RESUMEN
Previously we had discovered unusual enzymatic activity in the marine sponge Axinella polypoides, ATP N-glycosidase (Reintamm et al., 2003). We show here that the Ephydatia muelleri mRNA encoding protein with PNP_UDP_1 (phosphorylase superfamily) signature is the secreted ATP N-glycosidase. The functionality of the protein was established by recombinant expression in Pichia pastoris. In addition to the enzymatic domain, the full-length protein contains the N-terminal cysteine-rich domain belonging to the subfamily SCP_HrTT-1 (cd05559) of the SCP (sperm coating protein) superfamily (cl00133).
Asunto(s)
Clonación Molecular , Expresión Génica , Glicósido Hidrolasas , Poríferos , Animales , Glicósido Hidrolasas/biosíntesis , Glicósido Hidrolasas/química , Glicósido Hidrolasas/clasificación , Glicósido Hidrolasas/genética , Pichia/genética , Pichia/metabolismo , Poríferos/enzimología , Poríferos/genética , Dominios Proteicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/clasificación , Proteínas Recombinantes/genéticaRESUMEN
2',5'-Oligoadenylate synthetases (OASs) belong to the nucleotidyl transferase family together with poly(A) polymerases, CCA-adding enzymes and the recently discovered cyclic-GMP-AMP synthase (cGAS). Mammalian OASs have been thoroughly characterized as components of the interferon-induced antiviral system. The OAS activity and the respective genes were also discovered in marine sponges where the interferon system is absent. In this study the recombinant OASs from several multicellular animals and their closest unicellular relative, a choanoflagellate, were expressed in a bacterial expression system and their enzymatic activities were examined. We demonstrated 2-5A synthesizing activities of OASs from the marine sponge Tedania ignis, a representative of the phylogenetically oldest metazoan phylum (Porifera), from an invertebrate of the protostome lineage, the mollusk Mytilus californianus (Mollusca), and from a vertebrate species, a cartilaginous fish Leucoraja erinacea (Chordata). However, the expressed proteins from an amphibian, the salamander Ambystoma mexicanum (Chordata), and from a protozoan, the marine choanoflagellate Monosiga brevicollis (Choanozoa), did not show 2-5A synthesizing activity. Differently from other studied OASs, OAS from the marine sponge T. ignis was able to catalyze the formation of oligomers having both 2',5'- and 3',5'-phosphodiester linkages. Our data suggest that OASs from sponges and evolutionarily higher animals have similar activation mechanisms which still include different affinities and possibly different structural requirements for the activating RNAs. Considering their 2'- and 3'-specificities, sponge OASs could represent a link between evolutionarily earlier nucleotidyl transferases and 2'-specific OASs from higher animals.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/clasificación , Ambystoma mexicanum/metabolismo , Coanoflagelados/enzimología , Mytilus/enzimología , Filogenia , Poríferos/enzimología , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Ambystoma mexicanum/clasificación , Ambystoma mexicanum/genética , Secuencia de Aminoácidos , Animales , Evolución Biológica , Coanoflagelados/clasificación , Coanoflagelados/genética , Datos de Secuencia Molecular , Mytilus/clasificación , Mytilus/genética , Nucleotidiltransferasas/clasificación , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Poríferos/clasificación , Poríferos/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
2',5'-Oligoadenylate synthetases (2-5A synthetases, OAS) are enzymes that play an important role in the interferon-induced antiviral defense mechanisms in mammals. Sponges, the evolutionarily lowest multicellular animals, also possess OAS; however, their function is presently unclear. Low homology between primary structures of 2-5A synthetases from vertebrates and sponges renders their evolutionary relationship obscure. The genomic structure of vertebrate OASs has been thoroughly examined, making it possible to elucidate molecular evolution and expansion of this gene family. Until now, no OAS gene structure was available from sponges to compare it with the corresponding genes from higher organisms. In the present work, we determined the exon/intron structure of the OAS gene from the marine sponge Geodia cydonium and found it to be completely different from the strictly conserved exon/intron pattern of the OAS genes from vertebrates. This finding was corroborated by the analysis of OAS genes from another sponge, Amphimedon queenslandica, whose genome was recently sequenced. Our data suggest that vertebrate and sponge OAS genes have no direct common intron-containing ancestor and two (sub)types of OAS may be discriminated. This study opens new perspectives for understanding the phylogenesis and evolution of 2-5A synthetases as well as functional aspects of this multigene family.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , Exones/genética , Intrones/genética , Familia de Multigenes , Poríferos/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/química , Evolución Molecular , Genoma , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Poríferos/enzimología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , VertebradosRESUMEN
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.