©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutagenesis of apobec-1, the Catalytic Subunit of the Mammalian Apolipoprotein B mRNA Editing Enzyme, Reveals Distinct Domains That Mediate Cytosine Nucleoside Deaminase, RNA Binding, and RNA Editing Activity (*)

Andrew J. MacGinnitie , Shrikant Anant , Nicholas O. Davidson (§)

From the (1)Department of Medicine, University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Apolipoprotein (apo) B48 is synthesized by mammalian small intestine as a result of post-transcriptional RNA editing. This process is mediated by an enzyme complex containing a catalytic subunit, apobec-1, which is homologous to other cytidine deaminases, particularly in a domain (H/C)-(A/V)-E-(X)-P-C-(X)-C which coordinates zinc. apobec-1, expressed as a glutathione S-transferase fusion protein, demonstrates both apoB RNA editing and cytidine deaminase activity. His, Cys, and Cys, the putative zinc-coordinating residues, were mutated to Arg, Ser, and Ser, respectively, with loss of RNA editing activity and either great reduction or abolition of cytidine deaminase activity. Mutation of the catalytically active Glu residue to Gln and Pro to Leu abolished both cytidine deaminase and RNA editing activity. The conservative His Cys mutation, which should coordinate zinc, retained both editing and cytidine deaminase activity. Thus, zinc binding is required for both apoB RNA editing and cytidine deaminase activity. Mutation of the first four leucines within the heptad repeat of the leucine-rich region (LRR) of apobec-1 resulted in reduced RNA editing but preservation of wild-type cytidine deaminase activity. GST/APOBEC-1 was also demonstrated to cross-link to apoB RNA. Mutation of His Arg abolished RNA binding, while the Glu Gln and Cys Ser mutant proteins showed wild-type levels of RNA binding. The remaining mutants had reduced levels of activity. Overexpression of wild-type apobec-1 in McA 7777 cells resulted in a 5-6-fold increase in editing of endogenous apoB. Transfection of the His Cys, LRR, and Cys Ser mutants increased endogenous editing 2-3-fold, while Glu Gln and His Arg mutants acted as dominant negatives, reducing endogenous editing. These data suggest that apobec-1 has distinct functional domains which modulate activity in the context of the apoB mRNA editing enzyme.


INTRODUCTION

Tissue-specific production of mammalian apolipoprotein (apo)()B is regulated by a site-specific, post-transcriptional modification referred to as apoB mRNA editing(1) . This reaction involves the deamination of a cytidine residue in a CAA codon to produce uridine, generating an in-frame UAA stop codon and the production of a truncated apoB species, apoB48(2, 3) . ApoB mRNA editing occurs in the mammalian small intestine and in the liver of certain species, such as the rat and mouse, but is notably absent from the human liver which contains only unedited apoB mRNA and secretes only apoB100(1) . ApoB100 is secreted from the liver in association with very low density lipoproteins and, following a series of catabolic events, becomes the major protein component of low density lipoproteins, the principal transport vehicle for cholesterol in humans. ApoB48, which circulates in association with chylomicrons and their remnants, lacks the domains which mediate interaction with the LDL receptor(1) . As a result, lipoprotein particles containing apoB48 are directed to a different receptor pathway and undergo catabolic clearance much faster than particles containing apoB100(1) .

It is now apparent that apoB mRNA editing is mediated by an enzyme complex which includes a catalytic subunit and additional, yet to be identified, complementation factors(4, 5) . Each of these components has a distinct pattern of cellular expression. The catalytic subunit, an 27-kDa protein referred to as apobec-1, was originally identified by functional complementation from a rat small intestinal cDNA library (4). More recently, homologous gene products have been isolated from human and rabbit small intestine(6, 7, 8) . The distribution of apobec-1 is widespread in the rat but appears largely confined to the small intestinal enterocyte in humans and rabbits(4, 6, 7, 8, 9) . The tissue distribution of the complementation factors is presumed to be less restricted as evidenced by the ability of tissue extracts prepared from diverse sources to support in vitro apoB RNA editing in the presence of apobec-1. These sources include chicken enterocyte and human liver S100 extracts, which are derived from cells in which endogenous apoB mRNA is exclusively unedited(5) . The mechanism by which these complementation factors establish or maintain the operational integrity of the apoB mRNA editing enzyme is unknown, although their requisite involvement in both in vitro and in vivo apoB RNA editing has been documented by several groups(4, 5, 10, 11) . Among the possible mechanisms considered, however, is a specific function for these complementation factors in mediating apoB RNA binding. Indeed, several reports have emphasized the potential importance of proteins, present in nuclear and S100 extracts, which have been identified through UV cross-linking studies as being capable of binding to a mammalian apoB RNA template(12, 13, 14) . Among these are proteins of 60 kDa and 44 kDa, present in both rat intestinal and hepatic S100 extracts, and which appear to bind with 4-nucleotide specificity to a region at the 5` end of the mooring sequence, UGAUCAGUAUA(12, 14) . Other reports, however, have challenged these conclusions with the demonstration that binding of the 60-kDa protein occurs with a luciferase RNA template and also to an antisense apoB RNA(15) . As a result, neither the identity of these proteins nor proof that they comprise an indispensable component of the apoB mRNA editing enzyme has yet been established.

