Download Associated with Interstitial Lung Disease Asparaginyl

Anti-KS: Identification of Autoantibodies to
Asparaginyl-Transfer RNA Synthetase
Associated with Interstitial Lung Disease
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of June 15, 2017.
Michito Hirakata, Akira Suwa, Sonoko Nagai, Michael A.
Kron, Edward P. Trieu, Tsuneyo Mimori, Masashi Akizuki
and Ira N. Targoff
J Immunol 1999; 162:2315-2320; ;
http://www.jimmunol.org/content/162/4/2315
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Copyright © 1999 by The American Association of
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References
Anti-KS: Identification of Autoantibodies to
Asparaginyl-Transfer RNA Synthetase Associated with
Interstitial Lung Disease1
Michito Hirakata,2* Akira Suwa,* Sonoko Nagai,† Michael A. Kron,‡ Edward P. Trieu,§
Tsuneyo Mimori,* Masashi Akizuki,* and Ira N. Targoff§
A
utoantibodies directed against aminoacyl-transfer RNA
(tRNA)3 synthetases can be found in ;25–35% of patients with the chronic, inflammatory muscle disorders
polymyositis (PM) and dermatomyositis (DM) (1). Each member
of this family of enzymes catalyzes the formation of an aminoacyltRNA from a specific amino acid and its cognate tRNAs. Autoantibodies to five of these synthetases (histidyl-, threonyl-, alanyl-,
isoleucyl-, and glycyl-tRNA synthetases) have been identified in
patients with PM and DM (1–5). Among these “anti-synthetase
Abs,” anti-histidyl tRNA synthetase (anti-Jo-1) is the most common, found in 20 –30% of such patients (6 –10). Anti-threonyl
tRNA synthetase (anti-PL-7) and anti-alanyl tRNA synthetase (antiPL-12) Abs are less common, found in 3– 4% of all patients with
*Section of Rheumatology, Department of Internal Medicine, Keio University School
of Medicine, Tokyo, Japan; †Department of Respiratory Medicine, Graduate School
of Medicine, Kyoto University, Kyoto, Japan; ‡Section of Infectious Diseases, Department of Medicine, Michigan State University, East Lansing, MI 48824; and §Department of Medicine, Veterans Affairs Medical Center and University of Oklahoma
Health Science Center and Arthritis-Immunology Section, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104
Received for publication May 15, 1998. Accepted for publication October 29, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by grants from the Ministry of Health and Welfare
and the Ministry of Education (Grant-in-Aid for Scientific Research (C)-08670534,
10670426), Japanese Government, from the Cell Science Research Foundation, and
from the Keio University School of Medicine. This work was also supported by
Department of Veterans Affairs medical research funds to the Veterans Affairs Medical Center, Oklahoma City and National Institutes of Health Grant R29AI37668.
2
Address correspondence and reprint requests to Dr. Michito Hirakata, Section of
Rheumatology, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail address:
[email protected]
3
Abbreviations used in this paper: tRNA, transfer RNA; ILD, interstitial lung disease;
IPP, immunoprecipitation; AsnRS, asparaginyl-tRNA synthetase; PM, polymyositis;
DM, dermatomyositis.
Copyright © 1999 by The American Association of Immunologists
PM/DM (3, 4, 10 –12), while autoantibodies to isoleucyl-tRNA
synthetase (anti-OJ) and glycyl-tRNA synthetase (anti-EJ) are the
least common, occurring in , 2%(5, 13, 14). Isoleucyl-tRNA synthetase is the only one of these synthetase autoantigens that is a
component of the multienzyme synthetase complex, and some antiOJ sera also react with other components of the synthetase complex, but such additional reactivity does not change the immunoprecipitation (IPP) picture of anti-OJ. Thus, excluding the nine
synthetase activities that are part of the complex and the four other
described anti-synthetases, seven aminoacyl-tRNA synthetases exist for which autoantibodies have not been described, as determined by IPP of tRNA. The reason for this selectivity for certain
synthetases is not known. With the exception mentioned for antiOJ, it is extremely rare for a patient to have more than one
anti-synthetase (15).
