Nitric oxide synthase sequences in the marine fish Stenotomus chrysops and the sea urchin Arbacia punctulata, and phylogenetic analysis of nitric oxide synthase calmodulin-binding domains

The phylogenetic distribution and structural diversity of the nitric oxide synthases (NOS) remain important and issues that are little understood. We present sequence information, as well as phylogenetic analysis, for three NOS cDNAs identified in two non-mammalian species: the vertebrate marine teleost fish Stenotomus chrysops (scup) and the invertebrate echinoderm Arbacia punctulata (sea urchin). Partial gene sequences containing the well-conserved calmodulin (CaM)-binding domain were amplified by RT-PCR. Identical 375-bp cDNAs were amplified from scup brain, heart, liver and spleen; this sequence shares 82% nucleic acid and 91% predicted amino acid identity with the corresponding region of human neuronal NOS. A 387-bp cDNA was amplified from sea urchin ovary and testes; this sequence shares 72% nucleic acid identity and 65% deduced amino acid identity with human neuronal NOS. A second cDNA of 381 bp was amplified from sea urchin ovary and it shares 66% nucleic acid and 57% deduced amino acid identity with the first sea urchin sequence. Together with earlier reports of neuronal and inducible NOS sequences in fish, these data indicate that multiple NOS isoforms exist in non-mammalian species. Phylogenetic analysis of these sequences confirms the conserved nature of NOS, particularly of the calmodulin-binding domains.     

Expression of two vacuolar-type ATPase B subunit isoforms in swimbladder gas gland cells of the European eel: nucleotide sequences and deduced amino acid sequences

The poly(A)(+) RNA of swimbladder gas gland cells of the European eel Anguilla anguilla was isolated and used for cDNA synthesis. Using a pair of degenerate PCR primers directed towards the evolutionary highly conserved central part of the B subunit of vacuolar type H(+)-ATPase (V-ATPase) a fragment of 388 bp was amplified. By sequencing the cloned PCR products two different amplicons with a sequence identity of about 86% were obtained. BLASTN searches revealed a high degree of similarity of both to V-ATPase B subunits of other species. The sequences were completed by performing rapid amplification of cDNA ends PCR, subsequent cloning, and sequencing of the obtained products. The expression of two different isoforms of the V-ATPase B subunit is already demonstrated for Homo sapiens and Bos taurus. This is the first report that attributes the same phenomenon to a non-mammalian species, A. anguilla. The first isoform found in eel (vatB2) shows the highest degree of amino acid sequence homology with the human brain isoform (98.2%), the second one (vatB1) with the B subunit sequence of rainbow trout (Oncorhynchus mykiss) gill and kidney (98, 6%). The alignment of the deduced amino acid sequences of vatB1 and vatB2 shows that the highest sequence variation between these two isoforms is found at the amino-terminus, where vatB1 is nine amino acids shorter than vatB2, while at the carboxy-terminus it is two amino acids longer than vatB2. This has also been reported for the human and bovine kidney isoforms when compared with the brain isoforms. Northern blot analysis using specific hybridization probes revealed the expression of two mRNA's with lengths of about 2.9 kb and 3.5 kb for vatB1 and vatB2, respectively. For mammals, it is well known that V-ATPases containing the kidney isoforms of the B subunit are responsible for the extrusion of protons across the plasma membranes of several cell types. The fact that eel vatB1 seems to share structural features with the kidney isoforms in mammals supports the hypothesis that in gas gland cells a V-ATPase contributes to the acidification of the blood in the swimbladder.     

Alternative splicing generates two isoforms of the alpha 2 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein which displays developmental heterogeneity in the mammalian central nervous system. Here we describe 2 novel cDNA variants of the rat GlyR alpha 2 subunit and demonstrate that alternative splicing generates these 2 isoforms. The deduced protein sequences (alpha 2A and alpha 2B) exhibit 99% identity with the previously characterized human alpha 2 subunit. In situ hybridization revealed expression of both alpha 2A and alpha 2B mRNAs in the prenatal rat brain, suggesting that these variant proteins may have a role in synaptogenesis. Heterologous expression in Xenopus oocytes showed that the more abundantly expressed alpha 2A subunit forms strychnine-sensitive ion channels which resemble human alpha 2 subunit GlyRs in their electrophysiological properties.     