apobec-1 is a site-specific cytidine deaminase with homology to other cytidine and deoxycytidine deaminases which have been identified in species ranging from Escherichia coli to human(16) . The crystal structure of cytidine deaminase from E. coli predicts that deamination of the target substrate involves a single zinc ion, coordinated by one histidine and two cysteine residues, which then binds and activates a water molecule(17) . This tetrahedral complex then attacks C-4 of the pyrimidine ring, liberating the amino leaving group(17) . A conserved carboxylate amino acid (glutamate) is required for protonating both the leaving amino group and the ring nitrogen. These four residues are conserved between the E. coli cytidine deaminase and all homologs of apobec-1(4, 6, 7, 8, 17) . In addition, a proline occurring immediately before the first cysteine is fully conserved and is predicted to ensure that an -helix secondary structure element begins at the first zinc-coordinating cysteine(17) . Two recent studies have examined the effects of mutations of some (10) or all (7) of the zinc-coordinating residues in apobec-1, specifically in relation to in vitro apoB RNA editing. The more comprehensive analysis revealed that a His to Cys mutation retained 30% and a Pro to Ala mutation retained 18% of the wild-type editing activity, but the remaining mutations demonstrated greatly reduced editing activity(7) . The mechanism of the reduction in apoB RNA editing activity has been suggested to involve disruption of catalytic activity secondary to alterations in zinc chelation, in a manner analogous to that observed following incubation with 1,10-o-phenanthroline(11) . This inference remains untested, however, since cytidine deaminase activity was not determined in either of these recent studies(7, 10) . Additionally, other possibilities have been proposed to account for the alterations in apoB RNA editing activity associated with these mutations, such as disruption of substrate binding(7) . In this regard, studies presented in an accompanying manuscript (31) suggest that apobec-1 may function as an apoB RNA-binding protein.

In this study, we demonstrate that apobec-1 expressed in E. coli as a glutathione S-transferase (GST) fusion protein is competent to mediate in vitro apoB RNA editing when mixed with chicken intestinal extracts. In addition, purified GST/APOBEC-1 has cytidine deaminase activity and can be cross-linked to an apoB RNA fragment spanning the edited base. We have undertaken a systematic analysis of the structural determinants of apobec-1 which mediate apoB RNA editing, cytidine deaminase, and apoB RNA binding activity through construction of a series of mutant fusion proteins. These mutations involve both the putative zinc binding motif and the leucine-rich region in the carboxyl terminus of apobec-1(4) . Finally, we have created several stable cell lines of rat hepatoma cells (McA 7777) transfected with either wild-type or mutant apobec-1 expression plasmids and have characterized their effects upon endogenous apoB mRNA editing.


MATERIALS AND METHODS

Construction and Expression of pGEX/apobec-1

Full-length apobec-1 cDNA was generated by polymerase chain reaction (PCR) to include BamHI and SalI sites at the 5` and 3` ends, respectively, cloned in-frame into pGEX-4T3 and propagated in JM109 cells(18) . The entire coding sequence of apobec-1 was sequenced on both strands to eliminate the possibility of Taq-introduced mutations. Expression of fusion protein was conducted in lon RB791 cells (gift from K. Subramanian, Univ. of Illinois). A 50-ml culture was inoculated with the transformed RB791 cells and grown overnight. The culture was diluted to 500 ml in a 2-liter flask and incubated for 1 h. Isopropyl-1-thio--D-galactopyranoside (Sigma) was added to a final concentration of 0.1 mM. Cells were incubated for an additional 4 h with shaking, resuspended in phosphate-buffered saline, and disrupted by sonication, and Triton X-100 was added to 1% final concentration. The solution was mixed for 30 min at 4 °C, and the insoluble fraction was pelleted by centrifuging for 5 min at 10,000 g. Glutathione-agarose beads (Sigma) were added to the supernatant, mixed for 10 min at 4 °C, and washed three times with phosphate-buffered saline/1% Triton, and the fusion protein eluted in glutathione elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione). Protein concentrations were determined and purity was confirmed by SDS-PAGE analysis and Western blotting using both antipeptide antisera (9) and an antibody generated against the purified fusion protein. Following dialysis into the reaction buffer, in vitro conversion and primer extension analysis of apoB cRNA were performed as described(5) .

Site-directed Mutagenesis

Mutations were designed to minimize disruptions in local secondary structure, as predicted by the MacVector program (Kodak-IBI). All mutants were constructed by a two-step PCR method(19) . Those containing the correct mutation were subcloned into pGEX/apobec-1 using the SmaI and KpnI sites at nucleotides 132 and 540, respectively, except for the LRR mutation which was subcloned using the KpnI and SalI (external) sites. The entire subclone was then sequenced on both strands.

