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The transcriptional activator Sp1 a novel autoantigen.

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ARTHRITIS & RHEUMATISM
Vol. 40, No. 6, June 1997, pp 1085-1095
0 1997, Arnericdn College of Rheumatology
1085
THE TRANSCRIPTIONAL ACTIVATOR Spl, A NOVEL AUTOANTIGEN
TAMMY A. SPAIN, REN SUN, MARGARET GRADZKA, SU-FANG LIN:
JOSEPH CRAFT, and GEORGE MILLER
Objective. To identify one nuclear autoantigenic
protein within a complex of DNA binding proteins that
bind to GC-rich sequences in Epstein-Barr virus and
cellular DNA, and to describe the clinical characteristics of patients whose sera contained autoantibodies to
this novel autoantigen.
Methods. Antibodies to autoantigen Spl were
initially measured by an electrophoretic mobility shift
assay to detect DNA binding proteins. Nuclear extracts
and purified S pl protein were used in these assays.
Recognition of the autoantigen by autoimmune sera was
confirmed by immunoprecipitation and immunoblotting.
Results. The autoantigen was identified as Spl.
Anti-Spl was detected in sera from 8 (3%) of 230
patients. These sera contained antinuclear antibodies,
but lacked antibodies to double-stranded DNA or to
several extractable nuclear antigens. The patients whose
sera contained antibodies to Sp l were white women with
fatigue, arthritis, Raynaud’s phenomenon, malar rash,
and photosensitivity.
Conclusion. Sp l is the first described example of
an RNA polymerase I1 transcription activator as an autoantigen. The presence of Spl autoantibodies is associated
with undifferentiated connective tissue disease.
The present experiments were initiated as part of
a study to identify proteins that might play a role in
recombination events that are common to Epstein-Barr
virus (EBV) DNA and cellular DNA. Circular EBV
DNA is found during latency, and linear viral DNA is
present during lytic replication and is found in virions
(1-5). We previously identified a group of cellular
Supported by NIH grants CA-16038 and AI-22959 to Dr.
Miller, AR-40072 to Dr. Craft, and GM-14637 to Dr. Spain.
Tammy A. Spain, PhD, Ren Sun, PhD, Margaret Gradzka,
MD, Su-Fang Lin, PhD, Joseph Craft, MD, George Miller, MD: Yale
University School of Medicine, New Haven, Connecticut.
Address reprint requests to George Miller, MD, Yale University School of Medicine, Department of Pediatrics, Division of
Infectious Diseases, PO Box 208064, New Haven, CT 06520-8064.
Submitted for publication October 16, 1996; accepted in
revised form December 31, 1996.
proteins that recognized GC-rich motifs in the EBV
terminal repeats, at a locus responsible for the interconversion of linear and circular viral DNA (6). These same
proteins, called terminal repeat binding proteins
(TRBP), also bound to GC-rich sequences in repetitive
cellular DNA, within areas such as variable number
tandem repeats and Ig heavy chain class switch regions.
In efforts to further characterize TRBP, we found
that it was a conserved novel autoantigen that was
detected by serum antibodies from a group of patients
with undifferentiated connective tissue disorders. The
sera from these patients demonstrated antinuclear antibody (ANA) reactivity, but were nonreactive with
double-stranded DNA (dsDNA) or with several extractable nuclear antigens, including Sm, Ro, La, U1 RNP,
topoisomerase I, and Ku. Since the novel autoantigen
was a DNA binding protein, electrophoretic mobility
shift assay (EMSA) could be used to detect serum
antibodies to the autoantigen (7). In this assay, the
autoantibodies supershifted a DNA-protein complex.
Human B cell nuclear extract was the source of antigen
and duplex DNA probes were derived from GC-rich
sequences in the EBV terminal repeats. TRBP was
found to consist of 3 DNA-protein complexes, A, B, and
C, in order of increasing electrophoretic mobility. The
autoantisera supershifted TRBP complexes A and B, but
not C. Therefore, we sought to characterize the autoantigens contained in complexes A and B.
EMSA was used to define several properties of
TRBP. The autoantigenic factor was conserved through
evolution. Nuclear extracts from non-human primate,
bovine, and murine specimens were shown to form the
same DNA-protein complexes, and were supershifted
by autoantisera. In addition, DNA binding by the TRBP
autoantigen was sequence specific; mutations in the
EBV terminal repeat DNA reduced or eliminated binding to DNA. Moreover, DNA binding by TRBP was
augmented by the presence of zinc.