Molecular Cloning and Characterization of Two Isoforms of Trypsinogen from Anchovy Pyloric Ceca

Complementary DNA clones encoding two isoforms of trypsinogen were isolated from the pyloric ceca of anchovy by rapid amplification of cDNA ends (RACE). Nucleotide sequences of isolated clones encoded, in addition to characteristic signal and activation peptides, two isoforms of trypsin containing 220 and 221 amino acid residues. Both enzymes contained the catalytic triad of a serine protease, together with the residues determining substrate specificity. The anchovy trypsins showed a high amino acid identity of about 80% to those of other fish species. Southern blot analysis with a probe cross-reactive to both isoforms showed a complex genomic pattern. Northern blot analysis with the same probe revealed the highest expression of meassenger RNA in the pyloric ceca. Structural parameters possibly involved in higher catalytic properties of fish trypsin were examined by three-dimensional modeling, which included deletion in the autolysis loop, lack of Tyr-151 at the entrance of the S1 pocket, and distribution of charged residues. Received May 30, 2000; accepted August 15, 2000     

Duplication of phospholipase C-delta gene family in fish genomes

Fishes possess more genes than other vertebrates, possibly because of a genome duplication event during the evolution of the teleost (ray-finned) fish lineage. To further explore this idea, we cloned five genes encoding phosphoinositide-specific phospholipase C-delta (PLC-delta), designated respectively PoPLC-deltas, from olive flounder (Paralichthys olivaceus), and we performed phylogenetic analysis and sequence comparison to compare our putative gene products (PoPLC-deltas) with the sequences of known human PLC isoforms. The deduced amino acid sequences shared high sequence identity with human PLC-delta1, -delta3, and -delta4 isozymes and exhibited similar primary structures. In phylogenetic analysis of PoPLC-deltas with PLC-deltas of five teleost fishes (zebrafish, stickleback, medaka, Tetraodon, and Takifugu), three tetrapods (human, chicken, and frog), and two tunicates (sea squirt and pacific sea squirt), whose putative sequences of PLC-delta are available in Ensembl genome browser, the result also indicated that the two paralogous genes corresponding to each PLC-delta isoform originated from fish-specific genome duplication prior to the divergence of teleost fish. Our analyses suggest that an ancestral PLC-delta gene underwent three rounds of genome duplication during the evolution of vertebrates, leading to the six genes of three PLC-delta isoforms in teleost fish.     

Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of μ-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the  26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout μ-calpain and calpastatin were only partly purified; identity of trout μ-calpain was confirmed by labeling with antibodies to bovine skeletal muscle μ-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle μ- and m-calpain. Trout μ-calpain requires 4.4 ± 2.8 μM and trout m-calpain requires 585 ± 51 μM Ca²⁺ for half-maximal activity, similar to the Ca²⁺ requirements of μ- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.     

The isoforms of yeast cytochrome c oxidase subunit V alter the in vivo kinetic properties of the holoenzyme.

One of the nuclear-coded subunits of yeast cytochrome c oxidase is specified by a gene family composed of two genes, COX5a and COX5b. These genes are regulated differentially by oxygen and encode isoforms of subunit V, designated Va and Vb, which have only 66% primary sequence identity. Yeast cells require one or the other isoform for a functional cytochrome c oxidase (Trueblood, C. E., and Poyton, R. O. (1987) Mol. Cell Biol. 7, 3520-3526). To determine if these isoforms of subunit V alter the catalytic properties of holocytochrome c oxidase, we have analyzed various aspects of cytochrome c oxidase function in intact yeast cells that produce only one type of isoform. From measurements of room temperature turnover numbers and low temperature rates of ligand binding, single turnover cytochrome c oxidation, and internal electron transfer (heme a oxidation), we have found that isozymes which incorporate the Vb isoform have both higher turnover rates and higher rates of heme a oxidation than isozymes which incorporate Va. These findings support the conclusion that the isoforms of subunit V modulate cytochrome c oxidase activity in vivo and suggest that they do so by altering the rates of one or more intramolecular electron transfer reactions.     

