (P49746); TSP-4 (P35443) and TSP-5/COMP (P49747); from Mus musculus, TSP-1 (A40558); TSP-2 (Q03350); TSP-3 (U16175); TSP-4 (AF152393); TSP-5/COMP (AF033530); from Gallus gallus, TSP-2 (L81165; 72), and from Danio rerio, TSP-3 and TSP-4 (NP_775332 and NP_775333; ). Partial sequences predicted from cDNA included G. gallus TSP-1 (U76994; ), TSP-3 (L81165; ) and TSP-4 (L27263; ).
Identification of novel TSPS in fully-sequenced genomes of vertebrates and from expressed sequence tags
Human TSP-1 and TSP-5 were used as the query sequences in TBLASTX or BLASTP searches carried out at NCBI and UCSC Genome Bioinformatics portals against the fully-sequenced genomes and, as available, the genome-predicted proteins of the fish Takifugu rubripes (; assembly 3 with 5.7× coverage); Tetraodon nigroviridis (; assembly 1.1 with 8.3× coverage); Danio rerio ( and from August 2005, Zv5 with 5–7× coverage ); the amphibian Xenopus tropicalis, (; assembly 4.1, 7.65× coverage, searched via DOE Joint Genome Institute), and the bird Gallus gallus (; assembly 1 with 6.6× coverage). Accession and scaffold numbers used in this article are as of October 2005. Each matching sequence returned with an expectation value less than e= 0.0001 was used to query the GenBank non-redundant protein database, to establish the assignment as a TSP and to identify which of the mammalian TSPs 1–5 had the closest sequence identity. X. tropicalis sequences were also compared with available sequencs from Xenopus laevis : TSP-1 (P3544); TSP-3 (AAH48222) and TSP-4 (Z19091) . Sequences were also searched by TBLASTX against dbEST (database of expressed sequence tags) at NCBI for ESTs from the corresponding organism, to establish the existence of transcribed sequences corresponding to the open reading frame predicted from genomic DNA. In some cases, EST sequences and comparisons with known TSPs were used to extend or correct the genome-predicted sequences. Searches of dbEST for TSP ESTs in other fish species were carried out by limiting the query to the Entrez criteria Chondrichthyes or Teleostomi. Taxonomic classifications were based on the Tree of Life Project .
Analysis of domain architecture and oligomerization potential of novel TSPs
The domain architecture of the predicted novel TSP proteins was evaluated by searches against the Conserved Domain Database (CDD) database at NCBI , the Simple Modular architecture research tool (SMART) domain database at EMBL , and the InterPro database  via ExPasy , supplemented by manual inspection. Sequences were assigned to TSP sub-group A if they contained a vWF-C domain and TSP type 1 repeats and to TSP subgroup B if these domains were not present and the sequence included additional EGF-like domains . Sequences were analyzed for the presence of a coiled-coil region using the program COILS . Although most sequences in our set covered full-length TSPs, G. gallus TSP-3 is at present identified only as a partial cDNA that does not include the coiled-coils .
Multiple sequence alignment and phylogenetic trees
Multiple sequence alignments of the coiled-coil domains were prepared in TCOFFEE, that combines pairwise/global and local alignment methods into a single model . Alignments of the sixth type 3 repeat or the C-terminal region (i.e. the type 3 repeats and L-lectin domain) were prepared by the progressive, neighborhood-joining alignment method, CLUSTALW . The C-terminal region was also aligned by the TCOFFEE algorithm. The multiple sequence alignments are presented in Boxshade 3.2. For preparation of phylogenetic trees, gaps due to variations present in less than 10 % of the sequences were removed from the alignments. Unrooted trees were constructed either from the Phylip distance matrix output of the alignments in DRAWTREE, using UCSD Biology workbench 3 tools , or by the maximum-likelihood method, PHYML, using the WAG substitution model and 100 bootstrap cycles . Unrooted trees are presented in D.G. Gilbert's Phylodendron, version 0.8d .
Identification of syntenic relationships
The chromosomal locations of TSP-encoding genes were identified by TBLASTN searches of the physically-mapped genomes of the human (build 35.1) , mouse (build 34.1), , and chicken (build 1.1)  through the BLAST Genomes interface at NCBI, using in each case the TSP protein sequences encoded within the genome of interest as the queries. For each TSP gene in human, mouse and chicken, local syntenic genes were identified using the map viewer and Genemap Tables at NCBI. In the case of Tetraodon nigroviridis, positions of TSP-encoding genes were identified within the Genoscope physically-mapped shotgun scaffold sequences. This permitted their assignment to a chromosome and identification of the GenBank accession numbers for the neighboring predicted protein-coding sequences. The identification of each predicted protein was then accomplished by BLASTP searches of GenBank. Genomic locations and gene neighbors were also analyzed by BLAT search of the genome at UCSC Genome Bioinformatics. In the case of Takifugu rubripes, the predicted TSP protein sequences were mapped onto the genomic scaffolds by TBLASTN searches. Adjacent coding sequences on the scaffold were then identified by BLASTX searches of GenBank proteins and by viewing of genome-predicted proteins on the genome contigs at UCSC Genome Bioinformatics. In the case of D. rerio, initial identification of gene neighbors was made from the NCBI Genemap Table of the 2004 Zv4 assembly. Gene neighbors were re-confirmed on the contigs of the 2005 scaffold assembly Zv5 at Ensembl (EBI) . For identification of parologous TSP-encoding regions in the human genome, the database of "Paralogons in the human genome", version 5.28, was searched . In the figures, genes encoding known proteins are identified according to HUGO gene names where available. GenBank gene locus numbers, or accession numbers of the encoded proteins, are given for previously unknown genes. Because TSPs have not yet been assigned gene symbols in all the species studied here, they are all designated TSP-1, TSP-2, etc, in Figs. 3, 4, 5, 6.
basic local alignment search tool
cartilage oligomeric matrix protein
epidermal growth factor
expressed sequence tag
Online Mendelian inheritance in man
We thank Mario Caccamo, Wellcome Trust Sanger Institute, for advice on the Zebrafish v5 genome assembly. Supported by SCCOR P50 HL077107. Research in JCA's laboratory is supported by NIGMS, NIH.
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