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Novel autoantibodies directed against the common tertiary configuration of transfer RNA in a patient with interstitial lung disease.

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ARTHRITIS Rr RIIEUMArISM
Vol. 39, No. 8, August 1996, pp 1308-1312
0 1996, American Collegc of Kheurnatology
1308
NOVEL AUTOANTIBODIES DIRECTED AGAINST
THE COMMON TERTIARY CONFIGURATION OF
TRANSFER RNA IN A PATIENT WITH
INTERSTITIAL LUNG DISEASE
MAMI MATSUMURA, YASUO OHOSONE, KIYOMITSU MIYACHI, MASASHI AKIZUKI,
YASUO MATSUOKA, SHOICHIRO IRIMAJIRI, MIKIO SHIMIZU, and TSUNEYO MIMORI
Objective. To identify and characterize a novel
autoantibody, anti-WS, that binds total transfer RNA
(tRNA).
Methods. Serum from patient WS, who had polyarthritis, Sjogren’s syndrome, Raynaud’s phenomenon,
and interstitial pulmonary fibrosis, was used in this
study. Characteristics of anti-WS and antibody-reactive
determinants of tRNA were investigated by 32Pimmunoprecipitation using HeLa cell RNA and deletion mutants of tRNA transcribed in vitro.
Results. WS serum produced nucleolar and cytoplasmic staining on indirect immunofluorescence. 32P
immunoprecipitation assays demonstrated that this serum immunoprecipitated total tRNAs and 5.8s and 5s
ribosomal RNAs from 32P-labeled HeLa cell extract.
When deproteinized RNA was used as antigen source,
total tRNAs were still precipitated by WS serum. An
immunoprecipitation study, using various deletion mutants of Escherichiu coli tRNA, demonstrated that both
D and T I/J C loops were needed for antibody binding.
Substitution of nucleotide lSC with 18A of E coli
tRNAIrW, which is essential in the formation of the
tertiary “L” shape of tRNA, inhibited binding by
anti-WS antibodies.
Supported in part by grants from the Japanese iMinistry of
Education and by a Keio IJnivcrsity grant for the promotion of young
investigators.
Mami Matsurnura, MD, Yasuo Ohosonc, MD, Masashi A k zuki, MD, Tsuneyo Mimori, MI>: Keio University School of Mcdicinc,
Tokyo, Japan; Kiyomitsu Miyachi, MD: Autoimmune Discasc Ccntcr,
Health Scicnccs Research, Yokohama, Japan; YAWOMatsuoka, MD,
Shoichiro Irimajiri, MD: Kawasaki Municipal I lospital, Kawasaki,
Japan; Mikio Shimizu, PhD: Institute of Space and Astronautical
Scicncc, Sagarnihara, Japan.
Address reprint requests to Yasuo Ohosonc, MD, 35Shinanomachi, Shinjuku-ku, Tokyo, Japan.
Submitted for publication October 16, 1995; accepted in
revised form March 20, 1996.
Conclusion. Anti-WS antibodies are novel autoantibodies directed against tRNAs. The antibody binding site is the common L-shaped tertiary structure
conformed by the D loop and T J!,I C loop of tRNA,
suggesting that the antibodies are induced by a conserved sequence among all species. Furthermore, these
antibodies could be a marker for a newly recognized
subset of connective tissue disease.
Autoantibody production is one of the most
characteristic features in patients with connective tissue
diseases. Recent studies have demonstrated that RNAprotein complexes arc the major targets for such immune responses. Identification and characterization of
these autoantibody-antigen systems has provided clues
to characterize and diagnose patients with various connective tissue diseases and to investigate the biologic
function of autoantigens as well as mechanisms of
autoantibody production (1). On the othcr hand, some
patients produce autoantibodies directed against
“naked” RNAs. These include antibodies against U1
RNA (2), alaninc transfer RNA (tRNAA’”)( 3 ) , initiator
methionine tRNA (tRNAMe‘) (4), and ribosomal RNA
(rRNA) ( 5 ) . However, RNA autoantigens have not been
investigatcd as extensively as protein autoantigens.
In the present study, we identified novel autoantibodies directed against all species of tRNAs. Immunoprecipitation experiments using various deletion mutants of tRNAs indicated that these antibodies recognizc
the common L-shapcd structure of tRNAs, which is
highly conserved among species.
