Carbonic Anhydrases in Cnidarians: Novel Perspectives from the Octocorallian Corallium rubrum.

Although the ability to elaborate calcium carbonate biominerals was apparently gained independently during animal evolution, members of the alpha carbonic anhydrases (α-CAs) family, which catalyze the interconversion of CO2 into HCO3-, are involved in the biomineralization process across metazoans. In the Mediterranean red coral Corallium rubrum, inhibition studies suggest an essential role of CAs in the synthesis of two biominerals produced in this octocoral, the axial skeleton and the sclerites. Hitherto no molecular characterization of these enzymes was available. In the present study we determined the complete set of α-CAs in C. rubrum by data mining the genome and transcriptome, and measured their differential gene expression between calcifying and non-calcifying tissues. We identified six isozymes (CruCA1-6), one cytosolic and five secreted/membrane-bound among which one lacked two of the three zinc-binding histidines and was so referred to as a carbonic anhydrase related protein (CARP). One secreted isozyme (CruCA4) showed specific expression both by qPCR and western-blot in the calcifying tissues, suggesting its involvement in biomineralization. Moreover, phylogenetic analyses of α-CAs, identified in six representative cnidarians with complete genome, support an independent recruitment of α-CAs for biomineralization within anthozoans. Finally, characterization of cnidarian CARPs highlighted two families: the monophyletic cytosolic CARPs, and the polyphyletic secreted CARPs harboring a cnidarian specific cysteine disulfide bridge. Alignment of the cytosolic CARPs revealed an evolutionary conserved R-H-Q motif in place of the characteristic zinc-binding H-H-H necessary for the catalytic function of α-CAs.

Conserved functions for Mos in eumetazoan oocyte maturation revealed by studies in a cnidarian.

The kinase Mos, which activates intracellularly the MAP kinase pathway, is a key regulator of animal oocyte meiotic maturation. In vertebrate and echinoderm models, Mos RNA translation upon oocyte hormonal stimulation mediates "cytostatic" arrest of the egg after meiosis, as well as diverse earlier events [1-5]. Our phylogenetic survey has revealed that MOS genes are conserved in cnidarians and ctenophores, but not found outside the metazoa or in sponges. We demonstrated MAP kinase-mediated cytostatic activity for Mos orthologs from Pleurobrachia (ctenophore) and Clytia (cnidarian) by RNA injection into Xenopus blastomeres. Analyses of endogenous Mos in Clytia with morpholino antisense oligonucleotides and pharmacological inhibition demonstrated that Mos/MAP kinase function in postmeiotic arrest is conserved. They also revealed additional roles in spindle formation and positioning, strongly reminiscent of observations in starfish, mouse, and Xenopus. Unusually, cnidarians were found to possess multiple Mos paralogs. In Clytia, one of two maternally expressed paralogs accounted for the majority MAP kinase activation during maturation, whereas the other may be subject to differential translational regulation and have additional roles. Our findings indicate that Mos appeared early during animal evolution as an oocyte-expressed kinase and functioned ancestrally in regulating core specializations of female meiosis.

Comparative study on the cell toxicity and enzymatic activity of two northern scyphozoan species Cyanea capillata (L.) and Cyanea lamarckii (Péron & Léslieur).

Two species of venomous pelagic cnidaria are compared according to their enzymatic, cytotoxic and haemolytic potency. The widely distributed jellyfish Cyanea capillata and Cyanea lamarckii were collected in the North Sea at the coasts of the Orkney Island and the Island of Helgoland. Purified cnidocyst extracts from fishing and mesenteric tentacles were prepared and tested for their bioactivity. The haemolysis induced by toxins of C. capillata was determined with respect to organism size and toxigenic organs. The haemolytic activity of the related species C. lamarckii was documented for the first time. Dose dependent haemolytic activities have been detected by means of protein equivalents at concentrations above 20mug(protein)/mL. Extracts of fishing tentacle cnidocysts showed a less potent haemolytic activity compared to extracts of mesenteric tentacles. In vitro studies with permanent cells of a hepatoma cell line have shown a time and concentration dependent loss of cell vitality up to 90% at 33.3mug(protein)/mL (10mug(protein)/10(5) cells). Supplementing the cell based toxicity tests an enzyme assay was performed to measure a phospholipase A(2) (PLA(2)) activity. A PLA(2)-like activity could be demonstrated in cnidocysts extracts prepared from mesenteric and fishing tentacles of both jellyfish species.