Cytidine Deaminase Activity

Attempts at determination of cytidine deaminase activity using a previously described spectrophotometric assay (11) did not show consistent activity (data not shown). Accordingly, cytidine deaminase activity was determined using a thin layer chromatographic (TLC) assay, optimized for use with recombinant GST/APOBEC-1(20) . Briefly, the indicated amounts of purified GST/APOBEC-1 were incubated with 3.3 µCi of [H]deoxycytidine (24.8 Ci/mmol, DuPont NEN) and 250 µM cytidine in a total volume of 10 µl in a buffer containing 45 mM Tris, pH 7.5. After timed incubations (generally 2-4 h unless otherwise stated), the reaction was quenched by the addition of 2 µl of 10 µg/µl each deoxycytidine and deoxyuridine. Any insoluble material was removed by centrifugation for 2 min at full speed in a microcentrifuge, and 4 µl of the reaction mixture was applied to a polyethyleneimine-cellulose TLC plate (VWR). The plates were developed in 7:2 (v:v) isopropyl alcohol/10% HCl for up to 16 h. The corresponding deoxycytidine and deoxyuridine bands were visualized by exposure to (254 nm) UV light and scraped into Ultima Gold scintillation fluid (Packard) for quantitation by liquid scintillation spectroscopy (Packard LS 1500).

UV Cross-linking to ApoB RNA

A P-labeled rat apoB cRNA template (50,000 cpm at 2.5-3.0 10 cpm/µg) was incubated with 500 ng of wild-type or mutant GST/APOBEC-1 for 20 min at room temperature and then treated sequentially with RNase T1 (1 unit/µl final concentration) and heparin (5 mg/ml final concentration) for 5 min each, again at room temperature. The mixture was UV (254 nm)-irradiated for 1.5 min in a Stratalinker (Stratagene) cross-linker (energy = 250 mJ/cm) and then analyzed by 10% SDS-PAGE under denaturing conditions(21) .

Transfections

Mutant apobec-1 cDNAs were subcloned into the eukaryotic expression vector pCMV4(22) . Rat hepatoma cells (McA 7777) were cotransfected with pCMV4-apobec-1 constructs and pCMV-Neo (23) in a ratio of 20:1 using either lipofectamine or calcium phosphate. Stable transfectants were isolated by selection with 800 µg/ml G418, and individual colonies were isolated with cloning cylinders and expanded. Overexpression of the transgene was confirmed by Northern blot analysis of total RNA which was normalized to the abundance of glyceraldehyde-3-phosphate dehydrogenase mRNA(6) . Aliquots of RNA prepared from representative colonies were DNase-treated and used to determine the extent of endogenous apoB mRNA editing by reverse transcription and polymerase chain reaction amplification, followed by primer extension, as described previously(5) .

Oligonucleotides

The oligonucleotides used in this study are listed below. The underlined nucleotides represent restriction sites which were introduced for cloning purposes while the boldface, underlined nucleotides represent the mutations. apobec-1-outside 5` primer, AJM022, 5`-GTAGGATCCATGAGTTCCGAGACAGGC-3` (BamHI, then nt 1-18); His Arg-S, 5`-CCAACAAACGCGTTGAAGT-3` (nt 173-191); His Arg-AS, 5`-GACTTCAACGCGTTTGTTGGTG-3` (nt 192-171); His Cys-S, 5`-CACCAACAAATGCGTTGAAGTCAATTTC-3` (nt 171-198); His Cys-AS, 5`-GAAATTGACTTCAACGCATTTGTTGGTG-3` (nt 198-171); Glu Gln-S, 5`-CCAACAAACACGTTCAAGTCAATTTCATAG-3` (nt 173-202); Glu Gln-AS, 5`-CTATGAAATTGACTTGAACGTGTTTGTTGG-3` (nt 202-173); Pro Leu-S, 5`-CCTGGAGTCTCTGTGGGGAGTGCTCCAGGG-3` (nt 266-295); Pro Leu-AS, 5`-CACTCCCCACAGAGACTCCAGGAC-3` (nt 287-264); Cys Ser-S, 5`-GAGTCCCTCTGGGGAGT-3` (nt 270-286); Cys Ser-AS, 5`-GGAGCACTCCCCAGAGGGACTCCAGGAC-3` (nt 291-264); Cys Ser-S, 5`-TGGGGAGTCCTCCAGGG-3` (nt 279-295); Cys Ser-AS, 5`-GTAATGGCCCTGGAGGACTCCCCACAGG-3` (nt 302-261); apobec-1-outside 3` primer, AJM023, 5`-AGTGTCGACTTTCAACCCTGTGGCCCACAG-3` (SalI, then nt 687-667); 5` LZ outside primer, CH52, 5`-AAGGTACCCCCATCTGTGGGTGA-3` (KpnI, nt 504-526); Leu Ile-AS, 5`-AATATTTAAACAGGGTGGAATTCCTAAAATGATGCAGTAGAT-TTC-3` (nt 585-541); Leu Ile-S, 5`-CCACCCTGTTTAAATATTATAAGAAGAAAACAACCTCAAATCACG-3` (nt 568-612); 3` LZ outside primer, CH54, 5`-CTTCTAGATTCCTTGTGGCAGT-3` (XbaI, nt 173-191); primer extension primer, BT7, 5`-AGTCCTGTGCATCATAATTATCTCTAATATACTGA-3`; 5` reverse transcription PCR primer, ND1, 5`-ATCTGACTGGGAGAGACAAGTAG-3` (nt 6512-6534); 3` reverse transcription PCR primer, ND3, 5`-CACGGATATGATACTGTTCGTCAAGC-3` (nt 6786-6811).


