Production and purification of recombinant 2'-5' oligoadenylate synthetase and its mutants using the baculovirus system

Investigation of the structure-function relationship of the 2'-5' oligoadenylate [2-5 (A)] synthetases has been hampered by the lack of an efficient expression system for a recombinant enzyme. Here, we report that the 9-2 isozyme of murine 2-5 (A) synthetase can be efficiently expressed in insect cells using the baculovirus system. The recombinant protein was purified to apparent homogeneity, and its enzymatic activity was characterized. It had a high specific activity, required double-stranded RNA as a cofactor, and synthesized dimers to hexamers of 2-5 (A). The utility of our expression system was demonstrated by studying the properties of two previously reported mutant proteins. Both of these mutants, when produced in bacteria, are enzymatically inactive, although similarly produced wild-type protein is active. Unexpectedly, when expressed in insect cells, both mutant proteins were enzymatically as active as the wild-type protein. These results suggest that in the eukaryotic expression system described here, the mutant proteins can undergo appropriate modifications or folding that is required for attaining an enzymatically active conformation.     

Effects of mutating specific residues present near the amino terminus of 2'-5'-oligoadenylate synthetase

In this study, we investigated the role of specific amino acid residues present near the amino terminus of the 9-2 isozyme of 2'-5'-oligoadenylate synthetase. In vitro expression of deletion mutants showed that residues 1-9 are required for enzyme activity. Within this region, residues 3, 7, and 8 were found to be conserved among all known isozymes of 2'-5'-oligoadenylate synthetase. Mutation of these residues singly or in combination resulted in partial or total loss of enzyme activity. Substitution of the proline residue at position 7 by different residues caused a partial or complete loss of activity. The properties of the inactive P7Q mutant were further explored by expressing the protein in bacteria. The bacterially expressed protein was also enzymatically inactive. The mutant protein could bind the substrate ATP and the activator double-stranded RNA normally. Oligomerization properties of the protein were examined by an affinity-based interaction assay and by glycerol gradient centrifugation; there was no detectable difference between the wild type and the P7Q mutant. These results demonstrated the importance of the proline residue at position 7 in conferring enzyme activity to the protein without affecting its other properties.     

Enzymatic activity of 2'-5'-oligoadenylate synthetase is impaired by specific mutations that affect oligomerization of the protein

Previous studies from our laboratory have shown that deletion of residues 321 to 344 of the 9-2 isozyme of 2'-5'-oligoadenylate (2-5(A)) synthetase causes a loss of its enzyme activity (Ghosh, S. K., Kusari, J., Bandyopadhyay, S. K., Samanta, H., Kumar, R., and Sen, G. C. (1991) J. Biol. Chem. 266, 15293-15299). Sequence comparison of this region among the different isozymes of 2-5(A) synthetases revealed that the residues at positions 330 to 333 are highly conserved. Alanine-scanning mutagenesis of these residues demonstrated that the residues present at 331, 332, and 333 are important for activity but the proline at position 330 was dispensable. The triple mutant containing Ala residues at 331, 332, and 333 was completely inactive. Different double mutants were slightly active, and the three single mutants were partially active. The triple mutant was further characterized for delineating the nature of its defect. The mutant protein was enzymatically inactive irrespective of whether it was synthesized in rabbit reticulocyte lysate, Escherichia coli or Trichoplusia ni insect cells. It could bind double-stranded RNA and ATP as efficiently as the wild type protein. It was, however, defective in oligomerization. Gel filtration and sedimentation velocity analyses of in vitro synthesized proteins revealed that the wild type protein, but not the triple mutant, formed tetramers. The tetrameric fraction, but not the monomeric fraction of the wild type protein was enzymatically active. The failure of the triple mutant to participate in homomeric protein-protein interaction was confirmed by in vivo assays in insect cells. These results indicate that tetramerization of the protein is required for the enzymatic activity of the small 2-5(A) synthetases.     

Activation of 2'-5' oligoadenylate synthetase by single-stranded and double-stranded RNA aptamers

A number of small RNA molecules that are high affinity ligands for the 46-kDa form of human 2'-5' oligoadenylate synthetase have been identified by the SELEX method. Surface plasmon resonance analysis indicates that these RNAs bind to the enzyme with dissociation constants in the nanomolar range. Competition experiments indicate that the binding site for the small RNAs on the 2'-5' oligoadenylate synthetase molecule at least partially overlaps that for the synthetic double-stranded RNA, poly(I).poly(C). Several of the RNAs function as potent activators of 2'-5' oligoadenylate synthetase in vitro, although there is no correlation between binding affinity and ability to activate. The RNA aptamers having the strongest activation potential appear to have few base-paired regions. This suggests that 2'-5' oligoadenylate synthetase, which has previously been believed to be activated only by double-stranded RNA, can also be activated by RNA ligands with little secondary structure. Since 2'-5' oligoadenylate synthetase possesses no homology to other known RNA-binding proteins, the development of small specific ligands by SELEX should facilitate studies of RNA-protein interactions and may reveal novel features of the structure-function relationships involving this enzyme.     

