Identification of a novel member of 2H phosphoesterases, 2',5'-oligoadenylate degrading ribonuclease from the oyster Crassostrea gigas.

Several genes of IFN-mediated pathways in vertebrates, among them the genes that participate in the 2',5'-oligoadenylate synthetase (OAS)/RNase L pathway, have been identified in C. gigas. In the present study, we identified genes, which encode proteins having 2',5'-oligoadenylate degrading activity in C. gigas. These proteins belong to the 2H phosphoesterase superfamily and have sequence similarity to the mammalian A kinase anchoring protein 7 (AKAP7) central domain, which is responsible for the 2',5'-phosphodiesterase (2',5'-PDE) activity. Comparison of the genomic structures of C. gigas proteins with that of AKAP7 suggests that these enzymes originate from a direct common ancestor. However, the identified nucleases are not typical 2',5'-PDEs. The found enzymes catalyse the degradation of 2',5'-linked oligoadenylates in a metal-ion-independent way, yielding products with 2',3' -cyclic phosphate and 5'-OH termini similarly to the 3'-5' bond cleavage in RNA, catalyzed by metal-independent ribonucleases. 3',5'-linked oligoadenylates are not substrates for them. The preferred substrates for the C. gigas enzymes are 5'-triphosphorylated 2',5'-oligoadenylates, whose major cleavage reaction results in the removal of the 5'-triphosphorylated 2',3'-cyclic phosphate derivative, leaving behind the respective unphosphorylated 2',5'-oligoadenylate. Such a cleavage reaction results in the direct inactivation of the biologically active 2-5A molecule. The 2',5'-ribonucleases (2',5'-RNases) from C. gigas could be members of the ancient group of ribonucleases, specific to 2'-5' phosphodiester bond, together with the enzyme that was characterized previously from the marine sponge Tethya aurantium. The novel 2',5'-RNases may play a role in the control of cellular 2-5A levels, thereby limiting damage to host cells after viral infection.

Natural occurrence of 2',5'-linked heteronucleotides in marine sponges.

2',5'-oligoadenylate synthetases (OAS) as a component of mammalian interferon-induced antiviral enzymatic system catalyze the oligomerization of cellular ATP into 2',5'-linked oligoadenylates (2-5A). Though vertebrate OASs have been characterized as 2'-nucleotidyl transferases under in vitro conditions, the natural occurrence of 2',5'-oligonucleotides other than 2-5A has never been demonstrated. Here we have demonstrated that OASs from the marine sponges Thenea muricata and Chondrilla nucula are able to catalyze in vivo synthesis of 2-5A as well as the synthesis of a series 2',5'-linked heteronucleotides which accompanied high levels of 2',5'-diadenylates. In dephosphorylated perchloric acid extracts of the sponges, these heteronucleotides were identified as A2'p5'G, A2' p5'U, A2'p5'C, G2'p5'A and G2' p5'U. The natural occurrence of 2'-adenylated NAD(+) was also detected. In vitro assays demonstrated that besides ATP, GTP was a good substrate for the sponge OAS, especially for OAS from C. nucula. Pyrimidine nucleotides UTP and CTP were also used as substrates for oligomerization, giving 2',5'-linked homo-oligomers. These data refer to the substrate specificity of sponge OASs that is remarkably different from that of vertebrate OASs. Further studies of OASs from sponges may help to elucidate evolutionary and functional aspects of OASs as proteins of the nucleotidyltransferase family.

Expression and characterization of recombinant 2',5'-oligoadenylate synthetase from the marine sponge Geodia cydonium.

2',5'-oligoadenylate (2-5A) synthetases are known as components of the interferon-induced cellular defence mechanism in mammals. The existence of 2-5A synthetases in the evolutionarily lowest multicellular animals, the marine sponges, has been demonstrated and the respective candidate genes from Geodia cydonium and Suberites domuncula have been identified. In the present study, the putative 2-5A synthetase cDNA from G. cydonium was expressed in an Escherichia coli expression system to characterize the enzymatic activity of the recombinant polypeptide. Our studies reveal that, unlike the porcine recombinant 2-5A synthetase, the sponge recombinant protein associates strongly with RNA from E. coli, forming a heterogeneous set of complexes. No complete dissociation of the complex occurs during purification of the recombinant protein and the RNA constituent is partially protected from RNase degradation. We demonstrate that the sponge recombinant 2-5A synthetase in complex with E. coli RNA catalyzes the synthesis of 2',5'-phosphodiester-linked 5'-triphosphorylated oligoadenylates from ATP, although with a low specific activity. Poly(I).poly(C), an efficient artificial activator of the mammalian 2-5A synthetases, has only a minimal effect (an approximate two-fold increase) on the sponge recombinant 2-5A synthetase/bacterial RNA complex activity.