Anti-Jo-1 and other anti-synthetases have each been associated
with a similar syndrome marked by myositis with a high frequency
of interstitial lung disease (ILD) (50 – 80%) and arthritis (50 –90%)
(7–10, 16, 17), as well as an increase when compared with the
overall myositis population in Raynaud’s phenomenon (60%), fever with exacerbations (80%), and the skin lesion of the fingers
referred to as mechanic’s hands (70%) (8). Other associations,
such as an increase in sicca and sclerodactyly, have been observed
by some investigators (10). Although the similarities between patients with different anti-synthetases are most striking, certain differences have been observed that must be considered preliminary
due to the small number of patients with non-Jo-1 anti-synthetases
reported. One important difference is that patients with anti-PL-12
are more likely than anti-Jo-1 patients to have ILD and/or arthritis
either without myositis or with subclinical signs of muscle disease.
Absence of significant myositis over the full course of patients
with anti-Jo-1 is rare (,5%), although it may occur. Clinically
significant myositis was seen in 60% of U.S. patients with antiPL-12 (12, 18), whereas none of six Japanese patients with anti0022-1767/99/$02.00
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Autoantibodies to five of the aminoacyl-transfer RNA (tRNA) synthetases have been described, and each is associated with a
syndrome of inflammatory myopathy with interstitial lung disease (ILD) and arthritis. Serum KS, from a patient with ILD and
inflammatory arthritis without evidence of myositis, immunoprecipitated a tRNA that was distinct from that precipitated by any
described anti-synthetase or other reported tRNA-related Abs, along with a protein of 65 kDa. KS serum and IgG fraction each
showed significant (88%) inhibition of asparaginyl-tRNA synthetase (AsnRS) activity, but not of any of the other 19 aminoacyltRNA synthetase activities. Among 884 patients with connective tissue diseases tested, only two other sera were found to immunoprecipitate tRNAs and proteins of identical gel mobility. These two and KS showed identical immunodiffusion lines using HeLa
cell extract. The new sera significantly inhibited AsnRS without significant effects on other synthetases tested. Both patients had
ILD but neither had evidence of myositis. These data strongly suggest that these three sera have autoantibodies to AsnRS,
representing a sixth anti-synthetase. Anti-KS was more closely associated with ILD than with myositis. Further study of this Abs
might prove useful in dissecting the stimuli responsible for the genesis of anti-synthetase autoantibodies. The Journal of Immunology, 1999, 162: 2315–2320.
2316
Materials and Methods
Sera
Serum samples were obtained from 884 patients with connective tissue
diseases followed in clinics at Keio University in Tokyo and Kyoto University in Kyoto, Japan. These included 114 with PM/DM, 392 with SLE,
220 with systemic sclerosis, 56 with rheumatoid arthritis, and 102 patients
with ILD who had no evidence of myositis and did not meet criteria for
other conditions. Stored sera known to contain autoantibodies against synthetases for histidine, threonine, alanine, glycine, and isoleucine were used
as controls.
IPP
IPP from HeLa cell extracts was performed as previously described (5, 9).
A total of 10 ml of patient sera was mixed with 2 mg of protein A-Sepharose CL-4B (Pharmacia Biotech AB, Uppsala, Sweden) in 500 ml of IPP
buffer (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1% Nonidet P-40) and
incubated with end-over-end rotation (Labquake shaker; Lab Industries,
Berkeley, CA) for 2 h at 4°C. The IgG-coated Sepharose was washed four
times in 500 ml of IPP buffer using 10-s spins in a microfuge tube and was
resuspended in 400 ml of NET-2 buffer.
For analysis of RNAs, this suspension was incubated with 100 ml of
extracts, derived from 6 3 106 cells, on the rotator for 2 h at 4°C. The
Ag-bound Sepharose was then collected with a 10-s centrifugation in the
microfuge, washed four times with NET-2 buffer, and were resuspended in
300 ml of NET-2 buffer. To extract bound RNAs, 30 ml of 3.0 M sodium
acetate, 30 ml of 10% SDS, 2 ml of carrier yeast tRNA (10 mg/ml; Sigma,
St. Louis, MO) and 300 ml of phenol/chloroform/isoamyl alcohol (50:50:1;
containing 0.1% 8-hydroxyquinoline) were added to the Sepharose beads.
After agitation in a Vortex mixer and spinning for 1 min, RNAs were
recovered in the aqueous phase after ethanol precipitation and dissolved in
20 ml of electrophoresis sample buffer composed of 10 M urea, 0.025%
bromphenol blue, and 0.025% xylene cyanol-FF in TBE buffer (90 mM
Tris-HCl, pH 8.6, 90 mM boric acid, and 1 mM EDTA). The RNA samples
were denatured at 65°C for 5 min and then resolved in 7 M urea-10%
polyacrylamide gel, which was stained with silver (Bio-Rad Laboratories,
Hercules, CA). In certain experiments, cell extracts (6 3 106 cells/sample)
were deproteinized with phenol/chloroform/isoamyl alcohol before IPP
and tested in parallel with untreated extracts.
