©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
2-5A-dependent RNase Molecules Dimerize during Activation by 2-5A (*)

(Received for publication, November 23, 1994)

Beihua Dong Robert H. Silverman (§)

From the Department of Cancer Biology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

2-5A-dependent RNase is an interferon-inducible enzyme that requires 5`-phosphorylated, 2`,5`-linked oligoadenylates (2-5A) for its endoribonuclease activity against single-stranded RNAs. We demonstrate here that recombinant, human 2-5A-dependent RNase forms stable homodimers during its stimulation by 2-5A. The protein dimers were observed to form only upon binding to 2-5A, as shown using gel filtration chromatography and chemical cross-linking and after centrifugation in glycerol gradients. A monoclonal antibody to 2-5A-dependent RNase was prepared and used to probe the subunit structure of the enzyme in the presence or absence of 2-5A. Using oligoadenylates of different length, structure, and 5`-phosphorylation states we determined that conversion of 2-5A-dependent RNase from its monomeric, inactive form to its homodimeric, active form required the presence of functional 2-5A. These results demonstrate that the catalytically active form of 2-5A-dependent RNase is a homodimer.


INTRODUCTION

2-5A-dependent RNase is a uniquely controlled endoribonuclease that exists in cells of higher vertebrates as a component of the interferon-regulated 2-5A system (Kerr and Brown, 1978; Clemens and Williams, 1978; Slattery et al., 1979; Zhou et al., 1993). Interferon treatment of cells results in enhanced expression of the 2-5A-dependent RNase and the double-stranded RNA-activated synthetases that produce 5`-triphosphorylated, 2`,5`-linked oligoadenylates (2-5A) from ATP. Because double-stranded RNA is a frequent product of virus infection, the 2-5A system is often active in interferon-treated and virus-infected cells. For instance, the 2-5A system is implicated in the mechanism by which interferon inhibits encephalomyocarditis virus replication (Chebath et al., 1987; Rysiecki et al., 1989; Hassel et al., 1993). In addition, a role for the 2-5A system in cell growth control is suggested from the finding that murine cells expressing a dominant-negative form of 2-5A-dependent RNase are resistant to the anti-cellular activity of interferon (Hassel et al., 1993).

The 2-5A-dependent RNase is converted from its catalytically inactive state to its active form upon binding 2-5A, but little is known about the activation process per se. However, intriguing clues about the mode of regulation have emerged from the molecular cloning and expression of 2-5A-dependent RNase (Zhou et al., 1993; Hassel et al., 1993; Dong et al., 1994). For example, sequence analysis indicates the presence in the amino-terminal half of the ribonuclease of nine ankyrin-like protein-protein interaction domains (Hassel et al., 1993). The 2-5A binding domain requires 2 lysine residues present in P-loop motifs in the seventh and eighth ankyrin repeats (Zhou et al., 1993), whereas the ribonuclease function requires the carboxyl-terminal portion of the protein (Hassel et al., 1993). The presence of ankyrin repeats suggests that the catalytic function of the enzyme could be regulated through intra- or intermolecular protein/protein contacts.

Independent determinations of the size of the 2-5A-dependent RNase from various species, cell types, and tissues have yielded differing findings. Size estimations consistent with a monomeric form of 2-5A-dependent RNase were reported for the enzyme in mouse reticulocytes (Revel et al., 1979), rabbit reticulocytes (Wreschner et al., 1982), murine EAT cells (Floyd-Smith and Lengyel, 1986), human HeLa cells, and murine W7 cells (Nilsen et al., 1981), and for the recombinant human enzyme expressed in insect cells (Dong et al., 1994) (see Fig. 1). In contrast, a 185-kDa complex containing 2-5A-dependent RNase activity was reported from murine EAT cells (Slattery et al., 1979), whereas mouse spleen (Bisbal et al., 1989) was reported to contain 2-5A-dependent RNase activities which migrated as 185- and 40-kDa species. The 40-kDa activity has not been reported elsewhere and could represent a degradation product or novel form of 2-5A-dependent RNase. Salehzada et al.(1993) also reported a 160-kDa heterodimer that contained 2-5A-dependent RNase activity from human Daudi cells. Recently, Bayard et al.(1994) reported that murine 2-5A-dependent RNase covalently attached to a 2-5A analog exists largely as a 160-kDa species. To resolve these apparently contradictory findings, we have determined the effect of 2-5A binding on the subunit composition of 2-5A-dependent RNase. Using homogeneous, recombinant enzyme and a monoclonal antibody we provide clear evidence that the binding of functional 2-5A induces the formation of stable dimers of 2-5A-dependent RNase. These findings suggest a molecular mechanism in which activation and dimerization of 2-5A-dependent RNase are different aspects of the same process.


