The invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate Ciona intestinalis

The G-protein coupled cannabinoid receptors CB(1) and CB(2) are activated by Delta(9)-tetrahydrocannabinol, the psychoactive ingredient of cannabis, and mediate physiological effects of endogenous cannabinoids ('endocannabinoids'). CB(1) genes have been identified in mammals, birds, amphibians and fish, whilst CB(2) genes have been identified in mammals and in the puffer fish Fugu rubripes. Therefore, both CB(1) and CB(2) receptors probably occur throughout the vertebrates. However, cannabinoid receptor genes have yet to be identified in any invertebrate species and the evolutionary origin of cannabinoid receptors is unknown. Here we report the identification of CiCBR, a G-protein coupled receptor in a deuterostomian invertebrate - the urochordate Ciona intestinalis - that is orthologous to vertebrate cannabinoid receptors. The CiCBR cDNA encodes a protein with a predicted length (423 amino-acids) that is the intermediate of human CB(1) (472 amino-acids) and human CB(2) (360-amino-acid) receptors. Interestingly, the protein-coding region of the CiCBR gene is interrupted by seven introns, unlike in vertebrate cannabinoid receptor genes where the protein-coding region is typically intronless. Phylogenetic analysis revealed that CiCBR forms a clade with vertebrate cannabinoid receptors but is positioned outside the CB(1) and CB(2) clades of a phylogenetic tree, indicating that the common ancestor of CiCBR and vertebrate cannabinoid receptors predates a gene (genome) duplication event that gave rise to CB(1)- and CB(2)-type receptors in vertebrates. Importantly, the discovery of CiCBR and the absence of orthologues of CiCBR in protostomian invertebrates such as Drosophila melanogaster and Caenorhabditis elegans indicate that the ancestor of vertebrate CB(1) and CB(2) cannabinoid receptors originated in a deuterostomian invertebrate.     

Molecular cloning of 5-hydroxytryptamine (5-HT) type 1 receptor genes from the Japanese puffer fish,Fugu rubripes

To characterize the structure of Fugu G-protein coupled receptor family and its evolutionary divergence, we have cloned and sequenced the Fugu 5-HT type 1 receptor genes by Polymerase Chain Reaction (PCR) with degenerate primers followed by phage library screening. The analysis of the deduced amino acid sequences showed that F1Aα and F1Aβ have the highest homology to the human 5-HT1A receptor (71.5% and 63.7%, respectively). Another clone, F1D, showed highest (70.5%) homology to the human type 1D receptor. The amino acid residues that are important for ligand binding have been conserved in these Fugu genes. The phylogenetic tree analysis suggests that the duplication event of the Fugu type 1A receptor may have occurred after the divergence of Fugu and the tetrapod lineage.     

Fish possess multiple copies of fgfrl1, the gene for a novel FGF receptor

FGFRL1 is a novel FGF receptor that lacks the intracellular tyrosine kinase domain. While mammals, including man and mouse, possess a single copy of the FGFRL1 gene, fish have at least two copies, fgfrl1a and fgfrl1b. In zebrafish, both genes are located on chromosome 14, separated by about 10 cM. The two genes show a similar expression pattern in several zebrafish tissues, although the expression of fgfrl1b appears to be weaker than that of fgfrl1a. A clear difference is observed in the ovary of Fugu rubripes, which expresses fgfrl1a but not fgfrl1b. It is therefore possible that subfunctionalization has played a role in maintaining the two fgfrl1 genes during the evolution of fish. In human beings, the FGFRL1 gene is located on chromosome 4, adjacent to the SPON2, CTBP1 and MEAEA genes. These genes are also found adjacent to the fgfrl1a gene of Fugu, suggesting that FGFRL1, SPON2, CTBP1 and MEAEA were preserved as a coherent block during the evolution of Fugu and man.     

Long-term evolution of the CAZY glycosyltransferase 6 (ABO) gene family from fishes to mammals--a birth-and-death evolution model

Functional glycosyltransferase 6 (GT6) family members catalyze the transfer of galactose or N-acetylgalactosamine in alpha1,3 linkage to various substrates and synthesize structures related to the A and B histo-blood group antigens, the Forssman antigen, alphaGal epitope, and iGb3 glycolipid. In rat, mouse, dog, and cow genomes, we have identified three new mammalian genes (GT6m5, GT6m6, and GT6m7) encoding putative proteins belonging to the GT6 family. Among these, GT6m6 protein does not display major alterations of the GT6 motifs involved in binding of the divalent cation and the substrate. Based on protein sequence comparison, gene structure, and synteny, GT6 homologous sequences were also identified in bird, fish, and amphibian genomes. Strikingly, the number and type of GT6 genes varied widely from species to species, even within phylogenetically related groups. In human, except ABO functional alleles, all other GT6 genes are either absent or nonfunctional. Human, mouse, and cow have only one ABO gene, whereas rat and dog have several. In the chicken, the Forssman synthase-like is the single GT6 family member. Five Forssman synthase-like genes were found in zebrafish, but are absent from three other fishes (fugu, puffer fish, and medaka). Two iGb3 synthase-like genes were found in medaka, which are absent from zebrafish. Fugu, puffer fish, and medaka have an additional GT6 gene that we termed GT6m8, which is absent from all other species analyzed here. These observations indicate that individual GT6 genes have expanded and contracted by recurrent duplications and deletions during vertebrate evolution, following a birth-and-death evolution type.     