Optical bioluminescence and positron emission tomography imaging of a novel fusion reporter gene in tumor xenografts of living mice.

Noninvasive imaging of reporter gene expression using various imaging modalitiesis playing an increasingly important role in defining molecular events in the field of cancer biology, cell biology, and gene therapy. In this study, a novel reporter vector was constructed encoding a fusion protein comprised of a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) (tk), a positron emission tomography (PET) reporter gene, and renilla luciferase (rl), a bioluminescence optical reporter gene joined by a 20 amino acid long spacer sequence. We validated the activity of the two enzymes encoded by the fusion protein (tk(20)rl) in cell culture. Then, tumors stably expressing the tk(20)rl fusion gene were imaged both by microPET and optically using a cooled charge coupled device camera in xenograft-bearing living mice. Using a single fusion reporter (PET/optical) gene should accelerate the validation of reporter gene approaches developed in cell culture for translation into preclinical and clinical models.

Nurr1 affects pRL-TK but not phRG-B internal control plasmid in genetic reporter system.

In transcription assays, Renilla luciferase-expressing plasmids (more specifically pRL-TK) are commonly used as an internal control of transfection efficiency. Normalization of the experimental reporter gene transcription to the internal control reporter gene transcription minimizes variability of obtained results caused by differences in transfection efficiency between different samples of transfected cells. It is obvious that co-transfection with other plasmids or applied treatments should not affect the activity of the control reporter. Here we report that expression of the control Renilla luciferase encoded by pRL-TK plasmid was enhanced by co-transfection with vectors expressing orphan nuclear receptors Nur77 family (Nur77, Nurr1, Nor-1), leading to misinterpretation of the assay results. Further, we show that for Nurr1, phRG-B (a promoterless reporter plasmid containing synthetic Renilla luciferase gene) is a better control reporter vector than HSV-TK containing vectors. Finally, we noted the lack of effect of Nurr1 protein on the Fas Ligand promoter-driven transcription.

Poly(A)-independent regulation of maternal hunchback translation in the Drosophila embryo.

Development of the Drosophila abdomen requires repression of maternal hunchback (hb) mRNA translation in the posterior of the embryo. This regulation involves at least four components: nanos response elements within the hb 3' untranslated region and the activities of Pumilio (PUM), Nanos (NOS), and Brain tumor. To study this regulation, we have developed an RNA injection assay that faithfully recapitulates the regulation of the endogenous hb message. Previous studies have suggested that NOS and PUM can regulate translation by directing poly(A) removal. We have found that RNAs that lack a poly(A) tail and cannot be polyadenylated and RNAs that contain translational activating sequences in place of the poly(A) tail are still repressed in the posterior. These data demonstrate that the poly(A) tail is not required for regulation and suggest that NOS and PUM can regulate hb translation by two mechanisms: removal of the poly(A) tail and a poly(A)-independent pathway that directly affects translation.

Characterization of the coral allene oxide synthase active site with UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy: evidence for tyrosinate ligation to the ferric enzyme heme iron.

Coral allene oxide synthase (AOS), a hemoprotein with weak sequence homology to catalase, is the N-terminal domain of a naturally occurring fusion protein with an 8R-lipoxygenase. AOS converts 8R-hydroperoxyeicosatetraenoic acid to the corresponding allene oxide. The UV--visible absorption and magnetic circular dichroism spectra of ferric AOS and of its cyanide and azide complexes, and the electron paramagnetic resonance spectra of native AOS (high-spin, g = 6.56, 5.22, 2.00) and of its cyanide adduct (low-spin, g = 2.86, 2.24, 1.60) closely resemble the corresponding spectra of bovine liver catalase (BLC). These results provide strong evidence for tyrosinate ligation to the heme iron of AOS as has been established for catalases. On the other hand, the positive circular dichroism bands in the Soret region for all three derivatives of ferric AOS are almost the mirror image of those in catalase. In addition, the cyanide affinity of native AOS (K(d) = 10 mM at pH 7) is about 3 orders of magnitude lower than that of BLC. Thus, while these results conclusively support a common tyrosinate-ligated heme in AOS as in catalase, significant differences exist in the interaction between their respective heme prosthetic groups and protein environments, and in the access of small molecules to the heme iron.