Figure 1: 2-5A-dependent RNase forms a dimer in the presence of 2-5A as determined by gel filtration chromatography. 2-5A-dependent RNase was applied to a Superose 12 column in the presence (bullet) or absence () of p(3)A(2`p5`A)(2). 2-5A-dependent ribonuclease activity (in units) in the column fractions was determined (see ``Materials and Methods''). The positions of the protein markers and their sizes in kilodaltons are indicated (arrows).




MATERIALS AND METHODS

Purification of Recombinant, Human 2-5A-dependent RNase

Cloning and expression of human 2-5A-dependent RNase cDNA in insect cells was using a baculovirus vector as described by Dong et al.(1994). The 2-5A-dependent RNase was purified to homogeneity using three successive fast protein liquid chromatography columns.

Preparation of Monoclonal Antibody to 2-5A-dependent RNase

Female Balb/C mice (age about 8 weeks) were injected subcutaneously with 10 µg of purified human recombinant 2-5A-dependent RNase in complete Freund's adjuvant. Three subsequent subcutaneous injections were performed with 10 µg of 2-5A-dependent RNase per injection in incomplete Freund's adjuvant at 2-3-week intervals. The final boost was by intraperitoneal injection with 30 µg of 2-5A-dependent RNase without adjuvant 4 days prior to cell fusions and after antibody titers of the sera from the immunized mice were above 1:10,000 as determined by enzyme-linked immunosorbent assay. Spleen cells from two immunized mice were fused with Sp2/0 myeloma cells (from the American Type Culture Collection) at ratios of 7:1 using polyethylene glycol, M(r) 3,000-3,700 (catalog no. P-2906, Sigma). The fused cells were selected with hypoxanthine/aminopterin/thymidine (catalog no. H-0262, Hybri-Max, Sigma) in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 20% fetal bovine serum (Bio Whittaker), oxaloacetate-pyruvate-insulin (catalog no. O-5003, Sigma), glutamine, penicillin, and streptomycin in 96-well plates containing peritoneal feeder cells of BALB/C mice cultured for 1 day prior to addition of fused cells. Screenings for production of antibody against 2-5A-dependent RNase was performed beginning 8-10 days after cell fusions. Polyvinyl chloride plates (Falcon) were coated with either 100 µl (per well) of 20 µg/ml crude extract of insect cells expressing human 2-5A-dependent RNase (Dong et al., 1993) or with the same amount of control insect cell extract lacking 2-5A-dependent RNase. After incubation for 16 h at 4 °C, the plates were washed three times in 0.1% Tween 20 in saline. Blocking was with 2% bovine serum albumin in phosphate-buffered saline for 2 h at 37 °C followed by washing three times in 0.1% Tween 20 in saline. Supernatants from wells of hybridoma colonies were added to both the plates containing 2-5A-dependent RNase and control extract for 2 h at 37 °C, followed by three washes. Alkaline phosphatase-conjugated, anti-mouse IgG (Jackson ImmunoResearch) was used to detect antibody against 2-5A-dependent RNase by incubation for 1 h at 37 °C followed by four washes. p-Nitrophenyl phosphate in diethanolamine buffer (Pierce) was added, and the reaction was incubated at room temperature for 15-30 min, until a yellow color developed for the positive control containing crude antisera. Plates were analyzed at 405 nm using a microplate reader (Molecular Devices, Thermomax). Cells which screened positive were subcloned twice in succession by limiting dilution to obtain single cell clones. The hypoxanthine/aminopterin/thymidine medium was replaced with medium supplemented with hypoxanthine/thymidine (catalog no. H-0137, Hybri-Max, Sigma) during subcloning. Supernatants from expanded cultures were used to probe Western blots. The antibody titers for the cell supernatants were about 1:1,250 as determined by enzyme-linked immunosorbent assay.