PATIENT AND METHODS
Patient. The patient, WS, was a 61-ycar-old Japanese
woman who had presented at the age of 51 with polyarthritis,
1309
AUTOANTIBODIES DIRECTED AGAINST tRNA
Figure 1. Indirect immunofluorcscencc staining of WS serum (150
dilution) with Hep-2 cells. Nucleolar and cytoplasmic staining is
demonstrated. Antibody was detected at dilutions of <1:1,280.
used in an amount that yielded a level of radioactivity comparable with that obtained using whole cell extract.
Escherichia coli tRNA. Wild-type and various mutants
of B coli tRNA were constructed as described previously (6).
Template complementary DNAs (cDNAs) carrying the ‘IT
promoter and each tRNA gene were ligated into pUC19
vectors and transformed into E coli strain JM109. The template DNA sequences were confirmed by dideoxy sequencing.
These cDNAs were amplified by polymerase chain reaction.
Transcription of tRNA genes was performed in a reaction
mixture containing 40 mM Tris HC1, p H 8.1, 5 mM dithiothreitol, 2 mM spermidine, 10 mM MgCI,, 50 pghl bovine serum
albumin, nucleotide triphosphates (2 mM each), 20 mM
5‘-GMP, 0.2 mg/ml Bst NJ-digested template DNA, 2 units of
inorganic pyrophosphatase, and T7 RNA polymerase. The
amino acid acceptance of these synthesized tRNAs was confirmed by aminoacylation (6).
Native
Deproteinized
I
recurrent fever, Raynaud’s phenomenon, biopsy-proven Sjiigrcn’s syndrome, and slowly progressive bilateral interstitial
pulmonary fibrosis. During the 10 years of followup, no
sclerodactyly, digital pitting scars, or muscle disease was observed.
Indirect immunofluorescence. HEp-2 cells were fixed
in 3% paraformaldehyde on slides (Medical & Biological
Laboratorics, Nagoya, Japan) and incubated with diluted WS
serum for 30 minutes at room temperature. They were then
washed with phosphate buffered saline and incubated with
fluorescein isothiocyanate-labeled rabbit anti-human IgG.
Immunoprecipitation of RNA. Approximately 2 X lo7
HeLa cells were labeled with 32P-orthophosphate (370 kBq/ml;
ICN Biomedicals, Amsterdam, The Netherlands), at a cell
density of 2 X lo5 cells/ml in phosphate-free minimal essential
medium for 12 hours. Harvested cells were washed, resuspended in 1 ml of NET-2 buffer (50 mM Tris HCI, 150 mM
NaCI, 0.05% Nonidet P40, pH 7.4), and sonieated. After
centrifugation, the cleared cell supernatant was used as a
source of antigen. Two milligrams of protein A-Sepharose
CL4B (Pharmacia Biotech, Uppsala, Sweden) suspended in
500 pl of IPP buffer (10 mM Tris HCI, 500 mM NaC1, 0.1%
Nonidet P40, p H 8.0) was incubated with 10 p l of patient
serum for 1 hour at 4°C. After washing the IgG-coated beads,
cell supernatant (100 pl) was added and incubation was
continued for 2 hours at 4°C. The protein A-Sepharoseimmune complex precipitates were extcnsively washed 5 times
each with 500 pl of NET-2, and resuspended in 300 pl of
NET-2. KNAs in immunoprecipitates were extracted with 300
p1 of phenol/chloroform/isoamyl alcohol (50:50:1) containing
0.1 % hydroxyquinoline, precipitated with ethanol, resolved in
a 10% polyacrylamide gel containing 7M urea, and detected by
autoradiography.
To prepare deproteinized RNA, RNA was extracted
from labeled HeLa cell supernatants with phcnol/chloroform/
isoamyl alcohol, precipitated with ethanol, and dissolved in
NET-2, and radioactivity from incorporated 72Pwas measured.
In the immunoprecipitation study, deproteiniLed RNA was
1 2 3 4 5 6 7 8 9 1 0
Figure 2. “P immunoprccipitation assays. HcIa cells were labeled
with 3zP, and deproteinized KNA components were extracted with
phenol. ”P-labeled IIeLa cell extracts were used in lanes 1-5, and
3ZP-labelcdand dcprotcinizcd HcIa ccll extracts in lanes 6-10. Lanes
1 and 6, WS serum; lanes 2 and 7, antiribosomal P; lanes 3 and 8,
anti-U1 RNWanti-SS-A; lanes 4 and 9, anti-U1 RNP/anti-Sm; lanes 5
and 10. normal human serum. tKNA = transfer RNA.