RESULTS

Expression of apobec-1 in Bacteria and Demonstration of in Vitro ApoB RNA Editing Activity

apobec-1, expressed as a fusion protein with glutathione S-transferase (GST), was purified to greater than 80% homogeneity following a single round of glutathione-agarose affinity chromatography, with an apparent size of 60 kDa, as assessed by SDS-PAGE (see below). Known amounts of purified GST/APOBEC-1 protein were incubated with 20 µg of chicken intestinal S-100 extracts and a synthetic rat apoB RNA template. In vitro editing activity was demonstrated over a range of concentrations with maximal activity between 500 ng and 1 µg of GST/APOBEC-1 (Fig. 1A). Editing efficiency decreased above 1 µg of GST/APOBEC-1, findings consistent with our previous observations concerning the critical stoichiometry between complementation factors and catalytic subunit components of the apoB mRNA editing enzyme(5) . Preliminary studies conducted with preparations of apobec-1 isolated after thrombin cleavage of the GST moiety, yielded indistinguishable activity from the holo-GST/APOBEC-1 fusion protein (data not shown). Accordingly, the GST/APOBEC-1 fusion protein was used exclusively in the studies presented below.


Figure 1: Editing activity of wild-type GST/APOBEC-1 and expression and purification of wild-type and mutant proteins. A, in vitro apoB mRNA editing assay. The indicated amount of purified GST/APOBEC-1 was incubated with 20 µg of chicken intestinal extracts and 40 fmol of a 361-nucleotide synthetic rat apoB RNA spanning the edited nucleotide. The RNA was extracted, and the extent of editing was determined by primer extension assay. The products were separated on an 8% acrylamide sequencing gel. The relative mobilities of the edited (UAA) and unedited (CAA) bands are indicated. Relative levels of editing were determined by densitometric scanning and are indicated below each lane. B, 250 ng of purified wild-type and mutant protein was separated by 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The blot was incubated with a rabbit anti-GST/APOBEC-1 polyclonal antibody followed by a horseradish peroxidase-conjugated secondary antibody, with visualization by the enhanced chemiluminescence (ECL) method (Amersham). Molecular weight standards are indicated at left.



Mutations in Putative Zinc-binding and Leucine-rich Regions of apobec-1

Since apobec-1 has significant homology to cytidine deaminases found in species from E. coli to human, particularly in the zinc-binding regions, we made a series of mutations in this region to determine the effects on apobec-1 function. The three putative zinc-binding residues His, Cys, and Cys were all mutated to residues not known to bind zinc. Cys and Cys were mutated to Ser, and His was mutated to Arg. In addition, His was mutated to Cys, which is able to bind zinc. Glu, putatively involved in both proton transfer steps of the catalytic deamination reaction, was mutated to Gln whose amine group should preclude activity in proton transfer. Finally, Pro which lies immediately amino-terminal to the first Cys residue involved in zinc binding was mutated to Leu. This residue has been inferred, from the crystal structure of E. coli cytidine deaminase, to position the first zinc-binding Cys residue as the first residue in an helix(17) . apobec-1 also contains a carboxyl-terminal leucine-rich region (LRR), which, in the rat, consists of five leucine residues in a perfect heptad repeat(4) . A mutant apobec-1 (LRR mutant) was constructed in which the first four leucines of this heptad repeat were changed to isoleucine. All seven of these mutations (His Arg, His Cys, Glu Gln, Pro Leu, Cys Ser, Cys Ser, and LRR) were constructed by a two-step PCR mutagenesis procedure (19) and expressed as GST/APOBEC-1 fusion proteins in comparable yields and purity (Fig. 1B).

Editing Activity of Mutant GST/APOBEC-1 Proteins

All mutants were assayed by in vitro conversion using either 500 ng or 1 µg of purified fusion protein complemented with 20 µg of chick intestinal S-100 extract. Of the seven mutations tested, five eliminated editing activity (Fig. 2A). All assays were performed 3 or more times, with similar results. The five mutations that did not show editing activity (His Arg, Glu Gln, Pro Leu, Cys Ser, and Cys Ser) never demonstrated greater than 1% editing, which likely represents the lowest level of detectable activity in this assay. By contrast, the His Cys and LRR mutant proteins showed barely detectable levels of editing (<1.5% UAA), using either 500 ng or 1 µg of protein. Additional assays, performed using 2.5 µg of purified protein, revealed that both the His Cys and LRR mutant proteins retained editing activity (7.0 ± 2.0% UAA, n = 4, and 5.8 ± 1.7% UAA, n = 3, respectively). Maximal levels of apoB RNA editing activity, achieved with 2.5 µg of these mutant proteins, were approximately half those found with 1 µg of the wild-type protein (Fig. 2B). None of the other mutant proteins demonstrated editing activity following incubation with up to 5 µg of recombinant protein (data not shown). Additionally, there was no detectable editing activity with the inactive mutants and no augmentation of editing activity with either the wild-type or dysfunctional mutants following either preincubation or overnight dialysis into 50 µM zinc acetate (data not shown).


