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- Molecular cloning and functional characterization of porcine 2',5'-oligoadenylate synthetase 1b and its effect on infection with porcine reproductive and respiratory syndrome virus. [Journal Article]
- VIVet Immunol Immunopathol 2019; 209:22-30
- Porcine reproductive and respiratory syndrome virus (PRRSV) has previously been shown to increase porcine 2'-5'-oligoadenylate synthase (OAS) 1a expression, but the specific role of porcine OAS1b (pO…
Porcine reproductive and respiratory syndrome virus (PRRSV) has previously been shown to increase porcine 2'-5'-oligoadenylate synthase (OAS) 1a expression, but the specific role of porcine OAS1b (pOAS1b) in PRRSV replication remains unknown. In this study, we conducted sequence analysis of the porcine OAS1b gene and studied the effects of its overexpression or silencing on PRRSV replication. OAS1b, localized mainly in the cytoplasm, was found to contain conserved protein domains, such as the P-Loop and D-Box, indicating that its nucleotidyl transferase activity was complete and the antiviral effect depended on ribonuclease L (RNase L). OAS1b overexpression inhibited PRRSV replication, whereas small-interfering-RNA silencing of OAS1b resulted in increased virus titers. Additionally, OAS1b promoted expression of interferons as well as interferon-β promoter activity. These results lay the theoretical foundation for the development of new anti-PRRSV strategies.
- Differences in biochemical properties of two 5'-nucleotidases from deep- and shallow-sea Shewanella species under various harsh conditions. [Journal Article]
- BBBiosci Biotechnol Biochem 2019; 83(6):1085-1093
- Deep-sea Shewanella violacea 5'-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between th…
Deep-sea Shewanella violacea 5'-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between them being 70.4%. Here, SVNTase exhibited higher activity than SANTase with various inorganic salts, similar to the difference in their NaCl tolerance. In contrast, SVNTase activity decreased with various organic solvents, while SANTase activity was retained with the same concentrations of the solvents. Therefore, SVNTase is more robust than SANTase with inorganic salts, but more vulnerable with organic solvents. As to protein stability, SANTase was more stable against organic solvents and heat than SVNTase, which correlated with the differences in their enzymatic activities. We also found that SANTase retained higher activity for three weeks than SVNTase did in the presence of glycerol. These findings will facilitate further application of these enzymes as appropriate biological catalysts under various harsh conditions. Abbreviations: NTase: 5'-nucleotidase; SANTase: Shewanella amazonensis 5'-nucleotidase; SVNTase: Shewanella violacea 5'-nucleotidase; CD: circular dichroism.
- Purification and characterization of a platelet aggregation inhibitor and anticoagulant Cc 5_NTase, CD 73-like, from Cerastes cerastes venom. [Journal Article]
- JBJ Biochem Mol Toxicol 2017; 31(5)
- The present study is the first attempt to report the characterization of a nucleotidase from Cerastes cerastes venom. A 70 kDa 5'-nucleotidase (Cc-5'NTase) was purified to homogeneity. The amino acid…
The present study is the first attempt to report the characterization of a nucleotidase from Cerastes cerastes venom. A 70 kDa 5'-nucleotidase (Cc-5'NTase) was purified to homogeneity. The amino acid sequence of Cc-5'NTase displayed high homology with many nucleotidases. Its activity was optimal at pH 7 with a specific hydrolytic activity toward mono-, di-, and triphosphate adenylated nucleotides. Cc-5'NTase preferentially hydrolyzed ADP and obeyed Michaelis-Menten kinetics. Among the metals and inhibitors tested, Ni2+ and Mg2+ completely potentiated enzyme activity, whereas EGTA, PMSF, iodoacetamide, vanillic acid, vanillyl mandelic acid, and 1,10-phenanthroline partially abolished its activity. Cc-5'NTase was not lethal for mice at 5 mg/kg and exhibited in vivo anticoagulant effect. It also dose-dependently inhibited adenosine diphosphate-induced platelet aggregation by converting adenosine diphosphate to adenosine and prohibited arachidonic acid-induced aggregation but was not effective on fibrinogen-induced aggregation. Cc-5'NTase could be a good tool as pharmacological molecule in thrombosis diagnostic and/or therapy.