MATSUMURA ET AL
1310
Labeled tRNA
Figure 3. "P iiiimunoprecipitatioii assay, using mutant transfer RNAs (tRKAs). The 3' end of each tRKA was
labeled with 32P-pCp. The upper row demonstrates labeled and imrnunoprecipitatcd tKNAs with anti-WS. The
lower row demonstratcs labeled tRNAs. Faint bands with intensities not differing from those in experiments in
which normal human sera were used (not shown) were attributed to nonspecific binding.
Labeling of tRNA. The 3' ends of wild-type and mutant
E coli tRNAs were labeled with 32P-pCp (Amersham, Buckinghamshire, England) using T4 R N A ligase. Labeled tKNAs
wcrc incubated with protein A-Scpharose-bound anti-WS as
described above. Precipitated tRNAs were resolved by electrophoresis and detected by autoradiography.
KESULTS
In indirect immunofluorescence studies, WS serum demonstrated nucleolar and cytoplasmic staining
(Figure 1).
In immunoprecipitation experiments using labeled HeLa cell cxtracts as the antigen sourcc, WS
serum prccipitatcd all species of tKNA, in addition to
5.8s and S S rRNAs (Figurc 2, lane 1). Whcn dcprotcinizcd KNAs wcrc uscd as antigcn for immunoprecipitation, WS serum still immunoprecipitated total tRNAs
and 5.8s rRNA, but not 5s rKNA (Figure 2, lane 6).
This suggests that WS serum contains antibodies rcactive with all naked tRNAs as well as with 5.8s rRNA. To
identify the antibody binding site, tRNAs transcribed in
vitro from cloncd E coli tRNA gcncs and their deletion
mutants were prepared. All intact tKNAs for V (Val), D
(Asp), W (Trp), and S (Ser) werc cqually immunoprc-
cipitatcd (Figure 3 ) . Because thcsc lKNAs wcrc synthcsized using in vitro transcription, the results indicate that
antibodies in WS serum can rccognize unmodified nucleotides in native tRNAs. In S1 or S2, 1 (Sl) or 2 (S2)
nuclcotides in thc variable loop of tRNA"' were delctcd. In Vc, Dw, WG, Vw, and Sr, anticodons corrcsponding to V, D, W, V, and S, wcrc substituted for
thosc of C (Cys), W, G (Gly), W, and T (Thr), respectively, but rcmaining frames were left intact. All of these
mutant tRNAs wcrc precipitated by WS serum. These
results dcmonstratc that anti-WS antibodies recognize
the sharcd structurc among all tRNAs, but not the
anticodon or variable loops.
Minihelices arc mutant tRNAs which are reconstructed with the aminoacyl stcm and thc T 1+5C loop of
either tRNA"'p (mW) or tRNAA1"(mA). Thc minihclices pW, pV, and pD consist only of the aminoacyl stcm
of tRNATrp, tKNAV'", and tRNAAsp7respectively.
"Loop(-)" are mutant tRNAs which lack either the
anticodon loop of tRNAHlS(HA-), the T $ C loop of
tKNAV"' (V., c-), or the D loop of tRNA""' (VI,-).
Of these mutant tRNAs, only HA- was prccipitated
with anti-WS antibodies, suggesting that both the D and
AUTOANTIBODIES DIRECTED AGAINST tRNA
the T $ C loops were required for antibody binding.
Because it is known that basepairing between 18GL9Gin
the D loop and 5 5 ~ 6 in
C the T ~!,t C loop is most
important in forming thc tcrtiary L shape of tRNATrp,
'U, I4A, and "G of tRNA"'p were replaced with 'C, I4G,
and 18A to break the tertiary structure. This mutant
tRNA was not immunoprecipitated by anti-WS. These
results strongly suggest that anti-WS recognizes the
tcrtiary conformation of L-shapcd tRNA which is constructed with both the D and the T @ C loops.
DISCUSSION
Scvcral autoantibodies that immunoprecipitate
tRNAs have been previously identified. Howcver, most
of them target aminoacyl-tRNA synthetases and are
specifically found in patients with inflammatory myopathy associated with interstitial pulmonary fibrosis and
arthritis (7). In contrast to these antisynthetase antibodies, anti-WS prccipitated a broad spectrum of naked
tRNAs. In immunoblotting studies, WS serum bound
proteins with molecular weights of 65 kd, 45 kd, and 34
kd. However, when bound antibodies were eluted from
these polypeptides under acidic conditions (0.1M
glycine HCI, 0.5M NaCl, pH 3.0) and subsequently used
for RNA immunoprecipitation, these antibodies did not
immunoprecipitate any species of tRNA (results not
shown). These findings suggest that WS serum contains
a second population of antibodies directed against proteins which are unlikely to be associated with tRNAs.