For protein studies, Ab-coated Sepharose was mixed with 400 ml of
[35S]methionine-labeled HeLa extract derived from 2 3 105 cells, and rotated at 4°C for 2 h. After four washes with IPP buffer, the Sepharose was
resuspended in SDS-sample buffer (2% SDS, 10% glycerol, 62.5 mM
Tris-HCl, pH 6.8, 0.005% bromphenol blue). After heating (90°C for 5
min), the proteins were fractionated by SDS-10% PAGE gels, enhanced
with 0.5 M sodium salicylate, and dried. Labeled proteins were analyzed by
autoradiography.
Aminoacylation
Aminoacylation inhibition reactions were performed as described previously with minor modification (5, 23). First, 6 ml of HeLa cell extract
diluted 1/10 in Tris-buffered saline were incubated with 3 ml of a 1/10
dilution of serum for 2 h at 4°C. This was combined with 17 ml reaction
solution (50 mM Tris-HCl, pH 7.5, 0.02 M NaCl, 0.01 M MgSO4, 1 mM
DTT, containing 8 units of yeast tRNA), 3 ml of 14C-asparagine or
[3H]amino acid, and 1 ml of 200 mM cold amino acid. Then, 10 ml aliquots
were tested at 10 min and 20 min, spotted onto filter paper treated with 5%
TCA, then washed five times with 5% TCA, then with ethanol, then dried
for counting. Results of inhibition testing was expressed as the percent
inhibition of the average activity seen with the normals included in that
experiment: % inhibition 5 [(average cpm with normal serum) 2 (cpm
with test serum)] 3 100/(average cpm with normal serum). Inhibition of
.50% compared with the activity with normal serum was considered significant. In previous studies, although nonspecific effects on aminoacylation reactions by serum were common, nonspecific inhibition
was usually ,25% and inhibition .50% reliably reflected specific Ab
effects (5, 6, 11, 12, 23).
To test the AsnRS activity of purified KS antigen, the procedure was
similar except that 6 ml of purified Ag was substituted for the HeLa extract
and Tris-buffered saline was substituted for serum.
Purification of the KS Ag
Affinity chromatography was performed as previously described (23). The
KS Ag was purified from HeLa cell extracts. IgG fraction was purified
from 20 ml of KS serum using DEAE. KS IgG was coupled to Affi-gel
(Bio-Rad Laboratories) hydroxysuccinamide-agarose in 0.1 M bicarbonate
buffer, pH 8.3, with .90% coupling. The immunoadsorbent was washed
extensively, including with the intended eluting agent (3 M MgCl2). Later
experiments were performed using a second column prepared in a similar
manner. After HeLa extract was applied to the column in excess of adsorbing capacity, the column was extensively washed with 0.5 M NaCl in
0.05 M Tris buffer, pH 7.2, with 0.01 M sodium azide and 0.1 mM PMSF
and was eluted with 3 M MgCl2.
ELISA
ELISA was performed as previously described (6, 11). The immunoaffinity-purified KS Ag was diluted 80-fold in PBS for application to the microtiter well. Wells were blocked with 0.2% BSA in PBS. Sera were diluted 1:100 for testing with 2% BSA in PBS with 0.05% Tween 20. An
affinity-purified goat anti-human IgG conjugated to alkaline phosphatase
(Sigma) was used with p-nitrophenyl phosphate substrate.
Other
Ouchterlony double immunodiffusion was performed as described previously using HeLa cell extract as Ag (9). The IgG fraction of the patient sera
was purified by DEAE chromatography.
Cases
Case 1. In April of 1979 (at age 36), patient KS developed a nonproductive cough and shortness of breath. Chest radiography showed interstitial
fibrosis, and pulmonary function testing revealed a restrictive pattern. A
diagnosis of ILD was made, and prednisolone 40 mg/day was begun, resulting in dramatic improvement of respiratory symptoms. In the autumn of
1980, inflammatory arthritis developed, which was treated with aspirin. In
1988, she was admitted to Keio University Hospital because of worsening
polyarthritis and pulmonary hypertension due to fibrosis of both lower lung
fields. No muscle weakness was found, and the creatine kinase level was
normal (49 IU/L). Neither Raynaud’s phenomenon nor elevation of creatine kinase occurred at any point in her course.