Figure 2: In vitro apoB RNA editing activity of wild-type and mutant apobec-1 proteins. A, editing was assayed using 1 µg of purified fusion protein, 20 µg of chicken intestinal extract, and 40 fmol of synthetic apoB cRNA spanning the edited base. The relative mobilities of the edited (UAA) and unedited (CAA) bands are indicated. Relative levels of editing (%UAA) were determined by densitometric scanning and are indicated below each lane. B, as in A, except both 1 µg and 2.5 µg of purified protein were used. In both panels, values are the mean of 3-4 experiments.



Cytidine Deaminase Activity of GST/APOBEC-1

Conditions were optimized for the detection of cytidine deaminase activity using the recombinant GST/APOBEC-1, as shown in Fig. 3. Deaminase activity was linear between 0 and 250 ng, with smaller increases noted up to 1 µg (Fig. 3A). Deaminase activity of higher amounts of protein could not be examined since solvent migration of the TLC plates was altered with aberrant migration of the standards. Deaminase activity was linear with time up to 24 h at which time a plateau was reached and there was little further increase in deamination observed up to 72 h of incubation (Fig. 3B). Since deaminase activities of 10-15% are readily detectable by this assay, all further determinations were conducted following either 2- or 4-h incubations. GST/APOBEC-1 exhibited a broad temperature range, with maximal activity between 30 and 42 °C (Fig. 3C). Cytidine deaminase activity of the fusion protein was specifically inhibitable by tetrahydrouridine (THU) a competitive inhibitor of cytidine deaminases, with greater than 90% inhibition at 200 µM concentrations (Fig. 3D). Additionally, cytidine deaminase activity was inhibited by the addition of a 40-fold molar excess (10 mM) of either cytidine or deoxycytidine (Fig. 3D), suggesting that apobec-1 functions broadly as cytosine nucleoside deaminase as recently suggested(16) . Interestingly, although THU is a competitive inhibitor of cytidine deaminases, there was no inhibition noted of in vitro apoB RNA editing following inclusion of up to 200 µM THU (Fig. 3D, inset). Additionally, neither 10 mM cytidine nor 10 mM deoxycytidine had any inhibitory effect on apoB RNA editing (data not shown). We also examined cytidine deaminase activity of the mutant GST/APOBEC-1 proteins. The two mutant proteins that retained at least partial editing activity (His Cys and LRR) also demonstrated cytidine deaminase activity which was comparable to that of the wild-type protein (Fig. 3E). By contrast, the His Arg, Glu Gln, and Pro Leu mutations abolished cytidine deaminase activity, while the two Cys Ser mutations demonstrated deaminase activity which was only marginally above background. No alterations in the activity of cytidine deaminase could be demonstrated with any of these mutant proteins following preincubation with 50 µM zinc acetate (data not shown). Taken together, these data suggest that the functional requirements within apobec-1 for cytidine deaminase activity may be separable from those which determine apoB RNA editing.


Figure 3: Cytidine deaminase activity of wild-type and mutant GST/APOBEC-1 proteins. Cytidine deaminase activity was determined by incubation of purified fusion protein with H-labeled deoxycytidine and 250 µM cytidine unless otherwise specified. Reactions were quenched with 10 µg of deoxycytidine and deoxyuridine, and aliquots of each reaction were analyzed by thin layer chromatography. The dC and dU bands were visualized by 254 nm UV, excised, and quantitated by scintillation counting. Data are shown as the percent of radioactivity migrating in the dU band, corrected for a GST control. *, p < 0.001, using Student's two-tailed t test. A, standard curve of deamination versus amount of GST/APOBEC-1. Incubations were for 2 h. Data show mean ± S.D. of three experiments. B, time course of deamination. 500 ng of protein was incubated from 2-72 h, values representing the mean of two experiments. C, temperature optimum of cytidine deaminase activity. Incubation was with 500 ng of protein for 2 h, values representing the mean of two experiments. D, effect of inhibitors on cytidine deaminase activity. Inhibition experiments were performed with 500 ng of GST/APOBEC-1 for 2 h with the indicated concentrations of tetrahydrouridine (THU), cytidine, and deoxycytidine. Values are mean ± S.D. of 4 (THU) or 2 (cytidine and deoxycytidine) experiments. In vitro editing assays were performed with 1 µg of fusion protein, 20 µg of chicken intestinal S100 extracts, and the indicated amounts of THU. E, cytidine deaminase activity of wild-type and mutant protein. 500 ng of purified fusion protein was incubated for 4 h. Values represent the mean ± S.D. of three experiments.