- Identification of 2'-5'-Oligoadenylate Synthetase-Like Gene in Goose: Gene Structure, Expression Patterns, and Antiviral Activity Against Newcastle Disease Virus. [Journal Article]
- JIJ Interferon Cytokine Res 2016; 36(9):563-72
- 2'-5'-oligoadenylate synthetase-like (OASL) is a kind of antiviral protein induced by interferons (IFNs), which plays an important role in the IFNs-mediated antiviral signaling pathway. In this study…
2'-5'-oligoadenylate synthetase-like (OASL) is a kind of antiviral protein induced by interferons (IFNs), which plays an important role in the IFNs-mediated antiviral signaling pathway. In this study, we cloned and identified OASL in the Chinese goose for the first time. Goose 2'-5'-oligoadenylate synthetase-like (goOASL), including an ORF of 1527bp, encoding a protein of 508 amino acids. GoOASL protein contains 3 conserved motifs: nucleotidyltransferase (NTase) domain, 2'-5'-oligoadenylate synthetase (OAS) domain, and 2 ubiquitin-like (UBL) repeats. The tissue distribution profile of goOASL in 2-week-old gosling and adult goose were identified by Real-Time quantitative PCR, which revealed that the highest level of goOASL mRNA transcription was detected in the blood of adult goose and gosling. The mRNA transcription level of goOASL was upregulated in all tested tissues of duck Tembusu virus (DTMUV)-infected 3-day-old goslings, compared with control groups. Furthermore, using the stimulus Poly(I: C), ODN2006, R848, and lipopolysaccharide (LPS) as well as the viral pathogens DTMUV, H9N2 avian influenza virus (AIV), and gosling plague virus (GPV) to treat goose peripheral blood mononuclear cells (PBMCs) for 6 h, goOASL transcripts level was significantly upregulated in all treated groups. To further investigate the antiviral activity of goOASL, pcDNA3.1(+)-goOASL-His plasmid was constructed, and goOASL was expressed by the goose embryo fibroblast cells (GEFs) transfected with pcDNA3.1(+)-goOASL-His. Our research data suggested that Newcastle disease virus (NDV) replication (viral copies and viral titer) in GEFs was significantly reduced by the overexpression of goOASL protein. These data were meaningful for the antiviral immunity research of goose and shed light on the future prevention of NDV in fowl.
- Structure-function analysis of the OB and latch domains of chlorella virus DNA ligase. [Journal Article]
- JBJ Biol Chem 2011 Jun 24; 286(25):22642-52
- Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransfera…
Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.
- Functional dissection of the DNA interface of the nucleotidyltransferase domain of chlorella virus DNA ligase. [Journal Article]
- JBJ Biol Chem 2011 Apr 15; 286(15):13314-26
- Chlorella virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped prote…
Chlorella virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped protein clamp: a nucleotidyltransferase (NTase) domain and an OB domain (these two are common to all DNA ligases) as well as a distinctive β-hairpin latch module. The NTase domain, which performs the chemical steps of ligation, binds the major groove flanking the nick and the minor groove on the 3'-OH side of the nick. Here we performed a structure-guided mutational analysis of the NTase domain, surveying the effects of 35 mutations in 19 residues on ChVLig activity in vivo and in vitro, including biochemical tests of the composite nick sealing reaction and of the three component steps of the ligation pathway (ligase adenylylation, DNA adenylylation, and phosphodiester synthesis). The results highlight (i) key contacts by Thr-84 and Lys-173 to the template DNA strand phosphates at the outer margins of the DNA ligase footprint; (ii) essential contacts of Ser-41, Arg-42, Met-83, and Phe-75 with the 3'-OH strand at the nick; (iii) Arg-176 phosphate contacts at the nick and with ATP during ligase adenylylation; (iv) the role of Phe-44 in forming the protein clamp around the nicked DNA substrate; and (v) the importance of adenine-binding residue Phe-98 in all three steps of ligation. Kinetic analysis of single-turnover nick sealing by ChVLig-AMP underscored the importance of Phe-75-mediated distortion of the nick 3'-OH nucleoside in the catalysis of DNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3). Induced fit of the nicked DNA into a distorted conformation when bound within the ligase clamp may account for the nick-sensing capacity of ChVLig.
- PI-specific phospholipase C cleavage of a reconstituted GPI-anchored protein: modulation by the lipid bilayer. [Journal Article]
- BBiochemistry 2002 Jan 29; 41(4):1398-408
- Release of glycosylphosphatidylinositol- (GPI-) anchored ectoenzymes from the membrane by phosphatidylinositol- (PI-) specific phospholipases may play an important role in modulating the surface expr…
Release of glycosylphosphatidylinositol- (GPI-) anchored ectoenzymes from the membrane by phosphatidylinositol- (PI-) specific phospholipases may play an important role in modulating the surface expression and function of this group of proteins. To investigate how the properties of the host membrane affect anchor cleavage, porcine lymphocyte ecto-5'-nucleotidase (5'-NTase; EC 126.96.36.199) was purified, reconstituted into lipid bilayer vesicles of various lipids, and cleaved using PI-PLC from Bacillus thuringiensis (Bt-PI-PLC). Bt-PI-PLC activity was highly dependent on the chain length and unsaturation of the constituent phospholipids. Very high rates of cleavage were observed in fluid lipids with a low phase transition temperature (T(m)), in lymphocyte plasma membrane, and in a lipid mixture that formed rafts. Arrhenius plots of the rate of anchor cleavage in various lipids showed a characteristic break at the bilayer T(m), together with a discontinuity close to T(m). The activation energy for GPI anchor cleavage was substantially higher in gel phase bilayers compared to those in the liquid crystalline phase. The addition of cholesterol simultaneously abolished the phase transition and the large difference in cleavage rates observed above and below T(m). Inclusion of GM(1) and GT(1b) (components of lipid rafts) in the bilayer reduced the overall activity, but the pattern of the Arrhenius plots remained unchanged. Both gangliosides had similar effects, suggesting that bilayer surface charge has little influence on PI-PLC activity. Taken together, these results suggest that lipid fluidity and packing are the most important modulators of Bt-PI-PLC activity on GPI anchors.