Case 2. In 1990 (at age 61), patient NI noticed slight dyspnea on exertion.
In 1991, her chest radiograph showed bilateral interstitial fibrosis in the
lower lung fields, but she did not develop any other symptoms. The following year, a diagnosis of usual interstitial pneumonitis was made on the
basis of open lung biopsy. She did not have any muscle weakness, elevation of the creatine kinase level, arthritis, or Raynaud’s phenomenon.
Case 3. In 1966 (at age 44), patient KN was found to have a reticular
pattern on her chest radiograph, but was followed up without treatment. In
1968, she noticed dyspnea on exertion and fatigability. In 1983, at age 61,
she developed Raynaud’s phenomenon. At age 64, open lung biopsy was
performed, with histology showing usual interstitial pneumonitis, but she
did not satisfy criteria for any connective tissue diseases.
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PL-12 Abs fulfilled criteria for myositis (19). In the limited number of patients thus far observed, 2 of 10 anti-OJ patients had ILD
without detectable myositis and one had ILD with subclinical
myositis.
Most sera with any of the five reported anti-synthetases specifically inhibit the aminoacylation of the respective tRNAs, indicating inhibition of the enzymatic function of the synthetase (2, 4 – 6,
11). For example, anti-Jo-1 serum, IgG fraction, and affinity-purified IgG inhibit histidyl-tRNA synthetase activity and not that of
other synthetases (6). Only occasional anti-synthetase sera have
been exceptions, i.e., did not inhibit (12, 13). Such inhibition is not
consistently seen with animal antisera raised against synthetases
and suggests that autoantibodies target an active site of the enzyme
(20). The identification of the previous five anti-synthetases was
initially based on the demonstration that several sera that shared
the same Ab and immunoprecipitated the same tRNAs could inhibit the same synthetase enzyme and not others (2–5, 14). Later,
other methods were used to support these identifications, such as
demonstration for anti-Jo-1 and anti-EJ of reaction with enzymatically active recombinant protein (21, 22).
In this report, we describe a novel autoantibody that immunoprecipitates tRNA, and provide evidence that it reacts with asparaginyl-tRNA (AsnRS) synthetase.
AUTOANTIBODIES TO Asn-tRNA SYNTHETASE
The Journal of Immunology
2317
FIGURE 1. Immunoprecipitation for nucleic acids with anti-KS sera
and controls. Urea (7 M) and 10% PAGE of phenol-extracted immunoprecipitates from HeLa cell extract were developed with silver stain. TNA,
total nucleic acids, with the 5.8 and 5.0 S small ribosomal RNAs and the
tRNA region indicated. Sera used for immunoprecipitation include: lanes
1–5, anti-synthetase sera indicated, with Abs to Jo-1 (histidyl-tRNA synthetase), PL-7 (threonyl-tRNA synthetase), PL-12 (alanyl-tRNA synthetase), EJ (glycyl-tRNA synthetase), OJ (isoleucyl-tRNA synthetase);
lanes 6 – 8, anti-KS sera as indicated; and lane 9, control serum indicated
(NHS, normal human serum). The tRNA pattern with anti-KS sera is easily
distinguishable from that of other anti-synthetases.
Of 884 patients with connective tissue diseases or ILD and controls, sera from two other patients with ILD (KN and NI) immunoprecipitated tRNA bands that were identical to those of serum
KS each time they were analyzed (Fig. 1, lanes 7 and 8). Both of
these sera immunoprecipitated strong 65-kDa protein bands that
were also identical to that of serum KS, along with a faint 63-kDa
band (Fig. 2, lanes 7 and 8). Furthermore, by immunodiffusion
against HeLa cell extracts, a line of immunologic identity was seen
Results
Identification of a new tRNA-related Ab
Serum of patient KS was found to immunoprecipitate a strong
predominant nucleic acid band of tRNA size, accompanied by a
faster band (Fig. 1, lane 6). This gel pattern of tRNAs was clearly
distinguishable from the pattern of tRNAs precipitated by the five
described anti-synthetases (shown in Fig. 1 for five) or that associated with other identified tRNA-related autoantibodies. The predominant band was faster in migration than the Jo-1 RNA and
slower than the four major PL-12 bands. The additional faster band
was almost identical in migration to the slowest band of the EJ
RNAs. This serum also immunoprecipitated a very strong protein
band from [35S]methionine-labeled HeLa cell extracts (Fig. 2, lane
6) migrating at 65 kDa that was clearly different from the bands
immunoprecipitated by sera with the described anti-synthetases
(shown in Fig. 2 for five). A second, much fainter band was seen
at 63 kDa.