Superose 12 Chromatography and Enzyme Assay

Purified 2-5A-dependent RNase, 75 µg in 100 µl of buffer A (25 mM Tris-HCl, pH 7.4, 150 mM KCl, 10% glycerol, v/v, 1 mM EDTA, 0.1 mM ATP, 5 mM MgCl(2), 14 mM mercaptoethanol, and 1 µg/ml leupeptin), was applied to a fast protein liquid chromatography, Superose 12 gel-filtration column (HR 10/30; Pharmacia Biotech Inc.) at a flow rate of 0.3 ml of buffer A/min. Dimerization of 2-5A-dependent RNase was obtained by preincubating the enzyme with 100 µM of p(3)A(2`p5`A)(2) on ice for 2 h prior to loading to the column followed by elution in buffer A containing 1 µM of p(3)A(2`p5`A)(2). For comparison, 2-5A-dependent RNase was also applied and eluted in the absence of 2-5A. Ribonuclease activity was measured in serial dilutions of column fractions by measuring the conversion of poly(rU)-[P]pCp to trichloroacetic acid-soluble degradation products by filtration on glass fiber filters as described previously (Silverman, 1985; Dong et al., 1994). Column fractions lacking 2-5A were assayed after addition of 0.1 µM p(3)A(2`p5`A)(2) whereas fractions containing p(3)A(2`p5`A)(2) were assayed without the further addition of 2-5A. One unit of 2-5A-dependent RNase activity converts 50% of 12 nM (input amount) of poly(rU)pCp to acid-soluble breakdown products in 30 min at 30 °C. Marker proteins were beta-amylase (200 kDa), human transferrin (75.2 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa), all from Sigma.

Chemically Cross-linking 2-5A-dependent RNase with Dimethyl Suberimidate

The purified 2-5A-dependent RNase (Dong et al., 1994) was dialyzed against about 5,000 volumes of buffer B (25 mM HEPES, pH 7.4, 150 mM KCl, 14 mM beta-mercaptoethanol, 50 µM ATP, 5 mM MgCl(2), 1 mM EDTA, 10% glycerol and 1 µg/ml leupeptin) at 4 °C for 16 h with three changes. Solutions of 0.5 µM of dialyzed 2-5A-dependent RNase in 4 µl were incubated with 4 µl of different amounts of oligoadenylates on ice for 2 h. To obtain cross-linking, 4 µl of 12 mg/ml dimethyl suberimidate (DMS) (^1)(Pierce) in 0.4 M triethanolamine hydrochloride, pH 8.5, were added to the reactions followed by incubation at room temperature for 2 h. The protein was denatured by addition of 12 µl of 2% SDS, 2% beta-mercaptoethanol followed by incubation at 37 °C for 2 h. Reactions were mixed with 50% glycerol and 0.01% bromphenol blue prior to loading to SDS-7.5% polyacrylamide gels. Proteins were transferred to nitrocellulose filters, and blots were probed with a 1:50 dilution of supernatant containing monoclonal antibody to 2-5Adependent RNase followed with goat anti-mouse IgG-peroxidase (Life Technologies, Inc.) and enhanced chemiluminescence (Amersham Corp.).