In the present study, we have identified the
transcription activator Spl as an autoantigenic compo-
SPAIN ET AL
1086
nent of TRBP complex A, and we have provided a
clinical description of the patients whose sera reacted
with Spl. The characteristics of Spl fit those previously
described for TRBP. Spl binds specifically to GC-rich
elements in viral and cellular genomes (8-11). DNA
binding is mediated by zinc-coordinated tertiary structures of the protein, known as zinc fingers (12,13). Spl is
also evolutionarily conserved at the amino acid sequence
level (14-16). We have used EMSA, immunoprecipitation, and immunoblotting to demonstrate the presence
of Spl-specific autoantibodies in patients with undefined
connective tissue disease. We have also examined the
relative sensitivity of EMSA, immunoprecipitation, and
immunoblotting, and have found that EMSA is a much
more sensitive assay of autoantibody detection than the
other 2 methods.
buffer. Immunoprecipitation samples were prepared by resuspending the immunoprecipitate in 3-pellet volumes of 1X
Laemmli’s sample buffer. All samples were boiled for 5
minutes at 100°C. Samples were resolved by 8% SDS-PAGE
and electrophoretically transferred to nitrocellulose (0.45 pm;
Schleicher & Schuell, Keene, NH). Membranes were blocked
overnight in 5% skim milk and probed for 1 hour with
autoimmune sera or rabbit polyclonal antibodies. After 2
10-minute washes at room temperature with Tris saline (0.9%
weightivolume NaCl), the blot was incubated with ’251-labeled
protein A or horseradish peroxidase-conjugated anti-human
Ig, for chemiluminescence. The blot was then washed for 10
minutes in Tris saline at room temperature and autoradiographed .
Titration, quantitation, and analysis of anti-Spl activity. Autoantibodies were serially diluted in a rabbit serum that
is nonreactive with Spl. Antibody activity was quantitated by
phosphorimagery. Several formulae were used to quantify the
assay activity and the antibody titer. Supershift activity was
calculated as follows:
MATERIALS AND METHODS
%S
(%S + %A)‘
Reagents. Purified human Spl was obtained from
Promega (Madison, WI). Rabbit polyclonal anti-Spl was purchased from Santa Cruz Biotechnolagy ([PEP2]X TransCruz;
Santa Cruz, CA). Goat anti-human IgG was supplied by
Chemicon (Temecula, CA).
Cell lines and nuclear extract preparation. The EBVpositive human B cell line, Raji (17), and the EBV-negative
human B cell line, BJA-B (18), were grown in RPMI 1640
medium supplemented with 8% fetal bovine serum, penicillin,
streptomycin, and fungizone. Nuclear extracts were prepared
as previously described (6).
EMSA. The synthetic T oligonucleotide used in the
EMSA has the sequence 5’-CGAGATCGGGGTGGGGCATGGGGGATCCCG-3’. Nuclear extract and pure Spl protein
form a DNase-resistant footprint on the EBV terminal repeats
(as described above). Single-stranded oligonucleotides were
annealed and radiolabeled by filling in recessed 3‘ ends using
Klenow fragment of DNA polymerase I (Boehringer Mannheim, Indianapolis, IN) and [a”P]dCTP (Amersham, Arlington Heights, IL). The EMSA and supershift assays were
performed as previously described ( 6 ) , with the following
exceptions: 2.5 X lo4 counts per minute of 32P-labeled duplex
oligonucleotide T was brought to 5 nM of total duplex T
oligonucleotide with the addition of unlabeled T, and was
incubated with 5-10 pg of nuclear extract or 0.1 footprint units
of Spl. Supershift interference assays were carried out by
adding anti-human IgG to the supershift reaction, and incubating for 5 minutes at room temperature.
Immunoprecipitation. Purified Spl was immunoprecipitated by the method described by Craft and Hardin (l9),
except that unlabeled proteins were immunoprecipitated in the
presence of 5 nM duplex oligonucleotide T. Immunoprecipitation buffer was modified to contain a final salt concentration of
150 mM NaCI. Spl was detected in the immunoprecipitate by
probing immunoblots of the immunoprecipitate with rabbit
polyclonal anti-Spl.
Immunoblotting. Cellular extracts were prepared by
sonicating 10” cells in Laemmli’s sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) sample
where %S is the counts contained in the supershift band
divided by the total counts in the gel lane, multiplied by 100,
and %A is the counts contained in the Spl band divided by the
total counts in the gel lane, multiplied by 100. Supershift
activities were also corrected for background supershift obtained with nonreactive rabbit antiserum. Immunoprecipitation activity was calculated as the counts contained in the Spl
band divided by the counts contained in the Ig band. Immunoblotting activity was calculated as the counts contained in
the Spl band corrected for local averaging that was necessitated by large variations in background at different antibody
dilutions.