Serum KS showed a line by immunodiffusion against HeLa cell
extract that was nonidentical with that of anti-PL-7, anti-PL-12,
and anti-EJ and quite different from Jo-1 because the KS line was
present but the Jo-1 line was not appreciated clearly (Fig. 3A).
FIGURE 3. Ouchterlony double immunodiffusion of anti-KS sera and
other anti-aminoacyl tRNA synthetase sera. A, The precipitin line formed
between serum KS and HeLa cell extracts went through the lines of the
PL-7, PL-12, and EJ system and was quite different from Jo-1 because the
KS line was present but the Jo-1 line was not appreciated clearly, indicating
the immunologic distinctness of anti-KS from the previously described
systems including the Jo-1, PL-7, PL-12, and EJ. B, Furthermore, a line of
immunologic identity was seen between serum KS, serum NI, and serum
KN, confirming the presence of the same autoantibody in each serum by
immunodiffusion against HeLa cell extracts.
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FIGURE 2. Immunoprecipitation for proteins with anti-KS sera and
controls. Autoradiogram of 10% SDS-PAGE of immunoprecipitates from
[35S]methionine-labeled HeLa cell extract. Mr, molecular weight markers
of the sizes indicated to the left (kDa). The sera used for immunoprecipitation are the same as those in Fig. 1. The 65-kDa KS protein is easily
distinguished from that of the anti-synthetases.
2318
AUTOANTIBODIES TO Asn-tRNA SYNTHETASE
Table I. Inhibition of aminoacylation reactions for 20 amino acids by
KS serum and IgGa
between serum KS, serum NI, and serum KN, confirming the presence of the same autoantibody in each serum (Fig. 3B). Thus,
anti-KS Abs were found in 0.34% of 884 patients with connective
tissue disease and 2.94% of 102 patients with ILD.
None of these three sera immunoprecipitated any RNA after
deproteinization of the HeLa cell extracts, whereas control antiPL-12 sera consistently precipitated the PL-12 tRNA pattern from
the deproteinized extract (Fig. 4). This indicates that anti-KS did
not directly bind tRNAs, and the proteins of the KS Ag were required for antigenicity.
Identification of the KS Ag
IPP of a unique tRNA and a strong protein band by anti-KS suggested the possibility that it was a new anti-synthetase. This was
assessed by testing KS serum for the ability to inhibit each of the
20 aminoacyl-tRNA synthetases in turn. The enzyme source, HeLa
cell extract, was preincubated with serum, at a 1:100 final concentration in the reaction mixture, before using the extract in an in
vitro aminoacylation assay. Significant (.50%) inhibition of AsnRS was seen, with inhibition of 88% of the activity seen when
normal serum was added, but there was no significant inhibitory
effect on other aminoacyl-tRNA synthetases (range ,0 –29%) (Table I). To further demonstrate that the inhibition of AsnRS resulted
from Abs, the purified IgG fraction of KS serum was tested at a
NHS
(1:10)
NML-IgG
(6 mg/ml)
KS Serum
(1:10)
KS IgG
(6 mg/ml)
Relevant
Synthetaseb
Alanine
Arginine
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
14
3
0
0
0
0
0
0
0
24
24
17
0
30
14
8
20
0
0
4
88
20
7
3
19
0
0
0
3
5
1
0
0
6
0
29
0
4
0
0
88
0
0
0
0
0
0
0
0
8
16
7
0
15
6
4
0
0
91
2
2
2
2
2
2
89
99
66
2
95
2
2
2
2
48
2
2
2
a
The percent inhibition of each of the aminoacylation reactions as compared
normal serum is shown. The serum or IgG sample was added at the concentration
shown to twice the volume of HeLa cell extract containing the enzyme and was
preincubated. The final concentration in the reaction mixture was 10-fold diluted. The
extracts used for each reaction were the same or prepared similarly, and the amino
acid for each reaction was added in labeled form. NHS, normal human serum; NML,
normal; KS, prototype serum.
b
The “Relevant Synthetase” column shows the percent inhibition by a serum
known to have autoantibodies to the synthetase for the amino acid shown and tested
simultaneously. For example, results with an anti-PL-12 serum are shown in the
“alanine” row, and results with an anti-EJ serum are shown in the “glycine” row. A
single anti-OJ serum known to have both anti-IleRS and anti-LysRS activity was used
for both the “isoleucine” and “lysine” rows.
concentration of 0.6 mg/ml in the final reaction mixture. IgG
showed similar inhibition of AsnRS by 88% at 20 min compared
with the activity in the absence of IgG, whereas there was no
significant inhibition of other synthetases (,0 –16%). Normal control serum and anti-KS-negative myositis serum showed no significant inhibition of AsnRS, although sera with other antisynthetases inhibited their respective enzymes. Normal IgG
inhibited only 14%.