Isolation and structural assignment of 5-deoxytetrodotoxin from the puffer fish Fugu poecilonotus.

A novel 10,7-lactone type of tetrodotoxin analog, 5-deoxytetrodotoxin, was isolated from the puffer fish, Fugu poecilonotus, and its structure was assigned by spectroscopic methods.     

Characterization of the mitochondrial respiratory pathways in Candida albicans

cDNA and genomic clones encoding guanylate cyclase activating proteins (GCAP1 and GCAP2) in the Japanese puffer fish (Fugu rubripes) were identified by probing, respectively, a retinal cDNA library and a whole genomic cosmid library with human GCAP1 and GCAP2 cDNA probes. Clones were identified as GCAP1 and GCAP2 on the basis of amino acid identity with the equivalent frog sequences and their placement into GCAP1 and GCAP2 clades within a GCAP phylogenetic tree. The Fugu genes have an identical four exon/three intron structure to GCAP1 and GCAP2 genes from other vertebrates but the introns are smaller, with the result that the four exons spread over approximately 1 kb of DNA in each case. The two genes are separated on to separate cosmids. However, the results of Southern analysis of the cosmids and of genomic DNA are consistent with a tail-to-tail gene arrangement, as in other species, but with a surprisingly large intergenic separation of around 18.7 kb. Recombinant Fugu GCAP1 failed to activate human retinal guanylate cyclase (retGC) in vitro although CD spectroscopy shows that the protein is folded with a similar secondary structure to that of human GCAP1. The failure to activate may be due therefore to a lack of molecular compatibility in this heterologous assay system.     

Comparative genomics of the human and Fugu voltage-gated calcium channel alpha1-subunit gene family reveals greater diversity in Fugu

Extensive search for the orthologs of 10 human voltage-gated calcium channel (VGCC) alpha(1)-subunit genes in the Fugu genome sequence revealed 21 alpha(1)-subunit genes in the compact genome of Fugu. Subtype classification of the identified Fugu alpha(1) orthologs based on phylogenetic analysis, genomic organization and sequence comparison of the most divergent II/III loop and the C-terminal regions of the alpha(1)-subunits indicated extra copies of alpha(1S)-, alpha(1D)-, alpha(1F)-, alpha(1A)-, alpha(1E)-, alpha(1H)- and alpha(1G)-subunit genes. Phylogenetic analysis reveals that this is likely due to fish lineage specific alpha(1)-subunit subtype duplication. Sequence comparison shows that many of the structural features characteristic of VGCC and specific channel subtypes are also present in the Fugu alpha(1)-subunits. All the Fugu alpha(1)-subunits showed similar expression profile to that of the mammalian alpha(1)-subunits except for Fugu alpha(1S), alpha(1A), alpha(1B) and alpha(1H) which have a more widespread tissue distribution. These results indicate that Fugu, a lower vertebrate, has more extensive channel heterogeneity compared to human.     

Genomic Structure and Nucleotide Sequence of the p55 Gene of the Puffer Fish Fugu rubripes

The p55 gene, which codes for a 55-kDa erythrocyte membrane protein, has been cloned and sequenced from the genome of the Japanese puffer fish Fugu rubripes (Fugu). This organism has the smallest recorded vertebrate genome and therefore provides an efficient way to sequence genes at the genomic level. The gene encoding p55 covers 5.5 kb from the beginning to the end of the coding sequence, four to six times smaller than the estimated size of the human gene, and is encoded by 12 exons. The structure of this gene has not been previously elucidated, but from this and other data we would predict a similar or identical structure in mammals. The predicted amino acid sequence of this gene in Fugu, coding for a polypeptide of 467 amino acids, is very similar to that of the human gene with the exception of the first two exons, which differ considerably. The predicted Fugu protein has a molecular weight (52.6 kDa compared with 52.3 kDa) and an isoelectric point very similar to those of human p55. In human, the p55 gene lies in the gene-dense Xq28 region, just 30 kb 3′ to the Factor VIII gene, and is estimated to cover 20-30 kb. Its 5′ end is associated with a CpG island, although there is no evidence that this is the case in Fugu. The small size of genes in Fugu and the high coding homology that they share with their mammalian equivalents, both in structure and sequence, make this compact vertebrate genome an ideal model for genomic studies.     