RNA Binding Activity of Mutant GST/APOBEC-1 Proteins

Wild-type GST/APOBEC-1 can be demonstrated to bind to a synthetic rat apoB RNA template following UV cross-linking (Fig. 4A), whereas GST protein alone did not cross-link. Similar studies conducted with comparable amounts of the mutant GST/APOBEC-1 proteins showed a range of RNA cross-linking activity. The Glu Gln and Cys Ser mutant proteins demonstrated RNA cross-linking activity comparable to wild-type (Fig. 4B). By contrast, the His Arg mutant displayed barely detectable RNA binding activity, while the remaining mutant proteins (His Cys, Cys Ser, Pro Leu, and LRR) had diminished but demonstrable apoB RNA binding activity.


Figure 4: RNA binding activity of wild-type and mutant GST/APOBEC-1 proteins. 500 ng of protein was incubated with a P-labeled 105-nt synthetic rat apoB cRNA spanning the edited base for 20 min, followed by treatment with RNase T1 and UV cross-linking. The products were analyzed by denaturing 10% SDS-PAGE. Molecular weight markers (10) are shown to the left of each gel. A, lane 1, RNA alone; lane 2, RNA plus GST; lane 3, RNA plus GST/APOBEC-1. B, wild-type and mutant GST/APOBEC-1 proteins plus labeled RNA.



Activity of apobec-1 and Its Mutants in Rat Hepatoma Cells

Stable lines of McA 7777 cells were selected in G418 following transfection of the indicated expression vectors. Individual colonies were chosen for expansion and endogenous apoB mRNA editing examined following reverse transcription and polymerase chain reaction amplification. McA 7777 cells transfected with the empty vector yielded values for apoB mRNA editing of approximately 14% UAA, proportions which are strictly comparable to the parental clone (). Transfection with the wild-type apobec-1 in the sense orientation produced a greater than 5-fold increase in apoB mRNA editing while its expression in the antisense orientation reduced apoB mRNA editing to less than 50% of control levels (mean of 6% UAA, ). Cells transfected with the His Cys mutant demonstrated elevated levels of apoB mRNA editing (), consistent with the demonstration above that the His Cys mutant retains low levels of apoB RNA editing activity and wild-type levels of cytidine deaminase activity. Transfection with the Glu Gln which was demonstrated above to retain apoB RNA binding activity yet which is catalytically inactive, resulted in markedly decreased endogenous apoB mRNA editing, to a mean of 4% UAA (). One of two His Arg clones demonstrated a significant reduction in endogenous editing to about 2% UAA. This clone had approximately 10 times greater expression of the transgene than another His Arg clone in which endogenous editing was within the range seen with vector alone. An unanticipated finding was that transfection with the Cys Ser mutant yielded a 2-fold increase in endogenous apoB mRNA editing (). The explanation for this observation is not readily apparent, although the data presented above suggests that the Cys Ser mutation is not completely catalytically inactive, at least with regard to cytidine deamination, and retains weak apoB RNA binding activity. The LRR mutant, which is fully active as a cytidine deaminase and demonstrates reduced in vitro apoB RNA editing activity, also demonstrated an increase in endogenous apoB mRNA editing in a single clone examined (). Finally, both the Cys Ser and Pro Leu mutants produced variable but generally minor effects upon endogenous apoB mRNA editing.


DISCUSSION

The mammalian apoB mRNA editing enzyme is a multicomponent complex of which apobec-1 represents the catalytic subunit. The obligate requirement for additional complementation factors and the stringent nucleotide sequence configuration in the region flanking the edited base suggest that both target-site recognition combined with an optimal orientation of the catalytic subunit with respect to its substrate are necessary in order for apobec-1 to mediate apoB RNA editing. The present findings provide new insight into distinct functional domains of apobec-1 and suggest testable hypotheses concerning the molecular mechanism of apoB mRNA editing. The major conclusions of this study are summarized in .

apobec-1 was identified as an apoB RNA-specific cytidine deaminase and the presumed catalytic subunit of the apoB mRNA editing protein based upon several criteria. These include its homology to other known cytidine and deoxycytidine deaminases(16) , the ability of homogenates prepared from Xenopus oocytes injected with apobec-1 RNA to mediate cytidine deamination and, finally, the inhibition of in vitro apoB RNA editing following zinc chelation of S100 extracts with 1,10-o-phenanthroline(11) . Additional validation followed publication of the crystal structure of the E. coli cytidine deaminase, which conclusively demonstrated zinc coordination through a conserved motif, with a consensus: (H/C)-(A/V)-E-(X)-P-C-(X)-C (17). Further, mutational analysis of both recombinant adenosine and cytidine deaminase enzymes indicates a dramatic decrease in catalytic activity following disruption of the zinc liganding domain(24, 25, 26, 27) . The importance of the homologous region in apobec-1 has been examined using mutant proteins which were expressed in a variety of systems and assayed for their effects on apoB RNA editing. Driscoll and Zhang (10) found that extracts from COS-7 cells transfected with apobec-1 were competent to perform in vitro editing in the presence of complementation factors and that mutation of His to Arg, Pro to Leu, or Cys to Ser abolished this editing activity. Yamanaka et al.(7) carried out more extensive mutagenesis of the rabbit homolog, altering residues His, Val, Glu, Pro, Cys, and Cys each to Ala, and His to Cys. The His, Glu, and Cys mutations abolished editing, while the Cys mutation demonstrated approximately 1% editing, near the limits of detection for the in vitro conversion assay(7) . Their His Cys mutant demonstrated about one-third of the wild-type activity, while the conservative Val Ala mutant retained wild-type activity(7) . These findings are comparable to the current demonstration that GST/APOBEC-1 containing a His Cys mutation edited a rat apoB cRNA template with reduced efficiency. In contrast, the Pro Ala mutant studied by Yamanaka et al.(7) showed in vitro editing activity of about 20-25% of wild-type, findings in contrast to the present results and those of Driscoll and Zhang (10) in which in vitro RNA editing activity was abolished with the Pro Leu mutation. Among the possible explanations for this discrepancy are that an alanine residue can be better accommodated than the larger leucine residue or is less disruptive of other structural constraints.

