Amphioxus connectin exhibits merged structure as invertebrate connectin in I-band region and vertebrate connectin in A-band region

Connectin is an elastic protein found in vertebrate striated muscle and in some invertebrates as connectin-like proteins. In this study, we determined the structure of the amphioxus connectin gene and analyzed its sequence based on its genomic information. Amphioxus is not a vertebrate but, phylogenetically, the lowest chordate. Analysis of gene structure revealed that the amphioxus gene is approximately 430 kb in length and consists of regions with exons of repeatedly aligned immunoglobulin (Ig) domains and regions with exons of fibronectin type 3 and Ig domain repeats. With regard to this sequence, although the region corresponding to the I-band is homologous to that of invertebrate connectin-like proteins and has an Ig-PEVK region similar to that of the Neanthes sp. 4000K protein, the region corresponding to the A-band has a super-repeat structure of Ig and fibronectin type 3 domains and a kinase domain near the C-terminus, which is similar to the structure of vertebrate connectin. These findings revealed that amphioxus connectin has the domain structure of invertebrate connectin-like proteins at its N-terminus and that of vertebrate connectin at its C-terminus. Thus, amphioxus connectin has a novel structure among known connectin-like proteins. This finding suggests that the formation and maintenance of the sarcomeric structure of amphioxus striated muscle are similar to those of vertebrates; however, its elasticity is different from that of vertebrates, being more similar to that of invertebrates.     

Integrin evolution: insights from ascidian and teleost fish genomes.

Integrins are a family of alphabeta heterodimeric receptors essential to cell adhesion in all metazoans. In humans, the family consists of 18 alpha and 8 beta subunits that combine to form 24 dimers. Here, we present phylogenetic reconstructions for the alpha and beta integrin subunits based on sequences from 24 invertebrate and vertebrate species, including the fully sequenced genomes of the ascidian Ciona intestinalis (a urochordate) and the pufferfish Takifugu rubripes (a teleost). Both genomes contain integrin alpha subunits that have the inserted alphaI domain. As for the one alphaI domain containing integrin alpha subunit discovered earlier from the ascidian Halocynthia roretzi, the Ciona alphaI domains are missing the distinctive characteristics of mammalian collagen receptors and segregate from all vertebrate alphaI domain integrins in a phylogenetic tree, forming a new subgroup of alpha subunits with alphaI domains. Each of the pufferfish alphaI domain sequences does have characteristics of the collagen receptor alphaI domains, but no leukocyte-specific alphaI domains were found in pufferfish. Comparative protein modeling suggests that several of these fish alphaI domains are structurally compatible with binding to a GFOGER sequence in a collagen triple helix.     

Sodium dodecyl sulfate gel electrophoresis studies of connectin-like high molecular weight proteins of various types of vertebrate and invertebrate muscles

Using an SDS gel electrophoresis method, connectin, very high molecular weight (approximately 10(6) dalton) protein, was detected in an SDS extract of whole tissues of various types of muscles of vertebrates and invertebrates. Connectin bands were clearly recognized in all the types of striated muscles (skeletal and cardiac) of the vertebrates examined: rabbit, chicken, turtle, snake, newt, frog, and fish. This was also the case with skeletal muscle of prochordate, Amphioxus. In invertebrates, the situation was much complicated. Connectin-like protein bands were detected in C. elegans (nematode), but not in earthworm (annelid). Smaller sizes of proteins (approximately 10(6)) were faintly found in molluscan adductor muscles. In arthropods, connectin-like proteins were clearly detected in some muscles (e.g., claw muscles of crab and crayfish; leg muscles of several insects) but not at all in other muscles (e.g., tail muscles of crayfish and shrimp; thoracic muscles of some insects). These peculiar observations might be related to the presence of such specific elastic proteins as projectin in honeybee flight muscle. The present study has revealed that connectin is an elastic protein of vertebrate striated muscle, skeletal and cardiac muscles.     

Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.

The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled-coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin-like predecessor.     

Characterization of Brachyury-downstream notochord genes in the Ciona intestinalis embryo