Virus-induced cell death in plants expressing the mammalian 2',5' oligoadenylate system

The major components of the 2-5A system, responsible for the mammalian interferon-induced antiviral response, are the 2',5' oligoadenylate synthetase (2-5Aase) and 2',5' oligoadenylate (2-5A) dependent ribonuclease (RNase L). Transgenic tobacco plants expressing these two enzyme activities were produced by crossing the transgenic plants expressing RNase L with those expressing 2-5Aase. The double transgenic plants showed complete resistance against cucumber mosaic virus (CMV), infection with necrotic spots only forming on the virus-inoculated leaf. On the other hand, although plants inoculated with potato virus Y (PVY) formed necrotic spots on the inoculated leaf and virus amplification could not be detected, all plants died within 20 days of inoculation. The transgenic tobacco plants expressing either 2-5Aase or RNase L activity showed typical disease symptoms with CMV- or PVY-inoculation. These results suggest that the introduced 2-5A system is activated in tobacco cells by dsRNA, the replicating intermediates of RNA viruses, leading to death of the host cells, which has not been observed in mammalian cells.     

p59OASL, a 2'-5' oligoadenylate synthetase like protein: a novel human gene related to the 2'-5' oligoadenylate synthetase family

The 2'-5' oligoadenylate synthetases form a well conserved family of interferon induced proteins, presumably present throughout the mammalian class. Using the Expressed Sequence Tag databases, we have identified a novel member of this family. This protein, which we named p59 2'-5' oligoadenylate synthetase-like protein (p59OASL), shares a highly conserved N-terminal domain with the known forms of 2'-5' oligoadenylate synthetases, but differs completely in its C-terminal part. The C-terminus of p59OASL is formed of two domains of ubiquitin-like sequences. Here we present the characterisation of a full-length cDNA clone, the genomic sequence and the expression pattern of this gene. We have addressed the evolution of the 2'-5' oligoadenylate synthetase gene family, in the light of both this new member and new 2'-5' oligoadenylate synthetase sequence data from other species, which have recently appeared in the databases.     

Herpes simplex virus type 1 DNase: functional analysis of the enzyme expressed by recombinant baculovirus

Herpes simplex virus type 1 DNase (HSV-1 DNase) was expressed in insect cells by recombinant baculovirus (NPVUL12) and purified by a combination of anionic exchanger chromatography and gel filtration. Two polypeptides of 85 and 75 kD, whose ratio varied during purification, were induced 24 h after infection. The 75-kD protein was isolated and shown to possess catalytic activity. Gel filtration analysis indicated that the active form of the enzyme at an ionic strength of I = 0.3 is a dimeric protein with an apparent molecular weight of 130,000. The recombinant enzyme exhibited the overall characteristics of the native enzyme such as 5'-3' exonuclease and endonuclease activities with a preferred degradation of DNA. In the absence of extraneously added Mg2+, the enzyme was capable of removing mononucleotides from 5'-end-labeled DNA, but not from RNA and 3'-end-labeled DNA. The peculiar mechanism of double-strand DNA degradation suggests a specific role of HSV-1 DNase in DNA recombination processes during viral replication.     

Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control

In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). In mammals, the phosphorylation was shown to be carried out by eIF-2alpha kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2alpha kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2alpha kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced in Escherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2alpha on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In the Saccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2alpha kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2alpha. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2alpha kinase plays an important role in translational control from nematodes to mammals.     

Modification of ventricular tachycardia by carotid sinus massage

Imiquimod and its analogs belonging to a class of imidazoquinolinamines, activate immune system via cytokine induction, and have antitumor and antiviral effects in mammals. In this study, we showed that a related analog, designated S-28828, induced interferon (IFN) and macrophage activating cytokine(s) (macrophage activating factor, MAF) in chickens in vivo, ex vivo, and in vitro. IFN and MAF were detectable in the serum of chickens following oral administration. Serum IFN levels were the highest at 2 h after treatment. Although there was no detectable IFN in sera of chickens at 8, 24, and 48 h after treatment, high levels of interferon inducible enzyme, 2'-5' oligoadenylate synthase (2'5'OAS) were present at these time points. In vitro and ex vivo studies showed that spleen cells, bone marrow (BM) cells, and peripheral blood leukocytes (PBL) were capable of producing IFN and MAF, although spleen cells produced the highest levels. Our results suggest that S-28828 administered orally may be a useful immunoenhancing and antiviral agent for chickens.     

