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Enzyme assays for synthesis and degradation of 2-5As and other 2'-5' oligonucleotides.

Poulsen JB, Kjær KH, Justesen J, Martensen PM - BMC Biochem. (2015)

Bottom Line: The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication.Whereas synthesis is mediated by the 2'-5' oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7.The nuclease reactions make use of the isolated 2'-5' oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, DK-8000, Aarhus C, Denmark. JESP@ssi.dk.

ABSTRACT

Background: The 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are central to the interferon-induced antiviral 2-5A system. The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication. The 2-5A system is tightly controlled by synthesis and degradation of 2-5As. Whereas synthesis is mediated by the 2'-5' oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7.

Results: Here we present assay tools for identification and characterization of the enzymes regulating cellular 2-5A levels. A procedure is described for the production of 2'-5' oligoadenylates, which are then used as substrates for development and demonstration of enzyme assays measuring synthetase and nuclease activities, respectively. The synthetase assays produce only a single reaction product allowing for very precise kinetic assessment of the enzymes. We present an assay using dATP and the A(pA)3 tetramer core as substrates, which requires prior isolation of A(pA)3. A synthetase assay using either of the dNTPs individually together with NAD(+) as substrates is also presented. The nuclease reactions make use of the isolated 2'-5' oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities. A purified human 2'-5' oligoadenylate synthetase and a purified human phosphodiesterase 12 along with crude extracts expressing those proteins, are used to demonstrate the assays.

Conclusions: This paper comprises an assay toolbox for identification and characterization of the synthetases and nucleases regulating cellular 2-5A levels. Assays are presented for both enzyme families. The assays can also be used to address a broader cellular role of the OAS enzymes, based on the multiple substrate specificity intrinsic to these proteins.

No MeSH data available.


Single-product OAS assay using dNTPs and NAD+ as substrates. The purified human recombinant His-OAS1 enzyme was incubated with NAD+ and dNTP (dATP, dCTP, dGTP or TTP) for 30 min at 37 °C, and the reactants and product resolved using a HiTrap Q column. Each figure (a-d) includes three chromatograms that have been superimposed. The position of the reactants and product are indicated in the chromatograms. The substrates used were: (a) NAD+ and dATP, (b) NAD+ and dCTP, (c) NAD+ and dGTP and (d) NAD+ and TTP. Blue curves: Profiles from reactions using His-OAS1 together with NAD+ and dNTP. Red and green curves: Control reactions after incubation of His-OAS1 with either NAD+ or dNTP alone, respectively. The insert in (a) shows the structural formula of NAD+. The 2′ hydroxyl (OH) to be linked with dNMP during the reaction has been highlighted in red. Brown curves: experimental salt gradients. Chromatograms were obtained by using 254 nm as the absorbance wavelength. mAU, milli-absorbance unit and mS/cm, milliSiemens/centimeter
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Fig3: Single-product OAS assay using dNTPs and NAD+ as substrates. The purified human recombinant His-OAS1 enzyme was incubated with NAD+ and dNTP (dATP, dCTP, dGTP or TTP) for 30 min at 37 °C, and the reactants and product resolved using a HiTrap Q column. Each figure (a-d) includes three chromatograms that have been superimposed. The position of the reactants and product are indicated in the chromatograms. The substrates used were: (a) NAD+ and dATP, (b) NAD+ and dCTP, (c) NAD+ and dGTP and (d) NAD+ and TTP. Blue curves: Profiles from reactions using His-OAS1 together with NAD+ and dNTP. Red and green curves: Control reactions after incubation of His-OAS1 with either NAD+ or dNTP alone, respectively. The insert in (a) shows the structural formula of NAD+. The 2′ hydroxyl (OH) to be linked with dNMP during the reaction has been highlighted in red. Brown curves: experimental salt gradients. Chromatograms were obtained by using 254 nm as the absorbance wavelength. mAU, milli-absorbance unit and mS/cm, milliSiemens/centimeter

Mentions: We also made additional set-ups and optimized the assay using alternative ‘acceptor’ and ‘donor’ molecules as substrates for the OAS (Fig. 3). NAD+, a coenzyme present in all living cells is composed of two nucleotides one containing an adenine base (the 2′ nucleotide) and the other a nicotinamide base (Fig. 3a, inset). Both in terms of unavailability of free/reactive 5′ phosphates and the presence of a 2′ hydroxyl group, NAD+ closely mimics the A(pA)3 substrate. Consequently, we substituted the A(pA)3 substrate with NAD+ and carried out the reactions with all of the dNTPs (dATP, dCTP, dGTP and TTP) separately, which should allow for synthesis of single-product NAD-dNMP derivatives. Indeed the human His-OAS1 was found to produce NAD-dNMP derivatives albeit with variable efficiencies, resulting in nice and discrete peaks with retention volumes higher than NAD+ and lower than the dNTPs (Fig. 3). The specific enzyme activities were calculated to be 0.2 mmole/(sec*g) for NAD-pdA; 0.1 mmole/(sec*g) for NAD-pdC; 0.02 mmole/(sec*g) for NAD-pdG and 0.1 mmole/(sec*g) for NAD-pT.Fig. 3


Enzyme assays for synthesis and degradation of 2-5As and other 2'-5' oligonucleotides.