Glycerol Gradient Sedimentation of 2-5A-dependent RNase

Purified 2-5A-dependent RNase, 5 µl containing 0.3 µg, in buffer A or buffer B (for chemical cross-linking) was incubated for 2 h on ice in the presence or absence of 5 µM pA(2`p5`A)(3). Chemical cross-linking with DMS and denaturation of the protein was performed as described in the previous section. Internal protein standards were added as markers and reactions (in final volumes of 300 µl) were loaded to either 20-40% or 10-30% glycerol gradients in buffer A (gradient volume of 11.5 ml) prepared in a gradient apparatus (Hoefer, SG-50) using 14 times 89-mm centrifuge tubes. Centrifugation was at 247,600 times g for 40 h at 4 °C in an SW41 rotor (Beckman). Fractions (0.3 ml each) were taken, and the protein was separated by SDS-polyacrylamide gel electrophoresis. The monomeric and dimeric forms of 2-5A-dependent RNase were visualized by probing Western blots with monoclonal antibody against the RNase. The positions of the protein markers (beta-amylase at 200 kDa (Sigma), ferritin at 440 kDa (Pharmacia), and human transferrin at 75.2 kDa (Sigma)) were determined by staining with Coomassie Blue dye.

2-5A-dependent RNase Activity Assays

In solution ribonuclease assays with poly(rU)-[P]pCp as substrate were performed as described previously (Dong et al., 1994). Briefly, poly(rU) (Pharmacia) was labeled at the 3` termini with [P]pCp (3,000 Ci/mmol) and T4 RNA ligase as described previously (Silverman, 1985). 2-5A-dependent RNase (24 nM) was incubated in the presence or absence of various oligoadenylates and 12 nM of P-labeled RNA in final volumes of 25 µl at 30 °C. The trichloroacetic acid-insoluble fractions of RNA were determined by filtering on glass-fiber filters in the presence of carrier yeast tRNA by scintillation counting as described previously (Silverman, 1985).


RESULTS

Binding of 2-5A Causes Monomers of 2-5A-dependent RNase to Associate into Homodimers

To determine if 2-5A could affect the subunit structure of 2-5A-dependent RNase, gel filtration chromatography was performed on purified, recombinant human 2-5A-dependent RNase in the presence or absence of 2-5A. In the absence of activator, the 83.5-kDa RNase eluted in the volume expected for a monomer when compared to the elution volumes of marker proteins (Dong et al., 1994) (Fig. 1). In contrast, when p(3)A(2`p5`A)(3) was added to the ribonuclease preparation and to the column buffer there was a reduction in the retention volume for 2-5A-dependent RNase as monitored by ribonuclease assay (Fig. 1). By comparing the elution volumes with those of protein markers, we determined that in the presence of 2-5A, the peak of 2-5A-dependent RNase activity eluted in the volume expected for a protein of about 170 kDa.

Confirmation that 2-5A induced the formation of homodimers was obtained by monitoring 2-5A-dependent RNase after centrifugation in glycerol gradients. Purified RNase was preincubated in the absence or presence of pA(2`p5`A)(3) prior to loading on to glycerol gradients. Aliquots of the gradient fractions were analyzed after SDS-polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose filters and reacted with a monoclonal antibody against human 2-5A-dependent RNase (see ``Materials and Methods''). In the absence of 2-5A, the RNase migrated as a monomer (Fig. 2, upper panel). In contrast, after preincubation with 2-5A, the RNase migrated in two peaks corresponding in sizes to a monomer and a dimer (Fig. 2, lower panel). The lack of complete conversion to the dimeric form could be due to the dissociation of 2-5A from a portion of the RNase because of the absence of 2-5A in the gradient.


Figure 2: 2-5A induces homodimers of 2-5A-dependent RNase as shown after centrifugation in glycerol gradients. 2-5A-dependent RNase without 2-5A (upper panel) or preincubated with 5 µM pA(2`p5`A)(3) (lower panel) was centrifuged in 10-30% glycerol gradients prior to gel electrophoresis, transfer to nitrocellulose, and detection with monoclonal antibody against human 2-5A-dependent RNase (see ``Materials and Methods''). The positions and sizes (in kilodaltons) of the internal protein markers in the glycerol gradients are shown above the fraction numbers. To the right are indicated the RNase (arrow) and the positions and sizes of the protein markers in the gel.