Residues at the carboxy terminus of T4 DNA polymerase are important determinants for interaction with the polymerase accessory proteins

Three T4 DNA polymerase accessory proteins (44P/62P and 45P) stimulate the polymerase (pol) activity and the 3'-5' exonuclease (exo) activity of T4 DNA polymerase (43P) on long, double-stranded DNA substrates. The 44P/62P "clamp loader" facilitates the binding of 45P, the "sliding clamp", to DNA that is primed for replication. Using a series of truncated 43P mutants, we identified a region at the extreme carboxy terminus of the DNA polymerase that is required for its interaction with accessory proteins. Truncation mutants of 43P lacking the carboxy-terminal 3, 6, or 11 residues retained full pol and exo activity on short synthetic primer-templates. However, the ability of the accessory proteins to enhance these activities on long double-stranded DNA templates was drastically reduced, and the extent of the reduction in activity was greater as more residues were deleted. One of the truncation mutants (N881), which had 17 residues removed from the carboxy terminus, showed reduced binding affinity and diminished pol activity but enhanced exo activity upon incubation with a small primer-template. The exo activity of the N881 mutant, on short, single-stranded DNA was unchanged, however, compared to the wild-type enzyme. These results are consistent with inferences drawn from the crystal structure of a DNA polymerase from a related T-even phage, RB69, where the carboxy-terminal 12 residues (equivalent to the 11 residues of 43P from phage T4) protrude from the thumb domain and are free to interact with complementary surfaces of the accessory proteins. The structural integrity of the thumb region in the N881 mutant is probably perturbed and could account for its reduced binding affinity and pol activity when incubated with short, double-stranded DNA substrates.     

Probing the role of cysteine residues in the EcoP15I DNA methyltransferase

Chemical modification using thiol-directed agents and site-directed mutagenesis has been used to investigate the role of cysteine residues of EcoP15I DNA methyltransferase. Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethylmaleimide and iodoacetamide. 5, 5'-Dithiobis(2-nitrobenzoic acid) titration of the enzyme under nondenaturing and denaturing conditions confirmed the presence of six cysteine residues without any disulfides in the protein. Aware that relatively bulky reagents inactivate the methyltransferase by directly occluding the substrate-binding site or by locking the methyltransferase in an inactive conformation, we used site-directed mutagenesis to sequentially replace each of the six cysteines in the protein at positions 30, 213, 344, 434, 553, and 577. All the resultant mutant methylases except for the C344S and C344A enzymes retained significant activity as assessed by in vivo and in vitro assays. The effects of the substitutions on the function of EcoP15I DNA methyltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis. Our results clearly indicate that the cysteines at positions other than 344 are not essential for activity. In contrast, the C344A enzyme showed a marked loss of enzymatic activity. More importantly, whereas the inactive C344A mutant enzyme bound S-adenosyl-L-methionine, it failed to bind to DNA. Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive. Taken together, these results convincingly demonstrate that the Cys-344 is necessary for enzyme activity and indicate an essential role for it in DNA binding.     

Bisphenols that stimulate cells to release alkali metal cations: a structure-activity study

The laxative action of phenolphthalein (5) is believed to result from induction of potassium and water efflux from the colon epithelium. In cultured cells, K+ efflux is promoted by 5 and by a contaminant (1) present in commercial phenol red. Six compounds with chemical structures related to those of 5 and 1 were tested for ability to induce the release of 86Rb from COS-7 cells preloaded with this isotope: 4,4'-(9-fluorenylidene)diphenol (2), 4, 4'-(9-fluorenylidene)dianiline, 4, 4'-(9-fluorenylidene)bisphenoxyethanol, 1,1'-bi-2-naphthol, 4, 4'-biphenol, and bis(4-hydroxyphenyl)methane. With one exception these compounds were all inactive at a concentration of 10 microM. However, 2 caused profound 86Rb efflux at concentrations as low as 100 nM. Concentrations of 5 1-2 orders of magnitude higher were needed to achieve similar levels of activity. The three compounds known to be active in this experimental system share a common feature that is absent in all the inactive compounds: a five-membered ring structure, one of whose carbon atoms is disubstituted with p-hydroxyphenyl residues. Because 2 and 5 are readily available, comparative studies on the mechanism of action of these biphenols at the cellular level can now be undertaken.     

