Sayaka Yamagami

Four functionally distinct C-type natriuretic peptides found in fish reveal evolutionary history of the natriuretic peptide system

Natriuretic peptides (NPs) are major cardiovascular and osmoregulatory hormones in vertebrates. Although tetrapods generally have three subtypes, atrial NP (ANP), B-type NP (BNP), and C-type NP (CNP), some teleosts lack BNP, and sharks and hagfish have only one NP. Thus, NPs have diverged during fish evolution, possibly reflecting changes in osmoregulatory systems. In this study, we found, by cDNA cloning, four distinct CNPs (1 through 4) in the medaka (Oryzias latipes) and puffer fish (Takifugu rubripes), although to our knowledge no more than two CNPs have been isolated from a vertebrate species. Predicted mature CNP-1 was most similar, and CNP-4 was most dissimilar, to mammalian CNPs. However, synthetic CNP-4 most potently activated OlGC1, a medaka CNP-specific receptor (NPR-B) expressed in cultured cells, whereas CNP-1 and CNP-3 most activated OlGC7 and OlGC2, two medaka homologues of the ANP/BNP receptor (NPR-A), respectively. Linkage mapping in medaka followed by comparative genomic analyses among fishes and humans located four CNP genes in separate medaka chromosomes corresponding to human chromosomes 1, 2, 12, and 17. From conserved synteny, the following evolutionary history of NPs was evoked: (i) four CNP genes were generated by chromosomal duplications before the divergence of elasmobranchs; (ii) the CNP-3 gene generated ANP and BNP genes through tandem duplication before the divergence of tetrapods and teleosts; (iii) CNP-1 and -2 genes were retained in the teleost lineage but not in the tetrapod lineage; (iv) the CNP-3 gene disappeared from the tetrapod lineage after divergence of amphibians; and (v) the CNP-4 gene is retained in humans as CNP.

Diverse Forms of Guanylyl Cyclases in Medaka Fish – Their Genomic Structure and Phylogenetic Relationships to those in Vertebrates and Invertebrates

Abstract Fish species such as medaka fish, fugu, and zebrafish contain more guanylyl cyclases (GCs) than do mammals. These GCs can be divided into two types: soluble GCs and membrane GCs. The latter are further divided into four subfamilies: (i) natriuretic peptide receptors, (ii) STa/guanylin receptors, (iii) sensory-organ-specific membrane GCs, and (iv) orphan receptors. Phylogenetic analyses of medaka fish GCs, along with those of fugu and zebrafish, suggest that medaka fish is a much closer relative to fugu than to zebrafish. Analyses of nucleotide data available on a web site (http://www.ncbi.nlm.nih.gov/) of GCs from a range of organisms from bacteria to vertebrates suggest that gene duplication, and possibly chromosomal duplication, play important roles in the divergence of GCs. In particular, the membrane GC genes were generated by chromosomal duplication before the divergence of tetrapods and teleosts.

Expression and genomic organization of a medaka fish novel membrane form of guanylyl cyclase/orphan receptor.

Abstract A novel membrane guanylyl cyclase (membrane GC), OlGC8, was identified in the medaka fish Oryzias latipes by the isolation of full-length cDNA (4958 bp) and genomic DNA (14.3 kbp) clones. Phylogenetic analysis indicated that OlGC8 does not belong in any known vertebrate membrane GC sub-family. OlGC8 consists of an extracellular domain (214 residues), a transmembrane segment (19 residues), and an intracellular protein kinase-like domain (284 residues) and a cyclase catalytic domain (228 residues), although the extracellular domain is about half the length (around 450 residues) of other known vertebrate membrane GCs. OlGC8 transiently expressed in COS-7 cells exhibited only basal guanylyl cyclase activity. None of the known ligands (rat ANP, BNP, CNP, and C-ANF) and various medaka fish tissue extracts, which activated OlGC1, OlGC2, and OlGC7 differentially, stimulated basal activity, suggesting that OlGC8 is an orphan receptor. The OlGC8 gene consists of 24 exons and exists as a single copy on the medaka fish genome. Northern blot hybridization showed that a 5 kb-OlGC8 mRNA was expressed in the kidney and the testis at a high level and a 3.3 kb-OlGC8 mRNA was expressed only in the brain. The RNase protection, RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE), and reverse transcription-polymerase chain reaction (RT-PCR) analyses demonstrated that the 3.3 kb-OlGC8 mRNA detected in the brain is transcribed from the second transcription initiation site, and contains an intron at the position prior to the catalytic domain, the translation product of which appears to be a protein lacking the cyclase catalytic domain.

Expression of sperm-activating peptide IV receptor-associated membrane guanylyl cyclase in the testis of the sea urchin Diadema setosum

Abstract We obtained the full-length cDNA clone (DsPTGC04) encoding a membrane guanylyl cyclase expressed in the testis of the sea urchin Diadema setosum, the egg jelly of which contains sperm-activating peptide IV. The cDNA was 4305 bp long and an open reading frame predicted a protein of 1127 amino acids including an apparent signal peptide of 24 residues. The mature protein of 1103 amino acids is composed of a single transmembrane domain of 25 amino acids that divides the mature protein (Mw 123818) into an amino-terminal, extracellular domain of 484 amino acids and a carboxyl-terminal, intracellular domain of 594 amino acids, with the latter consisting of two clearly defined subdomains, a protein kinase-like and a cyclase catalytic. Four potential N-linked glycosylation sites are present in the extracellular domain and 4 presumed phosphorylatable serine residues are conserved in the cyclase catalytic domain. Northern blot analysis demonstrated that the 4.5 kb mRNA for DsPTGC04 is expressed only in the testis. Antibodies raised against two synthetic peptides, 800WVENPDERPN809 and 1080KPPPQKLSAEVMEAAANREIPEDL1103, corresponding to two carboxyl-terminal portions of DsPTGC04, reacted with a protein of about 120 kDa in D. setosum spermatozoa and testis, but not with any protein in the ovary, eggs, or intestine. Immunohistochemistry showed that both antibodies react with the mature spermatozoa in the testis.