The notochord has two major roles during chordate embryogenesis, as a source of inductive signals for the patterning of neural tube and paraxial mesoderm and as a supportive organ of the larval tail. Despite the recent identification of mutations that affect the notochord development in vertebrate embryos, little is known about genes that are expressed in the differentiating notochord itself. In the urochordate ascidian Ciona intestinalis, Brachyury (Ci-Bra) plays a key role in notochord differentiation. In a previous study, we isolated cDNA clones for nearly 40 potential Ci-Bra target genes that are expressed in notochord cells (H. Takahashi et al., 1999, Genes Dev. 13, 1519-1523). Here we characterized 20 of them by determining the complete nucleotide sequences of the cDNAs. These genes encode a broad spectrum of divergent proteins associated with notochord formation and function. Two genes encode ascidian homologs of the Drosophila Prickle LIM domain proteins and another encodes the ERM protein, all 3 of which appear to be involved in the control of cytoskeletal architecture. In addition, genes for netrin, leprecan, cdc45, ATP:citrate lyase, ATP sulfurylase/APS kinase, protein tyrosine phosphatase, beta4-galactosyltransferase, fibrinogen-like protein, divergent tropomyosin-like proteins, and Drosophila Pellino-like protein were identified. The observation of the netrin gene expression in the notochord may provide the first molecular evidence that the ascidian notochord is a source of signals as in vertebrates. In addition, the present information should be used to identify nonchordate deuterostome tissues homologous to the notochord as well as genes which are expressed in the notochord cells of vertebrate embryos.     

Computer-aided NMR assay for detecting natively folded structural domains

Structural genomics projects require strategies for rapidly recognizing protein sequences appropriate for routine structure determination. For large proteins, this strategy includes the dissection of proteins into structural domains that form stable native structures. However, protein dissection essentially remains an empirical and often a tedious process. Here, we describe a simple strategy for rapidly identifying structural domains and assessing their structures. This approach combines the computational prediction of sequence regions corresponding to putative domains with an experimental assessment of their structures and stabilities by NMR and biochemical methods. We tested this approach with nine putative domains predicted from a set of 108 Thermus thermophilus HB8 sequences using PASS, a domain prediction program we previously reported. To facilitate the experimental assessment of the domain structures, we developed a generic 6-hour His-tag-based purification protocol, which enables the sample quality evaluation of a putative structural domain in a single day. As a result, we observed that half of the predicted structural domains were indeed natively folded, as judged by their HSQC spectra. Furthermore, two of the natively folded domains were novel, without related sequences classified in the Pfam and SMART databases, which is a significant result with regard to the ability of structural genomics projects to uniformly cover the protein fold space.     

Fgf genes in the basal chordate <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

In vertebrates, a number of fibroblast growth factors (FGFs) have been shown to play important roles in developing embryos and adult organisms. However, the molecular relationships of the vertebrate FGFs are not yet completely understood, partly due to the divergence of their amino acid sequences. To solve this problem, we have identified six FGF genes in a basal chordate, the ascidian Ciona intestinalis. A phylogenetic analysis confidently assigned two of them to vertebrate FGF8/17/18 and FGF11/12/13/14, respectively. Based on the presence of the conserved domains within or outside of the FGF domains, we speculate that three of the other genes are orthologous to vertebrate FGF3/7/10/22, FGF4/5/6 and FGF9/16/20, respectively, although we cannot assign the sixth member to any of the vertebrate FGFs. A survey of the raw whole genome shotgun sequences of C. intestinalis demonstrated the presence of no FGF genes other than the six genes in the genome. The identification of these six FGF genes in the basal chordate gave us an insight into the diversification of specific subfamilies of vertebrate FGFs.     

Isolation and characterization of the iron-binding properties of a primitive monolobal transferrin from Ciona intestinalis

Transferrins are bilobal glycoproteins responsible for iron binding, transport, and delivery in many higher organisms. The two homologous lobes of transferrins are thought to have evolved by gene duplication of an ancestral monolobal form. In the present study, a 37.7-kDa primitive monolobal transferrin (nicatransferrin, or nicaTf) from the serum of the model ascidian species Ciona intestinalis was isolated by using an immobilized iron-affinity column and characterized by using mass spectrometry and N-terminal sequencing. The protein binds one equivalent of iron(III) and exhibits an electron paramagnetic resonance spectrum that is anion-dependent. The UV/vis spectrum of nicaTf has a shoulder at 330 nm in both the iron-depleted and the iron-replete forms, but does not display the approximately 460 nm tyrosine-to-iron charge transfer band common to vertebrate serum transferrins under the conditions investigated. This result suggests that iron may adopt a different binding mode in nicaTf compared with the more highly evolved transferrin proteins. This difference in binding mode could have implications for the physiological role of the protein in the ascidian. The genome of C. intestinalis has genes for both a monolobal and a bilobal transferrin, and the sequences of both proteins are discussed in light of the known features of vertebrate serum transferrins as well as other transferrin homologs.     