The Homodimeric Form of 2-5A-dependent RNase Can Be Chemically Cross-linked

The effect of 2-5A on the subunit structure of 2-5A-dependent RNase was further analyzed using the cross-linking reagent, DMS (Davies and Stark, 1970). DMS is a diimidoester that reacts with amino groups in proteins. Detection of the RNase was by probing with the monoclonal antibody rather than with silver staining because it was observed that DMS interferred with the latter method (data not shown). Addition of DMS to the RNase in the absence of activator did not result in a change in the subunit composition as determined by centrifugation in glycerol gradients and denaturing gel electrophoresis (Fig. 3, upper panel). However, in the presence of 2-5A, nearly all of the RNase was chemically cross-linked into homodimers. The RNase migrated in both the glycerol gradients and in denaturing gel electrophoresis as a dimer. These results further confirmed that 2-5A binding resulted in the conversion of the monomeric RNase to a dimer.


Figure 3: DMS covalently cross-links homodimers of 2-5A-dependent RNase. The cross-linking agent, DMS, was incubated with 2-5A-dependent RNase in the absence (upper panel) or presence (lower panel) of 5 µM pA(2`p5`A)(3) (see ``Materials and Methods''). Centrifugation of the 2-5A-dependent RNase was in 20-40% glycerol gradients followed by gel electrophoresis, transfer, and incubation with monoclonal antibody. Labeling of the figure was as described in the legend to Fig. 2.



Dimerization of 2-5A-dependent RNase Requires Functional 2-5A

To compare the ability of different oligoadenylates to induce dimers of the RNase, additional DMS cross-linking assays were performed. The monomeric and dimeric states of the RNase (at 0.25 µM) was determined by denaturing gel electrophoresis, transfer to nitrocellulose, and detection with the monoclonal antibody against the RNase (see ``Materials and Methods''). In the presence of only 0.06 µM of pA(2`p5A)(3), about half of the RNase was converted to dimer (Fig. 4A, lane 3). Conversion of almost all of the RNase to the dimeric form was obtained with 0.25 µM of pA(2`p5A)(3) (Fig. 4A, lane 5). Substantially higher amounts of A(2`p5A)(2) and A(2`p5A)(3), 12.5 µM and 1.25 µM, respectively, were required to obtain similar levels of the dimeric form of the RNase (Fig. 4A, lanes 8 and 10, respectively). On the other hand, the 3`,5`-linked oligoadenylate, pppA(3`p5`A)(2), and the 2`,5`-linked oligoadenylate, pppA2`p5`A, lacked the ability to dimerize the RNase monomers (Fig. 4B, lanes 1-6). In contrast, the authentic 2-5A molecules, pppA(2`p5`A)(2) and pppA(2`p5`A)(3), caused efficient dimerization at concentrations of 0.25 µM (Fig. 4B, lanes 9 and 12).


Figure 4: Dimerization of 2-5A-dependent RNase occurs only in the presence of functional activator. Covalent cross-linking of 2-5A-dependent RNase with DMS was performed after incubation in the presence of different amounts of various oligoadenylates (as indicated). A, Lane 1 was without DMS. After SDS-polyacrylamide gel electrophoresis, the proteins were transferred to nitrocellulose and probed with monoclonal antibody against 2-5A-dependent RNase. The positions of the monomer and dimer of 2-5A-dependent RNase are shown.



The relative amounts of 2-5A and RNase required to obtain dimers are apparent from these assays. Nearly complete conversion of the monomeric form of the enzyme to the dimeric form was observed when there was a 1:1 molar ratio of RNase to pA(2`p5`A)(3), i.e. 250 nM of each (lane 5). However, dimerization was also very efficient at a 2:1 ratio of [RNase] to [pA(2`p5`A)(3)] (Fig. 4A, lane 4). Similarly, efficient dimerization was obtained with 1:1 ratios of [p(3)A(2`p5`A)(2)] or [p(3)A(2`p5`A)(3)] to [RNase] (Fig. 4B, lanes 9 and 12, respectively).