In view of these results, sera KN and NI were similarly tested
and also showed specific inhibition of AsnRS compared with the
activity with normal serum (96 and 98%, respectively). There was
no significant inhibition of any of the 16 other synthetases tested
(KN range ,0 –9%; NI range ,0 –27%). IgG from these sera also
showed specific inhibition (87 and 64%, respectively) at a concentration of 0.3 mg/ml in the final reaction mixture.
The KS Ag was purified from HeLa cell extract by immunoaffinity chromatography using KS prototype serum. When 80-fold
diluted KS Ag was tested against KS serum and NI and KN IgG by
ELISA, all showed activity above controls (OD 1.19, 0.811, and
1.027, respectively, vs 0.227– 0.574 for normal or other antisynthetase sera), indicating that the affinity-purified material was
active antigenically. This Ag preparation was tested for AsnRS
enzymatic activity in an asparagine aminoacylation reaction, and it
was found to be highly active (8,059 cpm with KS Ag vs 126 cpm
without enzyme at 10 min), confirming that KS Ag is AsnRS.
Discussion
In the present study, a novel autoantibody was described directed
at AsnRS, the sixth in a series of autoantibodies to aminoacyltRNA synthetases. The evidence for its identification was similar
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FIGURE 4. Immunoprecipitation with anti-KS sera and controls for nucleic acids after deproteinization of the HeLa cell extracts. TNA, total
nucleic acids, with the 5.8 and 5.0 S small ribosomal RNAs and the tRNA
region indicated. None of three anti-KS sera (lanes 9 –11), anti-Jo-1 sera
(lanes 2–5), or anti-PL-7 serum (lane 6) immunoprecipitated any RNA
after deproteinization of the HeLa cell extracts, whereas control anti-PL-12
sera (lanes 7 and 8) consistently precipitated the PL-12 tRNA pattern from
the deproteinized extract.
Amino Acid
The Journal of Immunology
may prove to be associated with myositis in other populations. The
features that these three patients had can be considered to be within
the spectrum of the “anti-synthetase syndrome” that has been associated with other anti-synthetases. ILD is one of the major features of the anti-synthetase syndrome, and Raynaud’s phenomenon
and arthritis, as seen in some anti-KS patients, are also felt to be
part of the syndrome. The syndrome associated with anti-KS may
be one end of the spectrum of anti-synthetase patients. This highlights the clinical importance of looking for such Abs in patients
with ILD even if no signs of myositis or of connective tissue diseases are present.
This group of autoantibodies is unique in having a combination
of three characteristics: 1) they are directed at functionally related
enzymes (performing the same function for different amino acids);
2) they are associated with a similar syndrome; and 3) they are
mutually exclusive. Anti-KS Abs seem to follow this pattern. No
previously studied anti-synthetase serum has had evidence of Abs
to AsnRS, and none of the three anti-AsnRS sera reported here
showed signs of reaction with other synthetases. The mechanism
for this picture remains unknown. Several possible mechanisms
have been proposed, such as similar interaction with myositis-inducing viruses (through complexes with tRNA-like structures on
viral genomes (2) or anti-idiotypic mechanisms (4, 29)) or a similar pattern of surface expression. However, these proposed mechanisms remain speculative, and further studies could provide important clues for understanding the possible mechanisms for the
development of these Abs. Study of these Abs may provide insight
into the etiologic and pathogenetic mechanisms of myositis and
ILD.
Acknowledgments
We thank Ms. Mutsuko Ishida for expert technical assistance.
References
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and F. W. Miller. 1991. A new approach to the classification of idiopathic inflammatory myopathy: myositis-specific autoantibodies define useful homogeneous patient groups. Medicine 70:360.
9. Hirakata, M., T. Mimori, M. Akizuki, J. Craft, J. A. Hardin, and M. Homma.
1992. Autoantibodies to small nuclear and cytoplasmic ribonucleoproteins in Japanese patients with inflammatory muscle disease. Arthritis Rheum. 35:449.