A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP.

We have previously reported that Escherichia coli and mammalian cells containing a fusion protein consisting of the Renilla luciferase linked to Aequorea GFP exhibited luminescence resonance energy transfer (LRET) from luciferase to GFP in the presence of coelenterazine. In this paper, we describe the construction of two gene fusions in which the cDNA for insulin-like growth factor II (IGF-II) is connected to the cDNA for a "humanized" GFP, and the cDNA for insulin-like growth factor binding protein 6 (IGFBP-6) is linked to a cDNA encoding the Renilla luciferase (RUC). The expression of the fusion gene constructs in CHO cells resulted in single polypeptides with the molecular weights expected for IGF-II-GFP and IGFBP-6-RUC, respectively, based on the use of antibodies against GFP and Renilla luciferase. The secretion of IGF-II-GFP from CHO cells was verified by fluorescence microscopy and the presence of IGFBP-6-RUC in the culture medium was confirmed by luminometry. The interaction between the two known binding partners, IGF-II and IGFBP-6, was monitored by measuring LRET from the IGFBP-6-RUC protein to IGF-II-GFP in the presence of coelenterazine, using a low-light imaging system and spectrofluorometry. Based on these data, luciferase-to-GFP LRET holds great promise for the study of protein-protein interactions in eukaryotic cells in real time.

Suppression of gene expression by RNA interference in cultured plant cells.

Suppression by double-stranded RNA (dsRNA) of the expression of a target gene is known as RNA interference (RNAi). No quantitative analysis of the effects of RNAi on the expression of specific genes in cultured plant cells has been reported. However, as it is possible to produce populations of cultured plant cells that are uniform and divide synchronously for functional analysis of genes of interest, we performed a quantitative study of the effects of RNAi in such cells. We constructed dsRNA expression plasmids for a luciferase gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter by simply connecting sense and antisense sequences in a head-to-head manner. An RNAi effect was observed 24 hours after the introduction of dsRNA expression plasmids into tobacco BY-2 cells by electroporation. The simple system for suppression of specific genes in plant cells should be useful in attempts to elucidate the roles of individual genes in plant cells.

Renilla luciferase as a vital reporter for chloroplast gene expression in Chlamydomonas.

The use of luciferases as reporters of gene expression in living cells has been extended to the chloroplast genome. We show that the luciferase from the soft coral Renilla reniformis (Rluc) can be successfully expressed in the chloroplast of Chlamydomonas reinhardtii. Expression of the rluc cDNA was driven by the promoter and 5' untranslated regions of the atpA gene. Western analysis with an anti-Rluc antibody detected a single polypeptide of 38 kDa in the luminescent cells. This is 3 kDa larger than native Rluc, and suggests that translation of the chimeric mRNA begins at the atpA start codon, 29 codons upstream from the rluc start site. We also show that the luminescence of the transformants was sufficient to enable imaging of colonies using a cooled CCD camera.

Improved assay sensitivity of an engineered secreted Renilla luciferase.

We have previously reported the construction of a functional Renilla luciferase enzyme secreted by mammalian cells when fused to the signal peptide of human interleukin-2. The presence of three predicted cysteine residues in the amino acid sequence of Renilla luciferase suggested that its secreted form could contain oxidized sulfhydryls, which might impair enzyme activity. In this work, four secreted Renilla luciferase mutants were constructed to investigate this possibility: three luciferase mutants in which a different cysteine residue was replaced by an alanine residue, and one luciferase mutant in which all three cysteine residues were replaced by alanine residues. Simian cells were transfected with the genes encoding these mutant luciferases, as well as with the original gene construct, and cell culture media were assayed for bioluminescence activity. Only media containing a mutated luciferase with a cysteine to alanine substitution at position 152 in the preprotein showed a marked increase in bioluminescence activity when compared to media containing the original secreted Renilla luciferase. This increase in light emission was due in part to enhanced stability of the mutant enzyme. This new enzyme represents a significant improvement in the sensitivity of the secreted Renilla luciferase assay for monitoring gene expression.