Molecular cloning and expression analysis of three Mx isoforms of rock bream, Oplegnathus fasciatus

Complementary DNAs (cDNAs) corresponding to three isoforms of rock bream (Oplegnathus fasciatus) Mx (RbMx1, RbMx2 and RbMx3) were cloned using RACE reactions. Analysis of deduced amino acid sequences revealed that the tripartite GTP-binding domain, the dynamine family signature and the leucine zipper repeat were present in all three rock bream Mx isoforms. Cloning of genomic DNA sequence and Southern blot analysis showed that three rock bream Mx isoforms were encoded by different genomic loci, and they were not alternative splicing variants, although some alternative splicing variants were found in RbMx1 and RbMx2. When comparing amino acid sequence identity, RbMx1 shares about 60-70% identities with other fish Mx proteins, whereas both RbMx2 and RbMx3 share slightly high identity of 70-90%. As a result of expression analysis using RT-PCR, RbMx1 was constitutively expressed in the spleen and kidney of rock bream yearling, but RbMx2 and RbMx3 were rarely detected in both organs. When injected with synthetic double-stranded RNA polyinosinic:polycytidylic acid (poly I:C), expression of all rock bream Mx isoforms was up-regulated in spleen and head kidney. RbMx1 was continuously up-regulated throughout experimental period of 72 h but RbMx2 and RbMx3 were down-regulated to almost non-detectable level at 48 h post-injection.     

CK2-mediated phosphorylation of a type II regulatory subunit of cAMP-dependent protein kinase from the mollusk Mytilus galloprovincialis

Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only R(myt2) was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2alpha) itself was sufficient to phosphorylate R(myt2), but phosphorylation was enhanced by the presence of the regulatory subunit CK2beta. Even in the absence of CK2, R(myt2) was phosphorylated to a certain extent when it was incubated with GTP. This basal phosphorylation was partially abolished by the known inhibitors apigenin and emodin, which suggests the presence of a residual amount of endogenous CK2 in the preparation of purified R subunit. CK2-mediated phosphorylation significantly decreases the ability of R(myt2) to inhibit PKA catalytic (C) subunit activity in the absence of cAMP. On the other hand, the sequence of several peptides obtained from the tryptic digestion of R(myt2) showed that mussel protein contains the signature sequence common to all PKA family members, within the "phosphate binding cassette" (PBC) A and B. Moreover, the degree of identity between the sequences of peptides from R(myt2), as a whole, and those from type II R subunits was 68-75%, but the global identity percentage with type I R subunits was only about 30%, so that R(myt2) can be classified as a type II R subunit.     

An immunochemical analysis of the induction of cytochrome P-450 isoforms in the liver of fresh-water fishes by 3-methylcholanthrene, beta-naphthoflavone and aroclor 1254

The cytochrome P-450 isoforms have been studied in liver microsomes of some fish species from Lake Baikal. Using the inhibitory analysis of microsomal monooxygenase activities carried out by the specific polyclonal antibodies it has been shown that 3-methylcholanthrene, beta-naphthoflavone and arochlor 1254 induce isoforms immunologically related to cytochrome P-488c but not to the rat cytochrome P-450b in fish liver microsomes. The immunologic identity in isoforms of fish and rat cytochromes induced by methylcholanthrene has not been revealed. A possibility to use the method of the inhibitory analysis of fish microsomal activities by specific antibodies to the rat cytochrome P-450 isoforms for biomonitoring and biotesting of polycyclic hydrocarbons and polychlorinated biphenyls in aquatic systems is discussed.     

