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

Abstract

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.

Keywords: 2′,5′-linked heteronucleotides; 2′,5′-oligoadenylate synthetase; OAS; marine sponge; natural products.

Figure 1

The occurrence of 2′,5′-diadenylates in the form of tri-, di- and monophosphates, and “core” forms in perchloric acid extracts of T. muricata. HPLC analysis was performed by applying gradient 1 (see Experimental section). HPLC chromatograms correspond to approximately 6 mg of the sponge wet weight. Blue, red and green colored lines represent samples which were cultivated in laboratory tanks for different periods of time.

Figure 2

The occurrence of 2′,5′-linked nucleotides in a perchloric acid extract of T. muricata. HPLC analysis was performed by applying gradient 2 (see Experimental section). HPLC chromatogram corresponds to approximately 8 mg of the sponge wet weight. Blue line represents the nucleotide profile of a perchloric acid extract of the sample, red line–the product profile of the same sample after dephosphorylation of the extract. Inset: the analysis of the sample by applying HPLC gradient 1. The peaks P1-P3 were collected and analyzed: P1 involves A2′p5′G, P2–NAD2′p5′A, P3–A2′p5′U and A2′p5′C.

Figure 3

Determination of the primary structure and proof of 2′,5′-linkage in an oligoribonucleotide. Alkaline hydrolysis of an oligomeric nucleoside produces 2′- and 3′-monophosphates while the 3′-terminal base is found as a nucleoside. Digestion with snake venom phosphodiesterase produces 5′-monophosphates while the 5′-terminal base is found as a nucleoside. Ribonuclease T2 hydrolyzes any 3′,5′-internucleotide linkage between ribonucleotides, but oligomers with 2′,5′-internucleotide linkage are resistant to this nuclease.

Figure 4

Structures of compounds 1–6.

Figure 5

A correlation between the amounts of A2′p5′A and A2′p5′G in different samples (n = 64) of T. muricata. Dephosphorylated perchloric acid extracts of T. muricata were analyzed by HPLC. The amounts of 2′,5′-linked compounds were calculated from the same HPLC runs.

Figure 6

The occurrence of 2′,5′-linked compounds in a perchloric acid extract of C. nucula. HPLC analysis of a dephosphorylated sponge extract was performed by applying gradient 2 (see Experimental section). HPLC chromatogram corresponds to approximately 30 mg of the sponge wet weight. The peaks P1, P3, P4 and P5 were collected and analyzed: P1 involves A2′p5′G, P3-A2′p5′U and A2′p5′C, P4–G2′p5′U and P5–G2′p5′A.

Figure 7

The oligomerization of ATP in the presence of GTP (A, B) or UTP (C, D) by OAS from the extract of T. muricata (A, C) or C. nucula (B, D) under in vitro conditions. Substrates were used in the following concentrations: 0.86 mM ATP, 0.67 mM GTP, 0.76 mM UTP. Incubation time was 1 h for C. nucula and 17 h for T. muricata. HPLC analysis of dephosphorylated reaction mixtures was performed by applying gradient 1 (see Experimental section). The chromatographic peaks were collected and analyzed. The numbered compounds in panels are: A–1 (G2′p5′G2′p5′G + G2′p5′A2′p5′A), 2 (A2′p5′A2′p5′G), 3 (A2′p5′A2′p5′A); B–1 (G2′p5′G2′p5′A), 2 (G2′p5′G2′p5′A2′p5′A), 3 (G2′p5′G2′p5′G), 4 (G2′p5′A2′p5′A), 5 (A2′p5′G2′p5′G), 6 (A2′p5′A2′p5′G), 7 (G2′p5′A2′p5′G), 8 (A2′p5′G2′p5′A), 9 (A2′p5′A2′p5′A); C–1 (m/z 1207′1, the sequence of nucleotides not analyzed), 2 (U2′p5′A2′p5′U), 3 (U2′p5′A2′p5′A2′p5′A), 4 (U2′p5′A2′p5′A), 5 (A2′p5′A2′p5′U + A2′p5′A2′p5′A2′p5′U), 6 (A2′p5′A2′p5′A2′p5′A + A2′p5′A); D–1 (A2′p5′ ′A2′p5′U), 2 (A2′p5′A).