From the
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)
Tissue-specific production of mammalian apolipoprotein
(apo)
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
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
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.
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)
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
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
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
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
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.
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.
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.
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
(
)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) .
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.
-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.
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).
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.
-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.
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.
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.
, 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.
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) .
Table: In vivo
activity of mutant apobec-1 proteins
Table: Summary of activities associated with mutant
apobec-1 proteins
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.