Identification of cDNAs encoding two subtypes of vitamin D receptor in flounder, Paralichthys olivaceus

cDNAs encoding two subtypes of the vitamin D receptor (VDR) are cloned from a teleost (flounder, Paralichthys olivaceus). This is the first report of VDR subtypes in fish. Flounder VDR (fVDR) a and b share 86% identity at the amino acid level. With human (h), rat, mouse, quail, and Xenopus VDRs, fVDRa shares 72%, 71%, 71%, 69%, and 71% identity, and fVDRb shares 70%, 69%, 69%, 67%, and 68% identity, respectively. The peptide sequences of the DNA-binding domain (DBD) and hormone-binding domain (HBD) of both subtypes have particularly high homology to those of the tetrapods; e.g. 92% identity for DBP and 74% for HBD between fVDRa and hVDR. In an evolutionary tree constructed with peptide sequences of VDRs and related members of the nuclear receptor superfamily, fVDRa and b are more closely related to each other than to other molecules, and situated in the cluster of VDRs at a position which corresponds well with the evolutional position of fish in the vertebrates. Additional independent genome duplication which is thought to have occurred in ray-finned fish phylogeny may explain the existence of two subtypes of VDR in flounder.     

Sequence microheterogeneity of parvalbumin,the major fish allergen

The microheterogeneity of amino acid sequence observed in various allergens may affect immune response, but incidence of sequence microheterogeneity in allergens and its relation to their allergenicity are unclear. The occurrence of sequence microheterogeneity in major fish allergen, parvalbumin (PA), has been explored using bioinformatics approaches. 44% of 111 species with known PA sequence have PA isoforms. 41% of these species exhibit from 1 to 4 cases of PA sequence microheterogeneity, i.e. unique pairs of PA isoforms with sequence identity above 90%. 29% of 210 PA sequences studied are characterized by microheterogeneity. The occurrence of allergens among them is 2.5-fold higher than among other PAs. The incidence of sequence microheterogeneity within more allergenic β isoform of PA is 2.0-fold lower than that for its less allergenic α isoform. 39 residues affected by PA microheterogeneity are concentrated in the region of helices A, B, F, while helices D and E are the most conservative region. 44% and 11% of the microheterogeneous substitutions are located in the species-specific and cross-reactive IgE-binding epitopes of PAs, respectively. 45% and 48% of the substitution cases are predicted to cause notable changes in protein disorder propensity and protein stability, respectively. Hence, the increased allergenicity rate among PAs having microheterogeneous isoforms can be related to differences in their IgE-binding caused directly or in an allosteric manner. Overall, sequence microheterogeneity is shown to be inherent to many of PAs and likely contributes to PA allergenicity.     

Sequence analysis of the catalytic subunit of H(+)-ATPase from porcine renal brush-border membranes.

The catalytic subunit of the H(+)-ATPase from brush-border membranes of porcine renal proximal tubules was labeled with the hydrophobic SH-group reagent 10-N-(bromoacetyl)amino-1-decyl-beta-glucopyranoside (BADG) which irreversibly inhibits proton pump activity in the absence but not in the presence of ATP. The labeled protein was purified and digested with proteinases. After isolation and sequencing of proteolytic peptides two BADG-labeled cysteines were identified. The amino acid sequences of the obtained proteolytic peptides were homologous to the catalytic subunit of V-ATPases. From mRNA of porcine kidney cortex a catalytic H(+)-ATPase subunit was cloned. 181 of the 183 amino acids which overlap in the sequence derived from the cDNA and the proteolytic peptides were identical, and the two deviations are due to single base exchanges. A comparison of the amino acid sequence derived from the cloned cDNA with sequences of catalytic H(+)-ATPase subunits communicated by other laboratories revealed 98%, 96% and 94% identity with sequences from bovine adrenal medulla, from bovine kidney medulla and from clathrin-coated vesicles of bovine brain. Between 64% and 69% identity was obtained with sequences from fungi and plants. The data show that the catalytic subunit of V-ATPases is highly conserved during evolution. They indicate organ and species specificity in mammalians.