ATP N-glycosidase - a novel ATP-converting activity from a marine sponge Axinella polypoides.

A novel nucleosidase enzymatic activity was discovered in the marine sponge Axinella polypoides. This enzyme, designated as ATP N-glycosidase, converts adenosine-5'-triphosphate into adenine and ribose-5-triphosphate. The crude extract of A. polypoides was capable of hydrolysing 25 micro mol ATP.min-1 per g wet weight of sponge. The catalytic activity of a sponge crude extract per mg total protein is comparable with specific activities of purified plant adenosine and bacterial AMP nucleosidases. The preferred substrate for the novel enzyme is ATP but any compound containing adenosine-5'-diphosphoryl fragment is also cleaved. The biochemical properties (Km, Kip, environmental requirements) of ATP N-glycosidase show similarities with previously described adenine-specific nucleosidases; however, the pattern of its biochemical characteristics does not match with that of any of those enzymes.

Qualitative and quantitative aspects of 2-5A synthesizing capacity of different marine sponges.

2-5A synthetase is an important component of the mammalian antiviral 2-5A system. At present, the existence of 2-5A synthetase in the lowest animals, the marine sponges, has been demonstrated, although this enzyme has not been found in bacteria, yeast or plants. Here, we studied the 2-5A synthesizing capacity and the product profile of a variety of marine sponges belonging to Demospongia subclasses Tetractinomorpha and Ceractinomorpha. The 2-5A synthetase activity varied largely, in the range of four orders of magnitude, depending on the sponge species. Compared with the enzymes of the mammalian 2-5A synthetase family, the most active sponge species exhibited a surprisingly high 2-5A synthetase specific activity. Unlike the mammalian 2-5A synthetases that produce 2-5A oligomers in the presence of a double-stranded RNA activator, the 2-5A synthetase(s) from sponges were active without the addition of dsRNA. The sponge species differed in their product profiles. A novel product pool formed by Chondrosia reniformis was identified as a series of long 2-5A oligomers (up to 17-mers) with the prevalence of heptamers and octamers. The large variability of qualitative and quantitative characteristics of sponge 2-5A synthetases may refer to the occurrence of a variety of 2-5A synthetase isozymes in sponges.

2',5'-oligoadenylate synthetase from a lower invertebrate, the marine sponge Geodia cydonium, does not need dsRNA for its enzymatic activity.

Recently, the presence of 2',5'-linked oligoadenylates and a high 2',5'-oligoadenylate synthetase activity were discovered in a lower invertebrate, the marine sponge Geodia cydonium. It has been demonstrated that mammalian 2-5A synthetase isozymes require a dsRNA cofactor for their enzymatic activity. Our results show that, unlike mammalian 2-5A synthetases, the 2-5A synthetase from the sponge acts in a dsRNA-independent manner in vitro. A prolonged incubation of the G. cydonium extract with a high concentration of a micrococcal nuclease had no effect on the activity of the 2-5A synthetase. At the same time, the micrococcal nuclease was effective within 30 min in degrading dsRNA needed for the enzymatic activity in IFN-induced PC12 cells. These results indicate that the 2-5A synthetase from G. cydonium may be active per se or is activated by some other mechanism. The sponge enzyme is capable of synthesizing a series of 2-5A oligomers ranging from dimers to octamers. The accumulation of a dimer in the predominant proportion during the first stage of the reaction was observed, followed by a gradual increase in longer oligoadenylates. By its product profile and kinetics of formation, the sponge 2-5A synthetase behaves like a specific isoform of enzymes of the 2-5A synthetase family.