A dual-luciferase reporter system for studying recoding signals.

A new reporter system has been developed for measuring translation coupling efficiency of recoding mechanisms such as frameshifting or readthrough. A recoding test sequence is cloned in between the renilla and firefly luciferase reporter genes and the two luciferase activities are subsequently measured in the same tube. The normalized ratio of the two activities is proportional to the efficiency with which the ribosome "reads" the recoding signal making the transition from one open reading frame to the next. The internal control from measuring both activities provides a convenient and reliable assay of efficiency. This is the first enzymatic dual reporter assay suitable for in vitro translation. Translation signals can be tested in vivo and in vitro from a single construct, which allows an intimate comparison between the two systems. The assay is applicable for high throughput screening procedures. The dual-luciferase reporter system has been applied to in vivo and in vitro recoding of HIV-1 gag-pol, MMTV gag-pro, MuLV gag-pol, and human antizyme.

Purification and molecular cloning of an 8R-lipoxygenase from the coral Plexaura homomalla reveal the related primary structures of R- and S-lipoxygenases.

Lipoxygenases that form S configuration fatty acid hydroperoxides have been purified or cloned from plant and mammalian sources. Our objectives were to characterize one of the lipoxygenases with R stereospecificity, many of which are described in marine and freshwater invertebrates. Characterization of the primary structure of an R-specific enzyme should help provide a new perspective to consider the enzyme-substrate interactions that are the basis of the specificity of all lipoxygenases. We purified an 8R-lipoxygenase of the prostaglandin-containing coral Plexaura homomalla by cation and anion exchange chromatography. This yielded a colorless enzyme preparation, a band of approximately 100 kDa on SDS-polyacrylamide gel electrophoresis, and turnover numbers of 4000 min-1 of 8R-lipoxygenase activity in peak chromatographic fractions. The full-length cDNA was cloned by PCR using peptide sequence from the purified protein and by 5'- and 3'-rapid amplification of cDNA ends. The cDNA encodes a polypeptide of 715 amino acids, including over 70 amino acids identified by peptide microsequencing. A peptide presequence of 52 amino acids is cleaved to give the mature protein of 76 kDa; the difference from the estimated size by SDS-PAGE implies a post-translational modification of the P. homomalla enzyme. All of the iron-binding histidines of S-lipoxygenases are conserved in the 8R-lipoxygenase. However, the C-terminal amino acid is a threonine, as opposed to the isoleucine that provides the carboxylate ligand to the iron in all known S-lipoxygenases. These results establish that the 8R-lipoxygenase is related in primary structure to the S-lipoxygenases. A model of the basis of R and S stereospecificity is described.

Intraspecific recognition system in scleractinian corals: morphological and cytochemical description of the autolysis mechanism.

Intraspecific aggressive interactions have been observed in the scleractinian coral Stylophora pistillata. Extensive field observations suggest that intraspecific interactions result in the formation of necrosis in one of the two competing colonies. The formation of necroses is primarily restricted to those layers that are interspersed with polyps. In a second phase the non-living, central corallum is destroyed by foreign microorganisms. In some cases the necroses are resealed again, either by a living septal layer or by a calcareous plug. Evidence is presented indicating that the enzyme carbonic anhydrase has a role in these activities. First, it is involved in the apical growth of the colony. Using a semiquantitative assay procedure, the highest enzyme level was determined in polyps at the tips of the branches; lower levels were measured in polyps of the foot/trunk region. Secondly, the enzyme was found to be induced in those areas that are in close contact with non-isogeneic tissue. From these data we conclude that carbonic anhydrase is involved in the process of autolytic digestion in response to intraspecific contact of two non-clonemates.