All activities were standardized to the undiluted value
in each titration series. Assay activities were plotted against
dilutions. The dilution at which activity was considered to be
50% of the activity in undiluted serum was determined by
interpolation.
Clinical profiles of patients with antibodies to Spl.
The clinical records of patients with antibodies to Spl were
examined. The patients had been followed up medically for
2-42 months. Diagnoses were made according to the criteria
proposed for systemic lupus erythematosus (SLE) (20), scleroderma (21), and mixed connective tissue disease (22). Levels of
ANA were measured using indirect immunofluorescence on
HEp-2 cells (see below). Sera were screened at a dilution of
1:40; serial dilutions were performed on ANA-positive samples. Antibodies to dsDNA, Sm, Ro, La, UI RNP, and
topoisomerase I were measured using enzyme-linked immunosorbent assays (ELISAs) from Apotex (Arlington, TX),
according to the method of the manufacturer.
Patient sera and indirect antinuclear immunofluorescence. Patient sera were obtained from the repository in the
Section of Rheumatology, Department of Internal Medicine,
at Yale University School of Medicine. All sera had been
collected for the purpose of testing for ANA.
Fluorescent staining patterns were determined by indirect antinuclear immunofluorescence using the Quidel ANA
IFA kit (San Diego, CA) at a serum dilution of 1:40. HEp-2
substrate cells were provided with the ANA IFA kit. Fluores-
Spl, A NOVEL AUTOANTIGEN
Table 1. Results of screening of sera for antibodies against Spl using
the supershift assay*
Patient category
Number of
sera
ANA+
dsDNA/ENA+
dsDNA/ENANPC patients
Healthy individuals
Total
118
230
60
10
418
Number of
supershift-positive
sera
* Sera from antinuclear antibody (ANA)-positive patients, nasopharyngeal carcinoma (NPC) patients, and healthy individuals were tested
for activity against Spl by supershift assay. ANA-positive patients were
subdivided based on the presence (+) or absence (-) of antibodies
against double-stranded DNA (dsDNA) or against the extractable
nuclear antigens (ENA) La, Ro, Sm, U1 RNP, and topoisomerase I.
cence was visualized with a Zeiss Axioskop microscope (Atlantex Zieler, Avon, MA) at X 1,000 magnification.
1087
demonstrate that the supershift was due to IgG binding
to a component of TRBP complexes A and B.
Identification of transcriptional transactivator
Spl in TRBP complexes. In preliminary experiments,
one of the components of TRBP complex A was identified as Spl. Purified Spl protein bound to the DNA
sequences from the EBV terminal repeats, both in the
EMSA and in DNase footprinting assays (data not
shown). Point mutations that reduced binding by TRBP
to these DNA sequences also reduced binding by Spl
(data not shown). Monospecific antibody to Spl caused
a supershift of TRBP complex A (data not shown). To
Serum 6770: - + + + + - - L
o
8
In8
Anti-lgG (pg): - - mv,
mwt,
RESULTS
Identification of TRBP as an autoantigen. TRBP
was shown to consist of cellular proteins (6). Since
autoimmune sera are powerful reagents to identify and
characterize cellular proteins (23-25), a panel of 348
ANA-positive sera were screened by EMSA supershift
assay for antibodies to TRBP (Table 1). Eight sera were
reactive. All reactive sera were from a subgroup of 230
sera that lacked antibodies to dsDNA and to the autoantigens Sm, Ro, La, U1 RNP, and topoisomerase I.
Antibodies to TRBP were not found in the sera of 60
patients with nasopharyngeal cancer. Since the sera of
nasopharyngeal cancer patients have broad high-titer
reactivity to EBV proteins, TRBP was not likely to be
encoded or induced by EBV.
Influence of anti-Ig on TRBP supershift. To
confirm that the TRBP supershift was due to antibodies
in the patient sera, supershift binding reaction products
were incubated with anti-human Ig. If the supershift by
patient antisera was due to binding of Ig to TRBP,
anti-human Ig should alter the supershift, either by
decreasing the mobility of the complex or by interfering
with the formation of the supershift complex. This
supershift interference assay was also carried out with
anti-human IgG, IgA, and IgM individually and in a
mixture. Figure 1 shows that anti-human IgG interfered
with the supershift complex, but had no effect on the
shifted complexes alone. Anti-IgA and anti-IgM did not
affect the supershift complex, whereas anti-Ig and a
mixture of anti-IgG, anti-IgA, and anti-IgM interfered
with the supershift (data not shown). These results
A -
B-
P -
Lane:
i
z
3 4 5 6 7 8
Figure 1. Interference of anti-human IgG with the supershift produced by an autoimmune serum on terminal repeat binding protein
(TRBP). Raji nuclear extract was incubated with "P-labeled duplex T
oligonucleotide. Lane 1, unshifted complexes; lanes 2, 3, 4, and 5,
addition of autoimmune serum 6770; lanes 6,7, and 8, no autoimmune
serum added. Increasing amounts of goat anti-human IgG were added
to the reactions in lanes 3-8. Protein-DNA complexes and free
oligonucleotide were separated on a 4% non-denaturing polyacrylamide gel. A = TRBP complex A; B = TRBP complex B; S =
supershift complex; P = free probe.