- Preservation of global cardiac function in the rabbit following protracted ischemia/reperfusion using monophosphoryl lipid A (MLA). [Journal Article]
- JMJ Mol Cell Cardiol 1996; 28(1):197-208
- Monophosphoryl lipid A (MLA), a derivative of the minimal substructure of lipopolysaccharide (lipid A) possesses immunomodulatory activity of the parent lipid A yet enjoys reduced toxicity. It has pr…
Monophosphoryl lipid A (MLA), a derivative of the minimal substructure of lipopolysaccharide (lipid A) possesses immunomodulatory activity of the parent lipid A yet enjoys reduced toxicity. It has previously been reported that pretreatment with MLA reduces myocardial infarct size and stunning in dogs following ischemia and reperfusion. The aim of this study was to evaluate the ability of monophosphoryl lipid A (MLA) to preserve global cardiac function and peripheral hemodynamics in a rabbit model of prolonged regional ischemia (90 min), and reperfusion (6 h). An evaluation of potential mechanisms by which MLA may preserve cardiac function was also undertaken. Single dose pretreatment with MLA (35 micrograms/kg i.v.) 24 h prior to ischemia resulted in significant improvement in left ventricular developed pressure, dP/dt, rate-pressure product and mean arterial pressure during reperfusion (P < 0.05 v control). Although in this model of prolonged ischemia MLA pretreatment did not reduce infarct size (54.5 +/- 11.4% in control v 63.3 +/- 8.3% in MLA, P = N.S.), evaluation of myocardial adenylate and adenosine catabolite pools at the end of ischemia indicated a preservation of ATP and ADP and a decreased production of downstream adenosine catabolites including inosine, xanthine and uric acid. Adenosine kinase, but not 5'-nucleotidase (5'-NTase) or adenosine deaminase activity determined following reperfusion was 76% and 60% higher (P < 0.05) in non-risk and post-ischemic myocardium of MLA pretreated rabbits compared with controls. Although there was a trend toward lower tissue myeloperoxidase activity in post-ischemic myocardium from treated rabbits, the results were not significantly different from control animals. These results suggest that a 24-h pretreatment with MLA, without further treatment during ischemia or reperfusion was associated with: (1) preservation of global myocardial function during reperfusion; (2) preservation of myocardial high energy adenylates and reduced formation of adenosine catabolites during ischemia; (3) elevated myocardial adenosine kinase activity. Increased recycling of adenosine to phosphorylated nucleotides may result from MLA's affect on adenosine kinase, which could explain the drugs effect on adenylate and adenosine metabolite pools.
- Characterization of analog resistance and purine metabolism of adult rat-liver epithelial cell 8-azaguanine-resistant mutants. [Journal Article]
- MRMutat Res 1978; 49(1):103-15
- Adult rat-liver epithelial cultures were sensitive to the lethal effects of 8-azaguanine (AG), but lines contained variants resistant to AG. The frequency of retrievable AG-resistant colonies varied …
Adult rat-liver epithelial cultures were sensitive to the lethal effects of 8-azaguanine (AG), but lines contained variants resistant to AG. The frequency of retrievable AG-resistant colonies varied with both the concentration of AG used and the seeding density of the population under selection. Cells resistant to AG were also cross-resistant to 6-thioguanine and unable to grow in medium containing hypoxanthine, aminopterin and thymidine. Resistance was stable. AG resistance was due to a deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRTase) activity which was not caused by an inhibitor. In the assay for HGPRTase, a substantial amount of product appeared as inosine (In) in addition to inosine monophosphate (IMP). Purine nucleoside phosphorylase will generate In from hypoxanthine and, indeed, the cells did possess this activity. However, several findings indicated that the In was derived from IMP by catabolism by 5'-nucleotidase (NTase): (1) IMP decreased as In increased and (2) the inhibitors of NTase, adenosine monophosphate and thymidine triphosphate, reduced the generation of In by over 90% without inhibiting purine nucleoside phosphorylase. The cells possessed substantial NTase activity, 35% of which was located in the cytosol along with 69% of HGPRTase. Several lines of evidence suggested that the NTase activity limited the amount of 8-azaguanylic acid presented to the cells by catabolising the nucleotide and, thereby, reducing the toxicity of available AG.