Poulsen JB, Kjær KH, Justesen J, Martensen PM - BMC Biochem. (2015)

Single-product OAS assay using dNTPs and NAD+ as substrates. The purified human recombinant His-OAS1 enzyme was incubated with NAD+ and dNTP (dATP, dCTP, dGTP or TTP) for 30 min at 37 °C, and the reactants and product resolved using a HiTrap Q column. Each figure (a-d) includes three chromatograms that have been superimposed. The position of the reactants and product are indicated in the chromatograms. The substrates used were: (a) NAD+ and dATP, (b) NAD+ and dCTP, (c) NAD+ and dGTP and (d) NAD+ and TTP. Blue curves: Profiles from reactions using His-OAS1 together with NAD+ and dNTP. Red and green curves: Control reactions after incubation of His-OAS1 with either NAD+ or dNTP alone, respectively. The insert in (a) shows the structural formula of NAD+. The 2′ hydroxyl (OH) to be linked with dNMP during the reaction has been highlighted in red. Brown curves: experimental salt gradients. Chromatograms were obtained by using 254 nm as the absorbance wavelength. mAU, milli-absorbance unit and mS/cm, milliSiemens/centimeter
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Related In: Results  -  Collection

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Fig3: Single-product OAS assay using dNTPs and NAD+ as substrates. The purified human recombinant His-OAS1 enzyme was incubated with NAD+ and dNTP (dATP, dCTP, dGTP or TTP) for 30 min at 37 °C, and the reactants and product resolved using a HiTrap Q column. Each figure (a-d) includes three chromatograms that have been superimposed. The position of the reactants and product are indicated in the chromatograms. The substrates used were: (a) NAD+ and dATP, (b) NAD+ and dCTP, (c) NAD+ and dGTP and (d) NAD+ and TTP. Blue curves: Profiles from reactions using His-OAS1 together with NAD+ and dNTP. Red and green curves: Control reactions after incubation of His-OAS1 with either NAD+ or dNTP alone, respectively. The insert in (a) shows the structural formula of NAD+. The 2′ hydroxyl (OH) to be linked with dNMP during the reaction has been highlighted in red. Brown curves: experimental salt gradients. Chromatograms were obtained by using 254 nm as the absorbance wavelength. mAU, milli-absorbance unit and mS/cm, milliSiemens/centimeter
Mentions: We also made additional set-ups and optimized the assay using alternative ‘acceptor’ and ‘donor’ molecules as substrates for the OAS (Fig. 3). NAD+, a coenzyme present in all living cells is composed of two nucleotides one containing an adenine base (the 2′ nucleotide) and the other a nicotinamide base (Fig. 3a, inset). Both in terms of unavailability of free/reactive 5′ phosphates and the presence of a 2′ hydroxyl group, NAD+ closely mimics the A(pA)3 substrate. Consequently, we substituted the A(pA)3 substrate with NAD+ and carried out the reactions with all of the dNTPs (dATP, dCTP, dGTP and TTP) separately, which should allow for synthesis of single-product NAD-dNMP derivatives. Indeed the human His-OAS1 was found to produce NAD-dNMP derivatives albeit with variable efficiencies, resulting in nice and discrete peaks with retention volumes higher than NAD+ and lower than the dNTPs (Fig. 3). The specific enzyme activities were calculated to be 0.2 mmole/(sec*g) for NAD-pdA; 0.1 mmole/(sec*g) for NAD-pdC; 0.02 mmole/(sec*g) for NAD-pdG and 0.1 mmole/(sec*g) for NAD-pT.Fig. 3

Bottom Line: The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication.Whereas synthesis is mediated by the 2'-5' oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7.The nuclease reactions make use of the isolated 2'-5' oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, DK-8000, Aarhus C, Denmark. JESP@ssi.dk.

ABSTRACT

Background: The 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are central to the interferon-induced antiviral 2-5A system. The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication. The 2-5A system is tightly controlled by synthesis and degradation of 2-5As. Whereas synthesis is mediated by the 2'-5' oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7.

Results: Here we present assay tools for identification and characterization of the enzymes regulating cellular 2-5A levels. A procedure is described for the production of 2'-5' oligoadenylates, which are then used as substrates for development and demonstration of enzyme assays measuring synthetase and nuclease activities, respectively. The synthetase assays produce only a single reaction product allowing for very precise kinetic assessment of the enzymes. We present an assay using dATP and the A(pA)3 tetramer core as substrates, which requires prior isolation of A(pA)3. A synthetase assay using either of the dNTPs individually together with NAD(+) as substrates is also presented. The nuclease reactions make use of the isolated 2'-5' oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities. A purified human 2'-5' oligoadenylate synthetase and a purified human phosphodiesterase 12 along with crude extracts expressing those proteins, are used to demonstrate the assays.

Conclusions: This paper comprises an assay toolbox for identification and characterization of the synthetases and nucleases regulating cellular 2-5A levels. Assays are presented for both enzyme families. The assays can also be used to address a broader cellular role of the OAS enzymes, based on the multiple substrate specificity intrinsic to these proteins.

No MeSH data available.