The current studies included an examination of the effects of these mutations upon the activity of GST/APOBEC-1 as a cytosine nucleoside deaminase. GST/APOBEC-1 was not active in the spectrophotometric assay used by Navaratnam et al.(11) to demonstrate that apobec-1 expressed in Xenopus oocytes had cytidine deaminase activity. We therefore took advantage of the fact that many cytidine deaminases are bifunctional, accepting both ribonucleotides and deoxyribonucleotides, to adapt a more sensitive radiochemical assay (20). Cytidine deaminase activity of GST/APOBEC-1 was inhibitable by excess cytidine or deoxycytidine as well as by THU, a known inhibitor of cytidine deaminases, but none of these inhibitors interfered with apoB RNA editing activity. This may be because the complementation factors required for editing activity position apobec-1 so that it interacts specifically with the potentially edited nucleotide, while in the deaminase assay, any nucleotide is equally likely to encounter the active site of apobec-1. The major findings to emerge from these studies () are that mutations which disrupt the zinc-coordinating region of apobec-1 reduce or eliminate cytidine deaminase activity. However, the His Cys mutant, which manifests reduced apoB RNA editing activity, demonstrated wild-type levels of cytidine deaminase activity. These results may reflect the more stringent constraints for apoB RNA editing in which the active site must accommodate a cytidine in the context of an RNA template, while during cytidine deamination it must only accommodate the free nucleotide.

apobec-1 also contains a leucine-rich region which has been proposed to be involved in dimerization in other proteins(28) . Two studies have demonstrated that removal of the leucine-rich region results in loss of editing activity, but these findings require cautious interpretation since the entire carboxyl-terminal third of the protein was removed in these experiments(4, 7) . To begin to address the function of the leucine-rich region, a mutant protein was constructed in which the first four (of five) leucines in this heptad repeat were altered to isoleucine. The LRR mutant demonstrated reduced levels of apoB RNA editing activity in vitro, comparable to those found with the His Cys mutation. In addition, and similar to findings with the His Cys mutant, the LRR mutant also demonstrated wild-type levels of cytidine deaminase activity. This observation is consistent with the hypothesis that the leucine-rich region is involved in protein-protein interaction, either with the complementation factors and/or in homodimer formation(8) , and that disruption of this region would decrease apoB RNA editing as a result of inefficient assembly of the apoB mRNA editing enzyme. On the other hand, cytidine deaminase activity would be preserved since no other factors appear to be required for this activity, although known cytidine deaminases function as homopolymers(17) . Studies are currently underway to determine whether GST/APOBEC-1 functions as a monomeric or multimeric protein and whether the LRR mutation alters this intrinsic property.

Studies presented in an accompanying manuscript (31) have established that apobec-1 is an RNA-binding protein as evidenced by UV cross-linking to a rat apoB cRNA template as well as by electrophoretic mobility shift assay. The results now presented () suggest that certain residues within the zinc binding motif of apobec-1 (His, Cys) may be directly involved in RNA binding. On the other hand, the Cys Ser mutant, which is catalytically inactive, demonstrates wild-type levels of apoB RNA binding. No effect on RNA binding was demonstrated with the catalytically inactive Glu Gln mutant, suggesting that the role of this residue is confined to that of a general base in the deamination reaction. It should be noted, however, that disruption of zinc binding could cause global disruption of apobec-1 protein structure, decreasing RNA binding nonspecifically. In this regard, it bears emphasis that the zinc content of these mutant proteins remains to be established. These reservations make further interpretation of these experiments quite complicated since the relationship between apoB RNA binding and editing is unknown. Further analysis will be required to establish the precise functional domains which mediate apoB RNA binding, particularly since there is no canonical RNA binding motif within the predicted amino acid sequence of apobec-1.