Evolution of the chordate muscle actin gene

Summary The ascidians Styela plicata, S. clava, and Mogula citrina are urochordates. The larvae of urochordates are considered to morphologically resemble the ancestral vertebrate. We asked whether larval and adult ascidian muscle actin sequences are nonmusclelike as in lower invertebrates, musclelike as in vertebrates, or possess characteristics of both. Nonmuscle and muscle actin cDNA clones from S. plicata were sequenced. Based on 27 diagnostic amino acids, which distinguish vertebrate muscle actin from other actins, we found that the deduced protein sequences of ascidian muscle actins exhibit similarities to both invertebrate and vertebrate muscle actins. A comparison to muscle actins from different vertebrate and invertebrate phylogenetic groups suggested that the urochordate muscle actins represent a transition from a nonmusclelike sequence to a vertebrate musclelike sequence. The ascidian adult muscle actin is more similar to skeletal actin and the larval muscle actin is more similar to cardiac actin, which indicates that the divergence of the skeletal and cardiac isoforms occurred before the emergence of urochordates. The muscle actin gene may be a powerful probe for investigating the chordate lineage. Offprint requests to: C.R. Tomlinson     

Local database and the search program for proteomic analysis of sperm proteins in the ascidian Ciona intestinalis

Separation of proteins by two-dimensional electrophoresis and following mass spectrometry (MS) is now a conventional technique for proteomic analysis. For proteomic analysis of a certain tissue with a limited information of primary structures of proteins, we have developed an analytical system for peptide mass fingerprinting in gene products in the testis of the ascidian Ciona intestinalis. Ciona sperm proteins were separated by two-dimensional gel electrophoresis and the tryptic fragments were subjected to MALDI-TOF/MS. The mass pattern was searched against on-line databases but resulted in less identification of these proteins. We have constructed a MS database from Ciona testis ESTs and the genome draft sequence, along with a newly devised, perl-based search program PerMS for peptide mass fingerprinting. This system could identify more than 80% of Ciona sperm proteins, suggesting that it could be widely applied for proteomic analysis for a limited tissue with less genomic information.     

Conservation of inter-residue interactions and prediction of folding rates of domain repeats

Domains are the main structural and functional units of larger proteins. They tend to be contiguous in primary structure and can fold and function independently. It has been observed that 10–20% of all encoded proteins contain duplicated domains and the average pairwise sequence identity between them is usually low. In the present study, we have analyzed the structural similarity between domain repeats of proteins with known structures available in the Protein Data Bank using structure-based inter-residue interaction measures such as the number of long-range contacts, surrounding hydrophobicity, and pairwise interaction energy. We used RADAR program for detecting the repeats in a protein sequence which were further validated using Pfam domain assignments. The sequence identity between the repeats in domains ranges from 20 to 40% and their secondary structural elements are well conserved. The number of long-range contacts, surrounding hydrophobicity calculations and pairwise interaction energy of the domain repeats clearly reveal the conservation of 3-D structure environment in the repeats of domains. The proportions of mainchain–mainchain hydrogen bonds and hydrophobic interactions are also highly conserved between the repeats. The present study has suggested that the computation of these structure-based parameters will give better clues about the tertiary environment of the repeats in domains. The folding rates of individual domains in the repeats predicted using the long-range order parameter indicate that the predicted folding rates correlate well with most of the experimentally observed folding rates for the analyzed independently folded domains.     

Rapidly evolving lamins in a chordate, Oikopleura dioica, with unusual nuclear architecture

Metazoan lamins are implicated in the organization of numerous critical nuclear processes. Among chordates, the appendicularian, Oikopleura dioica, has an unusually short life cycle involving rapid growth through extensive recourse to endoreduplication, a characteristic more associated with some invertebrates. In some tissues, this is accompanied by the formation of elaborate, bilaterally symmetric nuclear morphologies associated with specific gene expression patterns. Lamin composition can mediate nuclear shape in spermiogenesis as well as during pathological and normal aging and we have analyzed the O. dioica lamin and intermediate filament (IF) complement, comparing it to that present in other deuterostomes. O. dioica has one lamin gene coding two splice variants. Both variants share with the sister class ascidians a highly reduced C-terminal tail region lacking the immunoglobulin fold, indicating this derivation occurred at the base of the urochordate lineage. The OdLamin2 variant has a unique insertion of 63 amino acids in the normally short N-terminal region and has a developmental expression profile corresponding to the occurrence of endocycling. O. dioica has 4 cytoplasmic IF proteins, IF-A, C, Dalpha, and Dbeta. No homologues to the ascidian IF-B or F proteins were identified, reinforcing the suggestion that these proteins are unique to ascidians. The degree of sequence evolution in the rod domains of O. dioica cytoplasmic IF proteins and their closest ascidian and vertebrate homologues was similar. In contrast, whereas the rate of lamin B rod domain sequence evolution has also been similar in vertebrates, cephalochordates and the sea urchin, faster rates have occurred among the urochordates, with the O. dioica lamin displaying a far greater rate than any other lamin.