Isolation and characterization of a <Emphasis Type="Italic">Bacillus</Emphasis> species capable of producing tetrodotoxin from the puffer fish <Emphasis Type="Italic">Fugu obscurus</Emphasis>

We obtained puffer fish Fugu obscurus from Wudi, China and analyzed the level of tetrodotoxin (TTX) toxictiy by mouse bioassay. The ovary showed the highest potency (125 MU/g), followed by the liver, intestine, and skin. A TTX-producing strain, namely, W-3, was isolated from the ovary of puffer fish F. obscurus. After culturing at 28 °C for 48 h, toxins were extracted from the liquid medium and analyzed by mouse bioassay, high-performance liquid chromatography, and gas chromatography-mass spectrometry. The results showed that strain W-3 produced TTX and related compounds. Physiological and biochemical characterization and 16S rRNA analysis indicated that this strain represents a novel species within the Bacillus genus; we named this strain as Bacillus sp. W-3. Our results suggested that marine bacteria play a role in the production of TTX in puffer fish F. obscurus.     

Fugu genome analysis provides evidence for a whole-genome duplication early during the evolution of ray-finned fishes

With about 24,000 extant species, teleosts are the largest group of vertebrates. They constitute more than 99% of the ray-finned fishes (Actinopterygii) that diverged from the lobe-finned fish lineage (Sarcopterygii) about 450 MYA. Although the role of genome duplication in the evolution of vertebrates is now established, its role in structuring the teleost genomes has been controversial. At least two hypotheses have been proposed: a whole-genome duplication in an ancient ray-finned fish and independent gene duplications in different lineages. These hypotheses are, however, based on small data sets and lack adequate statistical and phylogenetic support. In this study, we have made a systematic comparison of the draft genome sequences of Fugu and humans to identify paralogous chromosomal regions ("paralogons") in the Fugu that arose in the ray-finned fish lineage ("fish-specific"). We identified duplicate genes in the Fugu by phylogenetic analyses of the Fugu, human, and invertebrate sequences. Our analyses provide evidence for 425 fish-specific duplicate genes in the Fugu and show that at least 6.6% of the genome is represented by fish-specific paralogons. We estimated the ages of Fugu duplicate genes and paralogons using the molecular clock. Remarkably, the ages of duplicate genes and paralogons are clustered, with a peak around 350 MYA. These data strongly suggest a whole-genome duplication event early during the evolution of ray-finned fishes, probably before the origin of teleosts.     

Prediction of the prototype of the human Toll-like receptor gene family from the pufferfish,<Emphasis Type="Italic"> Fugu rubripes</Emphasis>, genome

The insect Toll family of proteins and their mammalian counterparts seemingly shared one common ancestor and evolved independently. Here we demonstrated that the prototype of the mammalian-type (M-type) Toll family is shared by the fish and humans. According to the draft of the pufferfish Fugu genome project, the signature Toll-IL-1 receptor homology domain (TIR domain) has been conserved during evolution. FuguTLR2, 3, 5, 7, 8 and 9 members correspond structurally to respective mammalian TLRs. One Fugu TLR showed equally high amino acid identity to human TLR1, 6 and 10, and we named it FuguTLR1. Fugu rubripes has genes for TLR21 and 22, which are unique to fish. One possible interpretation of these findings is that TLR1, 2, 3, 4, 5, 7, 8, 9, 21 and 22 existed in the ancestral genome common to fish and mammals, and that TLR4 was lost in the fish lineage, while TLR21 and 22 were lost in the mammalian lineage. Strikingly, a solitary ascidian, Halocynthia roretzi, has only a few Toll-like proteins, which, like Caenorhabditis elegans Toll, represent primitive ones before the expansion of the Toll family. Therefore, the expansion of TLR genes should have occurred earlier than fish, but not C. intestinalis, separated evolutionarily from mammals. These results infer that the appearance of the M-type innate system was completed before or concomitant with the appearance of acquired immunity. We interpret the present data to mean that the differences of TLRs identified in this study between fishes and humans may be rather peripheral, partially due to selection pressure exerted by pathogens in distinct environments.     

FRG1, a gene in the FSH muscular dystrophy region on human chromosome 4q35, is highly conserved in vertebrates and invertebrates

The human FRG1 gene maps to human chromosome 4q35 and was identified as a candidate for facioscapulohumeral muscular dystrophy. However, FRG1 is apparently not causally associated with the disease and as yet, its function remains unclear. We have cloned homologues of FRG1 from two additional vertebrates, the mouse and the Japanese puffer fish Fugu rubripes, and investigated the genomic organization of the genes in the two species. The intron/exon structure of the genes is identical throughout the protein coding region, although the Fugu gene is five times smaller than the mouse gene. We have also identified FRG1 homologues in two nematodes; Caenorhabditis elegans and Brugia malayi. The FRG1 protein is highly conserved and contains a lipocalin sequence motif, suggesting it may function as a transport protein.     

Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalis.

Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 +/- 0.97 nmol x mg(-1) protein and a K(d) of 14.6 +/- 0.33 nm for [(3)H]saxitoxin in equilibrium binding assays. [(3)H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 microm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish.     

Genomics and mapping of teleostei (bony fish)

Until recently, the Human Genome Project held centre stage in the press releases concerning sequencing programmes. However, in October 2001, it was announced that the Japanese puffer fish (Takifugu rubripes, Fugu) was the second vertebrate organism to be sequenced to draft quality. Briefly, the spotlight was on fish genomes. There are currently two other fish species undergoing intensive sequencing, the green spotted puffer fish (Tetraodon nigroviridis) and the zebrafish (Danio rerio). But this trio are, in many ways, atypical representations of the current state of fish genomic research. The aim of this brief review is to demonstrate the complexity of fish as a group of vertebrates and to publicize the 'lesser-known' species, all of which have something to offer.     

A Tetrodotoxin-Producing Vibrio Strain, LM-1, from the Puffer Fish Fugu vermicularis radiatus

Identification of tetrodotoxin (TTX) and its derivatives produced from a Vibrio strain in the intestine of the puffer fish Fugu vermicularis radiatus was performed by thin-layer chromatography, electrophoresis, high-performance liquid chromatography, and gas chromatography-mass spectrometry, together with a mouse bioassay for toxicity. It was demonstrated that the isolated bacterium produced TTX, 4-epi-TTX, and anhTTX during cultivation, suggesting that Vibrio strains are responsible for the toxification of the puffer fish.     

A shifted repertoire of endocannabinoid genes in the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

The zebrafish has served as a model organism for developmental biology. Sequencing its genome has expanded zebrafish research into physiology and drug-development testing. Several cannabinoid pharmaceuticals are in development, but expression of endocannabinoid receptors and enzymes remains unknown in this species. We conducted a bioinformatics analysis of the zebrafish genome using 17 human endocannabinoid genes as a reference set. Putative zebrafish orthologs were identified in filtered BLAST searches as reciprocal best hits. Orthology was confirmed by three in silico methods: phylogenetic testing, synteny analysis, and functional mapping. Zebrafish expressed orthologs of cannabinoid receptor 1, transient receptor potential channel vanilloid receptor 4, GPR55 receptor, fatty acid amide hydrolase 1, monoacylglycerol lipase, NAPE-selective phospholipase D, abhydrolase domain-containing protein 4, and diacylglycerol lipase alpha and beta; and paired paralogs of cannabinoid receptor 2, fatty acid amide hydrolase 2, peroxisome proliferator-activated receptor alpha, prostaglandin-endoperoxide synthase 2, and transient receptor potential cation channel subtype A1. Functional mapping suggested the orthologs of transient receptor potential vanilloid receptor 1 and peroxisome proliferator-activated receptor gamma lack specific amino acids critical for cannabinoid ligand binding. No orthologs of N-acylethanolamine acid amidase or protein tyrosine phosphatase, non-receptor type 22 were identified. In conclusion, the zebrafish genome expresses a shifted repertoire of endocannabinoid genes. In vitro analyses are warranted before using zebrafish for cannabinoid development testing.     

Characterization of the MHC class I region of the Japanese pufferfish (Fugu rubripes)

A BAC map of the Japanese pufferfish (Fugu) MHC class I region was constructed using a mixture of sequence scanning and sequence-tagged site mapping methodologies. The Fugu MHC class Ia genes are linked to genes which are found within the human classical MHC class II and extended class II regions, a situation which has been found in the MHC of all teleosts mapped so far. The 300-kb contig comprises 24 MHC-related genes and is bounded by six non-MHC genes, which are thought to represent an evolutionary breakpoint within the region. Comparative analysis with both human and zebrafish MHC maps indicates two blocks of genes (KNSL2, ZNF297, DAXX, TAPBP, FLOTILLIN; and PSMB8, PSMB10, PSMB9, ABCB3, FABGL, BRD2, COL11A2, RXRB) which have remained linked over 400 million years and may represent an ancestral arrangement of the vertebrate MHC. Zebrafish and Fugu diverged between 100-200 million years ago and differences exist between these two fish species. The position and number of MHC class Ia genes is not conserved between species, there is an inversion of a block of nine genes centering on the PSMB cluster, and additional genes are present in zebrafish coding for a transport-associated protein and a beta proteasome subunit. The extent of these differences has implications for the extrapolation of fish model organism data to commercial aquaculture species. The data presented here represent the most extensive analysis of a fish MHC class Ia region described so far and clearly delimit the extent of this region in Fugu and, potentially, all teleosts.