For comparison, we measured the abilities of the same oligoadenylates to activate the catalytic activity of 2-5A-dependent RNase against poly(rU)-[P]pCp (Dong et al., 1994) (Table 1). Under the conditions of the assay, which included 24 nM 2-5A-dependent RNase, a 50% decrease of intact poly(U) was obtained with about 0.3 nM pA(2`p5`A)(3), p3A(2`p5`A)(2), or p3A(2`p5`A)(3). In contrast, 4 nM A(2`p5`A)(3) and 110 nM A(2`p5`A)(2) were necessary to obtain the same level of RNase activity (Table 1). However, pppA(3`p5`A)(2) and pppA2`p5`A were completely lacking in the ability to activate the RNase. Therefore, there was a relationship between the abilities of various 2`,5`-linked oligoadenylates to activate and induce homodimers of the 2-5A-dependent RNase.




DISCUSSION

We demonstrate here by three independent methods that binding of 2-5A to 2-5A-dependent RNase induces dimerization of the enzyme. The results are consistent with a model in which only 2-5A-dependent RNase molecules which are bound to 2-5A can form such dimers (Fig. 4). These findings may explain results from a number of different laboratories that the 2-5A-dependent RNase exists as either a 85- or 170-kDa protein (Revel et al., 1979; Wreschner et al., 1982; Floyd-Smith and Lengyel, 1986; Nilsen et al., 1981; Dong et al., 1994; Slattery et al., 1979; Bisbal et al., 1989; Bayard et al., 1994). In at least some instances, the larger species of the enzyme may have resulted from dimerization caused by endogenous 2-5A. In other situations, however, 2-5A-dependent RNase may interact with other proteins. In Slattery et al.(1979) addition of 2-5A did not alter the migration of the 185-kDa peak of 2-5A-dependent RNase activity during gel filtration. Therefore, in that study perhaps the enzyme was already in its homodimeric form. The close correlation between the ability of different oligoadenylates to activate and dimerize the enzyme suggests that the association of 2-5A-dependent RNase molecules into homodimers is an essential feature of the activation process per se. For instance, pppA(3`p5`A)(2) and pppA2`p5`A fail to either activate or dimerize the RNase. All of the 2`,5`-oligoadenylates which activate the RNase also induce dimers to form. In general, the more efficient the ability of the 2`,5`-oligoadenylate to activate the catalytic function of the enzyme, the greater the ability to induce dimers. How might the binding of 2-5A to the enzyme lead to dimerization? Perhaps, in the absence of 2-5A, the nine ankyrin-like repeats interact with the catalytic domain to block ribonuclease activity. The ankyrin domains could also prevent dimerization by blocking access to the interaction sites. Binding of 2-5A to the P-loop motifs present in the seventh and eighth ankyrin repeats (Hassel et al., 1993) could release the ankyrin clamp, thus allowing the RNase to dimerize into its active form. It is possible that the catalytic function of the enzyme requires both subunits of the dimeric enzyme. In this regard, we previously reported that a carboxyl-terminal truncation in the RNase produced a potent dominant negative inhibitor of the wild type RNase (Hassel et al., 1993). Perhaps the inhibition could have resulted, at least in part, from the formation of inactive heterodimers of the enzyme. In this model, the principal function of 2-5A could be to alter the structure of the monomeric protein to allow the active, dimeric form of the enzyme to form.


FOOTNOTES

*
This investigation was supported by United States Public Health Service Grant 5R01 CA44059, awarded by the Department of Health and Human Services, National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Dept. of Cancer Biology, NN1-06, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-9650; Fax: 216-445-6269.

(^1)
The abbreviation used is: DMS, dimethyl suberimidate.


ACKNOWLEDGEMENTS

We thank George R. Stark, Bryan R. G. Williams, and Aimin Zhou (Cleveland) for discussions and Paul F. Torrence (Bethesda) for gifts of oligoadenylates.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.