However, these results do not exclude the possibility that
WS serum contains other autoantibodies to tRNAassociated proteins, and further studies to identify these
proteins and their association with tRNA are needed.
Targoff et a1 described an antibody (anti-Fcr)
which immunoprecipitates heterogeneous tRNAs (8).
The Fer antigen was identified as elongation factor l a , a
48-kd translation factor that binds to aminoacyl-tRNhs.
From 35S-methionine-labeled HeLd cell extracts, WS
serum immunoprecipitated 3 proteins (65 kd, 34 kd, and
14 kd), but not a 48-kd protein (results not shown).
Several autoantibodics which recognize RNA itself
have been described previously. These include antibodies against U1 RNA (2), tKNAA1"(3), tRNAM"
(4), and rKNA (5). Antiribosome antibodies which
precipitate 5.8s and 5s rRNAs have been found in
patients with systemic lupus crythcniatosus and rheumatoid arthritis (9).
In this study, WS serum precipitated 5.8s rKNA
and tRNAs, but not SS rRNA, from deprotcinizcd
RNAs. Moreover, in immunoblotting experiments, WS
1311
serum did not react with 3 acidic ribosomal phosphoproteins, Po (38 kd), P, (19 kd), and P2 (17 kd), with the
ribosomal small subunit protein, S10 (20 kd) (lo), or
with the ribosomal large subunit protein, L12 (20 kd)
( l l ) , which are the main antigens of antiribosome
antibodies (results not shown). These findings suggest
that WS serum contains at least 2 populations of autoantibodies against RNAs: 1 that recognizes tRNAs and 1
that recognizes 5.8s rRNAs. However, the possibility
that WS scrum might contain antibodies to the L5
(approximate rnolccular wcight 35 kd)/SS RNA complex
(12), or that a cross-reaction bctwecn rRNA and tRNA
occurs, is not excluded by thc rcsults of this study.
Anti-PL-12 antibodies contain 2 populations of antibodies: 1 recognizes alanyl-tRNA synthetase and the other is
directed against the anticodon region of tRNAAla. Although thc prcscnt study did not determine whether WS
serum contained antisynthetase antibodies, the molccular weights of the 3 polypcptidcs prccipitated by WS
serum wcrc diffcrent from those of polypeptides precipitated by previously reported antisynthetase antibodies.
Transfer RNAs have quite similar structures
among species (13). Most tRNAs consist of approximately 76 nucleotide residues and form the typical
"cloverleaf" secondary structure. They consist of anticodon, D, T @ c, and variable loops and the acceptor
stem. As a result of basepairing of 'G1'G in the D loop
with 5 5 ~ 6 inC the T $ C loop, the L shape is formed as
a tertiary structure by 2 helices: 1 helix consists of the
acceptor stem and T $ C loop, and the other consists of
the D loop and anticodon loop. In this study, substitution or deletion within the anticodon loop and variable
loop did not affect antibody binding, whereas the prescnce of both D and T rlr C loops was required for
antibody binding. Moreover, when "G, which is essential to maintaining the L shape of tRNA, was replaced by
"A, such tRNA was not recognized by WS serum. These
results strongly suggcst that anti-WS antibodies are
directed against the common L-shaped tertiary structure
of tRNAs.
Although most of the autoreactive epitopes on
RNAs are highly sequence specific, several conformational epitopes have been demonstrated (14). The
epitope recognized by anti-WS appears to be one of such
conformational epitopes on RNAs. Since the structure
of tRNA is highly conserved, and anti-WS antibodies
recognize both mammalian and bacterial tRNAs, this
fact raises the possibility that the target molecule of
anti-tRNA autoantibodies is of bacterial origin.
It should be noted that patient WS had polyarthralgia, pulmonary fibrosis, and Raynaud's phenomc-
1312
MATSUMURA ET AL
non, all of which have been known to be associated with
autoantibodies to aminoacyl-tRNA synthetases (7), although she did not have myositis. Friedman et a1 described 10 patients with antisynthetases and interstitial
lung disease in the absence of myositis (15). Recently,
we identified another patient who had anti-WS antibodies, Raynaud's phenomenon, and interstitial pulmonary
fibrosis, but not myositis (Ohosonc Y ct al: manuscript
in preparation). It is possiblc that anti-WS antibodies
may be associated with a distinctive clinical subset of
connective tissue diseases. Although the immunologic
significance of autoantibodies to tRNA in tcrms of the
pathogcncsis of such clinical features is not yet understood, routine examination for thcse antibodies and
gathering of more defined clinical data on patients with
such autoantibodies will be important.
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