Expression, purification, and properties of the aldehyde dehydrogenase homologous carboxyl-terminal domain of rat 10-formyltetrahydrofolate dehydrogenase

We expressed the NH2-terminal domain of the multidomain, multifunctional enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), using a baculovirus expression system in insect cells. Expression of the 203-amino acid NH2-terminal domain (residues 1-203), which is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.2), resulted in the appearance of insoluble recombinant protein apparently due to incorrect folding. The longer NH2-terminal recombinant protein (residues 1-310), which shares 32% identity with Escherichia coli L-methionyl-tRNA formyltransferase (EC 2.1.2.9), was expressed as a soluble protein. During expression, this protein was released from cells to the culture medium and was purified from the culture medium by 5-formyltetrahydrofolate-Sepharose affinity chromatography followed by chromatography on a Mono-Q column. We found that the purified NH2-terminal domain bears a folate binding site, possesses 10-formyltetrahydrofolate hydrolase activity, and exists as a monomer. Titration of tryptophan fluorescence showed that native FDH bound both the substrate of the reaction, 10-formyl-5, 8-dideazafolate, and the product of the reaction, 5,8-dideazafolate, with the same affinities as its NH2-terminal domain did and that both proteins bound the substrate with a 50-fold higher affinity than the product. Neither the NH2-terminal domain nor its mixture with the previously purified COOH-terminal domain had 10-formyltetrahydrofolate dehydrogenase activity. Formation of complexes between the COOH- and NH2-terminal domains also was not observed. We conclude that the 10-formyltetrahydrofolate dehydrogenase activity of FDH is a result of the action of the aldehyde dehydrogenase catalytic center residing in the COOH-terminal domain on the substrate bound in the NH2-terminal domain and that the intermediate domain is necessary to bring the two functional domains together in the correct orientation.     

Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and lambda repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.     

Purification and characterization of a recombinant human thyroid peroxidase expressed in insect cells

Thyroid peroxidase (TPO) is an essential enzyme for thyroid hormone biosynthesis and is an autoantigen against which antibodies are found in a number of autoimmune thyroid disorders. Large quantities of pure TPO are essential for understanding its structure and role in normal thyroid function and thyroid diseases. In this study, we describe the production of human TPO (hTPO) using a baculovirus expression vector in insect cells. TPO was sequentially extracted from insect cells using various buffers and the protein was purified to homogeneity on a C4 reversed-phase semipreparative column using high-performance liquid chromatography. The purified protein was identified as hTPO by enzyme-linked immunosorbent assay, Western blot, and amino acid sequence analyses. Carbohydrate analysis of the recombinant hTPO showed that the protein is glycosylated and mannose is the major oligosaccharide. We have extended the carbohydrate analysis by establishing the occurrence of N-acetyl galactosamine which suggested that the recombinant hTPO might contain O-glycosyl moieties. Purified hTPO reacted specifically with sera from patients with Hashimoto's thyroiditis. Crude as well as purified hTPO did not show any enzymatic activity when produced in Sf9 insect cells grown in serum free medium. In contrast, hTPO produced in the presence of 10% fetal bovine serum containing 1 microgram/ml of haematin was enzymatically active. However, the enzymatic activity of the recombinant hTPO was lower than that often found with hTPO purified from thyroid tissue. Availability of purified hTPO in relatively large quantities should allow further structural and immunological studies.     

The prosequence of thermolysin acts as an intramolecular chaperone when expressed in trans with the mature sequence in Escherichia coli

The zinc metalloendopeptidase, thermolysin (EC 3.4.24.27) produced by Bacillus thermoproteolyticus serves as a model of important physiological enzymes such as neprilysin, angiotensin converting enzyme and endothelin converting enzyme. Thermolysin is synthesised as a pre-proenzyme, with an N-terminal prosequence of 204 residues and a mature sequence of 316 residues. The prosequence facilitates the folding of the denatured mature sequence in vitro and the cleavage of the peptide bond linking the pro and mature sequences occurs by an autocatalytic, intramolecular process. With the aim to study the role of the prosequence in vivo and to produce active mutants for structural studies, the mature sequence of thermolysin has now been expressed in Escherichia coli, either alone or with the prosequence as an independent polypeptide, i.e. in trans form. In addition, the mature sequence of an inactive mutant in which Glu143 involved in the catalytic process was replaced by Ala has also been expressed in trans with the prosequence. The results show that the pro-sequence is required to obtain active thermolysin and that a covalent link with the mature sequence is not necessary for the correct folding of the protease in vivo. Moreover, when expressed in E. coli (in trans with the prosequence), the yield of correctly folded E143A mutant was similar to that of the wild-type protease, whereas no mature enzyme was detected when it was expressed as a pre-proenzyme in Bacillus subtilis. These results demonstrate that the thermolysin prosequence acts as an intramolecular chaperone in vivo and open the way to structural studies of catalytic site mutants produced in large quantities in E. coli.