10. Marguerie, C., C. C. Bunn, H. L. C. Beynon, R. M. Bernstein, J. M. B. Hughes,
A. K. So, and M. J. Walport. 1990. Polymyositis, pulmonary fibrosis and autoantibodies to aminoacyl-tRNA synthetase enzymes. Q. J. Med. 77:1019.
11. Targoff, I. N., F. C. Arnett, and M. Reichlin. 1988. Antibody to threonyl-transfer
RNA synthetase in myositis sera. Arthritis Rheum. 31:515.
12. Targoff, I. N., and F. C. Arnett. 1990. Clinical manifestations in patients with
antibody to PL-12 antigen (alanyl-tRNA synthetase). Am. J. Med. 88:241.
13. Targoff, I. N., E. P. Trieu, and F. W. Miller. 1993. Reaction of anti-OJ autoantibodies with components of the multi-enzyme complex of aminoacyl-tRNA synthetases in addition to isoleucyl-tRNA synthetase. J. Clin. Invest. 91:2556.
14. Targoff, I. N., E. P. Trieu, P. H. Plotz, and F. W. Miller. 1992. Antibodies to
glycyl-transfer RNA synthetase in patients with myositis and interstitial lung
disease. Arthritis Rheum. 35:821.
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to that provided for other anti-synthetases, including the IPP of a
distinctive set of restricted tRNAs differing from those precipitated
by other anti-synthetases, a protein of a size consistent with that
expected of the synthetase, and specific inhibition of the enzyme
target by IgG from each patient that shows the Ab, without inhibiting other synthetases. In addition, immunoaffinity-purified KS Ag
shows enzymatic activity as AsnRS. Although the background in
the ELISA was high, all three sera showed higher binding with the
Ag. These findings show human AsnRS to be ;65 kDa, similar in
size to other forms of AsnRS that have been characterized (bacterial AsnRS at 53 kDa and yeast AsnRS at 51 kDa, and Brugia
malayi at 63 kDa (24 –26)). Recently Hartlein et al. tested our
prototype serum KS against a recombinant form of human AsnRS,
and the serum demonstrated reactivity, providing further proof of
the identification of the KS Ag as AsnRS (M. Hartlein, et al.,
personal communication).
Aminoacyl-tRNA synthetases are divided into class I and class
II synthetases based on several properties shared by members of
the class, including: sequence motifs (signature sequences); molecular structures (Rossman dinucleotide binding fold and parallel
b-sheet regions for class I vs extensive anti-parallel b-sheet regions for class II); and the site of initial aminoacylation (class I at
the 29 OH of the terminal ribose vs class II at the 39 OH of the
terminal ribose) (27, 28). Among higher eukaryotes, nine synthetase activities, most of which are class I enzymes, are associated
into a multienzyme complex. Including AsnRS, five of six synthetase Ags are class II aminoacyl tRNA synthetases, each found
free and uncomplexed in the cell cytoplasm. Anti-OJ sera immunoprecipitate the full multienzyme complex with nine synthetase
activities, but most anti-OJ sera react primarily with isoleucyltRNA synthetase, a class I synthetase (13). A very small number of
sera have anti-OJ by IPP but appear to be equally or more strongly
reactive with lysyl-tRNA synthetase, another class I synthetase.
However, anti-OJ is one of the least common anti-synthetases, and
thus, most anti-synthetase Abs, and most anti-synthetase sera, react
with uncomplexed class II synthetases. The reason for this preference is unknown. Possibly, such Ags can be expressed on the
surface or presented more easily.
Six synthetases remain that have not been found to be Ags by
IPP and are not in the multienzyme complex. This, along with the
fact that anti-Jo-1 is more common than all other anti-synthetases
together, clearly indicates that synthetases are not randomly targeted. If Abs to these other six synthetases occur, they must be
extremely rare. Thousands of myositis and connective tissue disease sera, and hundreds of ILD sera, have been tested by us and
others by IPP (18, 19). This would detect tRNAs precipitated by
anti-synthetases, but unidentified Abs that immunoprecipitate
tRNA are very uncommon. However, if autoantibodies to these
synthetases existed that did not immunoprecipitate tRNA, which
can be seen with some animal antisera to synthetases, they may not
have been detected.