In an attempt to establish in vivo physiologic relevance of these structural alterations in apobec-1, rat hepatoma (McA 7777) cells were stably transfected with expression plasmids encoding the various mutant apobec-1 proteins. McA 7777 cells were chosen since they demonstrate consistent, low levels of endogenous apoB mRNA editing(29, 30) . A number of findings emerged from these studies (), among them that expression of the Glu Gln mutant exerts a strong dominant negative effect, with three independent clones yielding values for endogenous apoB mRNA editing between 2 and 6% UAA, compared to an average of 14% UAA for vector-transfected clones (range 10-20%). A single His Arg clone also produced a marked reduction in RNA editing, to about 2% UAA. This clone had significant overexpression of the mutant apobec-1 RNA as compared to another His Arg clone which demonstrated no effect on endogenous editing. This suggests that the His Arg mutant acts as a dominant negative modifier, conditional upon its expression at high levels, an explanation consistent with the predicted loss of zinc coordination in this mutant. The extent of the dominant negative suppression in both the Glu Gln and His Arg compares with the results of overexpression of antisense apobec-1, in which endogenous apoB mRNA editing was reduced to a mean of 6% UAA. By contrast, overexpression of the wild-type apobec-1 led to greatly increased levels of editing, with two clones demonstrating 82 and 91% editing of endogenous apoB mRNA. These results suggest that the levels of apobec-1 protein are limiting for endogenous apoB mRNA editing by McA 7777 cells. Furthermore, the ability to increase editing activity to levels comparable to those found in the small intestine suggests that McA 7777 cells contain an abundance of complementation factors. Driscoll et al.(10) transiently transfected McA 7777 cells with apobec-1 and obtained 27% editing, roughly a 2-fold increase over control levels. These values should be compared to the 5-6-fold increase reported in the current studies. However, since Driscoll et al.(10) utilized transient transfection, only a portion of the cells would have expressed apobec-1. Transfection of the LRR, His Cys, and Cys Ser mutants all increased the levels of endogenous apoB RNA editing, but to a lesser extent than following transfection of the wild-type apobec-1. In the case of the LRR and His Cys mutations, the results confirm the in vitro data that they are competent to edit, albeit less efficiently than the wild-type. The 2-fold increase in endogenous apoB mRNA editing following transfection of the Cys Ser mutant into McA 7777 cells was unexpected and could represent different requirements for in vivo and in vitro activity. It is possible, for example, that this mutant protein is forming an active heterodimer with the wild-type protein in McArdle cells, increasing endogenous apoB RNA editing, and this hypothesis is currently under investigation. As alluded to above, Yamanaka et al. found that a Cys Ala mutation in the rabbit homolog retained extremely low, but detectable, in vitro editing activity(7) .

These results suggest the existence of distinct functional domains within apobec-1 which may interact to modulate apoB mRNA editing in vivo. Confirmation that the zinc-binding region of apobec-1 is crucial to catalytic function both in apoB RNA editing and as a cytidine deaminase suggests that future determination of the zinc content of these mutant proteins be given high priority in order to distinguish between different mechanisms of metal coordination within the active site of the enzyme. In addition, it remains to be established whether both RNA binding and cytidine deaminase activity are necessary functional requirements for apoB mRNA editing. Resolution of this question will require the development of mutants or reagents which specifically inhibit RNA binding. This and other issues will be the focus of future reports.

  
Table: In vivo activity of mutant apobec-1 proteins

Mutant and wild-type apobec-1 cDNAs as well as antisense apobec-1 cDNA were cotransfected along with pCMV-Neo into rat hepatoma (McA 7777) cells. Stable transfectants were selected in G418 and RNA isolated from selected clones, amplified by reverse transcription PCR, and endogenous apoB mRNA editing was determined by primer extension. Clones were selected for overexpression of the transgene by Northern blotting with loading normalized by hybridization to glyceraldehyde-3-phosphate dehydrogenase.


  
Table: Summary of activities associated with mutant apobec-1 proteins

Activities of wild-type and mutant GST/APOBEC-1 proteins using in vitro assays. For in vitro editing, cytidine deamination, and RNA binding, wild-type levels of activity are designated by +++, low activity by +, and complete absence of activity by -. +/- indicates activity marginally above background. Effects of the mutations on endogenous apoB mRNA editing in McA 7777 cells are represented by arrows, which indicate both the direction and magnitude of change. A horizontal arrow denotes no significant change in editing.



FOOTNOTES

*
These studies were supported by National Institutes of Health Grants HL-38180, HL-18577, and DK-42086 (to N. O. D.), Training Grants HD-07136 (to S. A.), and GM-07281 and HL-07237 (to A. J. M.). 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 correspondence and reprint requests should be addressed: MC 4076, Dept. of Medicine, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637. Tel.: 312-702-6480; Fax: 312-702-2182.

The abbreviations used are: apoB, apolipoprotein B; apobec-1, apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1; GST, glutathione S-transferase; PCR, polymerase chain reaction; THU, tetrahydrouridine; LRR, leucine-rich region; PAGE, polyacrylamide gel electrophoresis; nt, nucleotide(s).


ACKNOWLEDGEMENTS

We thank Toru Funahashi, Christos Hadjiagapiou, Susan Skarosi, Trish Glascoff, and Annalise Hausman for outstanding technical assistance and Federico Giannoni for discussions and the provision of cell extracts.


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