Enzymatic 5-hydroxylation of L-dopa by a tyrosinase isolated from the sea anemone Metridium senile.

A particulate tyrosinase has been extracted and purified from tentacles of the sea anemone Metridium senile. The purified enzyme had properties in common with both mushroom and vertebrate tyrosinase and catalyzed three different reactions: oxidation of catechols, hydroxylation of L-tyrosine with L-dopa as cofactor and 5-hydroxylation of L-dopa. 5-Hydroxylation of L-dopa by an animal tyrosinase has not been reported earlier. The reaction could be analyzed under reducing conditions when the much faster oxidation of L-dopa to dopaquinone was inhibited. The conditions required for the accumulation of L-dopa and 5-hydroxydopa observed in vivo in tentacles of Metridium are discussed.

Primary structure of triosephosphate isomerase from Bacillus stearothermophilus.

1. Triosephosphate isomerase from Bacillus stearothermophilus is a dimeric enzyme comprising two chemically identical polypeptide chains. 2. The nearly complete amino acid sequence of the subunit polypeptide chain has been established from sequences of tryptic, chymotryptic and lysine-blocked tyrptic fragments of S-[2-14C]carboxymethylated enzyme. Overlaps not established by experimental data have been provisionally established from considerations of sequence homology with previously established sequences for the rabbit, chicken and coelacanth enzymes. The nearly complete sequence of the 249 residues is as follows. (See Text). 3. Comparison of the thermophile and chicken muscle enzymes shows that 40% of the residues are in identical sequence. 4. Correlation of the sequence of the thermophile enzyme with the three-dimensional structure of the muscle enzyme shows that residues in the catalytic site and in the subunit interface are strongly conserved. Possible correlations between sequence changes and thermal stabilisation of the dimeric structure are also noted.

Purification and properties of Renilla reniformis luciferase.

Luciferase from the anthozoan coelenterate Renilla reniformis (Renilla luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5.) catalyzes the bioluminescent oxidation of Renilla luciferin producing light (lambdaB 480 nm, QB 5.5%), oxyluciferin, and CO2 (Hori, K., Wampler, J.E., Matthews, J.C., and Cormier, M.J. (1973), Biochemistry 12, 4463). Using a combination of ion-exchange, molecular-sieve, sulfhydryl-exchange, and affinity chromatography, luciferase has been purified, approximately 12 000-fold with 24% recovery, to homogeneity as judged by analysis with disc and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and ultracentrifugation. Renilla luciferase is active as a nearly spherical single polypeptide chain monomer of 3.5 X 10(4) daltons having a specific activity of 1.8 X 10(15) hp s-1 mg-1 and a turnover number of 111 mumol min-1 mumol-1 of enzyme. This enzyme has a high content of aromatic and hydrophobic amino acids such that it has an epsilon280nm 0.1% of 2.1 and an average hydrophobicity of 1200 cal residue-1. The high average hydrophobicity of luciferase, which places it among the more hydrophobic proteins reported, is believed to account, at least in part, for its tendency to self-associate forming inactive dimers and higher molecular weight species.

The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is suggested that, under anaerobic conditions, muscles of marine invertebrates form lactate and/or octopine or succinate (or similar end product) according to the activities of the enzymes present in the muscles (see above). The muscles investigated possess low activities of cytosolic glycerol 3-phosphate dehydrogenase, which indicates that glycerol phosphate formation is quantitatively unimportant under anaerobic conditions, and low activities of mitochondrial glycerol phosphate dehydrogenase, which indicates that the glycerol phosphate cycle is unimportant in the re-oxidation of glycolytically produced NADH in these muscles under aerobic conditions. Conversely, high activities of glutamate-oxaloacetate transaminase are present in some muscles, which indicates that the malate-aspartate cycle may be important in oxidation of glycolytically produced NADH under aerobic conditions. 3. High activities of nucleoside diphosphate kinase were found in muscles that function for prolonged periods under anaerobic conditions (e.g...