All three patients with anti-KS autoantibodies had ILD, some
with other associated features of connective tissue disease including Raynaud’s phenomenon and arthritis, but none with any evidence of myositis or myopathy such as weakness or elevated creatine kinase. Each of the five previous anti-synthetases were first
identified as myositis-associated autoantibodies and then found to
be associated with ILD. However, as noted above, a small number
of patients may have ILD without clinical evidence of myositis,
and this is more common with some anti-synthetases (anti-PL-12
and, from available evidence, anti-OJ) (18). In a recent report,
none of six Japanese patients who had anti-PL-12 Abs fulfilled
criteria for myositis (19). In this aspect, anti-KS appears to resemble anti-PL-12 more than anti-Jo-1. Also, like anti-PL-12, anti-KS
2319
2320
15. Gelpi, C., E. Kanterewicz, J. Gratacos, I. N. Targoff, and J. L. Rodriguez-Sanchez.
1996. Coexistence of two antisynthetases in a patient with the antisynthetase syndrome. Arthritis Rheum. 39:692.
16. Yoshida, S., M. Akizuki, T. Mimori, H. Yamagata, S. Inada, and M. Homma.
1983. The precipitating antibody to an acidic nuclear protein antigen, the Jo-1, in
connective tissue diseases: a marker for a subset of polymyositis with interstitial
pulmonary fibrosis. Arthritis Rheum. 26:604.
17. Bernstein, R. M., S. H. Morgan, J. Chapman, C. C. Bunn, M. B. Mathews,
M. Turner-Warwick, and G. R. V. Hughes. 1984. Anti-Jo-1 antibody: a marker
for myositis with interstitial lung disease. Br. Med. J. 289:151.
18. Friedman, A. W., I. N. Targoff, and F. C. Arnett. 1996. Interstitial lung disease
with autoantibodies against aminoacyl-tRNA synthetase in the absence of clinically apparent myositis. Semin. Arthritis Rheum. 26:459.
19. Hirakata, M., K. Nakamura, Y. Okano, A. Suwa, S. Inada, M. Akizuki, and
J. A. Hardin. 1995. Anti-alanyl tRNA synthetase (PL-12) antibodies are associated with interstitial lung disease in Japanese patients. Arthritis Rheum. 38:S321
(Abstr.).
20. Miller, F. W., K. A. Waite, T. Biswas, and P. H. Plotz. 1990. The role of an
autoantigen, histidyl-tRNA synthetase, in the induction and maintenance of autoimmunity. Proc. Natl. Acad. Sci. USA 87:9933.
21. Raben, N., R. C. Nichols, J. Dohlman, P. McPhie, V. Sridhar, C. Hyde, R. L. Leff,
and P. H. Plotz. 1994. A motif in human histidyl-tRNA synthetase which is
shared among several aminoacyl-tRNA synthetases is a coiled-coil that is essential for enzymatic activity and contains the major autoantigenic epitope. J. Biol.
Chem. 269:24277.
AUTOANTIBODIES TO Asn-tRNA SYNTHETASE
22. Ge, Q., E. P. Trieu, and I. N. Targoff. 1994. Primary structure and functional
expression of human glycyl-tRNA synthetase, an autoantigen in myositis. J. Biol.
Chem. 269:28790.
23. Targoff, I. N., F. C. Arnett, L. Berman, C. A. O’Brien, and M. Reichlin. 1989.
Anti-KJ: a new antibody associated with the myositis/lung syndrome that reacts
with a translation-related protein. J. Clin. Invest. 84:162.
24. Anselme, J., and M. Hartlein. 1989. Asparaginyl-tRNA synthetase from Escherichia coli has significant sequence homologies with yeast aspartyl-tRNA synthetase. Gene 84:481.
25. Seignovert, L., M. Hartlein, and R. Leberman. 1996. Asparaginyl-tRNA synthetase from Thermus thermophilus HB8 sequence of the gene and crystallization
of the enzyme expressed in Escherichia coli. Eur. J. Biochem. 239:501.
26. Kron, M., K. Marquard, M. Hartlein, S. Price, and R. Leberman. 1995. An immunodominant antigen of Brugia malayi is an asparaginyl-tRNA synthetase.
FEBS Lett. 374:122.
27. Eriani, G., M. Delarue, O. Poch, J. Gangloff, and D. Moras. 1990. Partition of
tRNA synthetases into two classes based on mutually exclusive sets of sequence
motifs. Nature 347:203.
28. Cusack, S., M. Hartlein, and R. Leberman. 1991. Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases. Nucleic Acids Res. 19:3489.
29. Plotz, P. H. 1983. Autoantibodies are anti-idiotype antibodies to antiviral antibodies. Lancet 2:824.
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