SPAIN ET AL
1088
A
--$ggFg;BwJ3f;z$
Serum #
Lane: - 1
2
3
4
5
6
7
8
910111213
141516
S T
L
A B-
C -
Nuclear Extract
Serum #: Lane: 1
-
KU KU (-)
50 55 57
2 3 4 5
sL
sl
Sp1
nuclear extracts (Figure 2A) also were found to supershift purified Spl (Figure 2B). The TRBP supershiftnegative sera did not supershift purified Spl. Furthermore, the degree to which a given serum supershifted
the TRBP complexes correlated with the amount of
Spl supershifted by that serum. Therefore, these results
demonstrate that Spl is an autoantigenic component
of TRBP.
Distinguishing autoantisera with antibodies to
Spl from those with antibodies to Ku, by EMSA supershift assay. Ku was identified as a component of TRBP
complex C in the EMSA with duplex oligonucleotide T
(Figure 3). Autoantisera with antibodies to Ku (26)
supershifted complex C, but did not affect complexes A
or B (Figure 3, lanes 3 and 4). Conversely, antisera that
-
A -
BC-
P-
spl
Figure 2. Concordance of recognition of TRBP complexes and Spl by
autoimmune sera. A panel of antinuclear antibody-positive, doublestranded DNNextractable nuclear antigen-negative autoimmune sera
that were either supershift positive (lanes 3-10) or supershift negative
(lanes 11-16) were used to supershift A, Raji nuclear extract or B, Spl.
In both A and B, lane 1 contains probe only and lane 2 contains probe
and specified protein only. Complexes and free probe were separated
on 4% non-denaturing polyacrylamide gels. C = TRBP complex C;
Spl = Spl shift complex; see Figure 1 for other definitions.
P determine whether the Spl protein is an autoantigen, a
group of 14 autoantisera-wiih and without reactivity to
TRBP (Figure 2A) were examined for their
to
supershift purified SPl (Figure 2B)- The 8 autoantisera
that contained antibody to TRBP from unfractionated
Figure 3. Supershift of TRBP complex C with Ku-reactive sera. Ku 50
and Ku 55, both reactive to Ku, were used to supershift Raji nuclear
extract. (-)57 is a human serum that does not recognize Ku (negative
control). See Figure 1 for definitions.
Spl, A NOVEL AUTOANTIGEN
reacted with Spl (Figure 2B, lanes 3-10) supershifted
TRBP complexes A and B, but failed to cause a supershift of complex C. Autoantisera lacking antibodies to
Ku or Spl (e.g., Figure 2A, lanes 11-16 and Figure 3,
lane 5 ) did not supershift complexes A, B, or C. These
results show that the supershift assay could distinguish
among different DNA binding autoantigens and thus,
could discriminate the different DNA binding proteins
that are components of the TRBP complexes.
Detection of Spl by autoantisera using immunoblotting and immunoprecipitation. A single autoantiserum, 7336, detected Spl on immunoblot (Figure 4A,
lane 4). The 95-kd Spl protein co-migrated with a 95-kd
protein present in B cells that was detected by the same
autoantiserum. These same proteins were detected by a
monospecific antibody to Spl (Figure 4, lanes 1 and 2).
The remainder of the autoantisera that were reactive
with Spl by supershift assay (Figure 2B) failed to detect
Spl on immunoblot (data not shown).
However, all 8 human sera in which Spl was
detected by EMSA also showed recognition of Spl by
immunoprecipitation followed by immunoblotting with
antibody to Spl (Figure 4B). Sera 7336, 6388, and 6770,
which produced the most complete supershift (Figure
2B, lanes 3-5), were also more efficient at immunoprecipitating Spl (Figure 4B, lanes 2-4). Serum 7405, which
was weakly reactive by immunoprecipitation (Figure 4B,
lane 5 ) , was also relatively weak by supershift assay
(Figure 2B, lane 6). The capacity of the autoantibodies
to detect Spl by EMSA and immunoprecipitation, and
the failure of the majority of them to react by immunoblotting, suggests that most of the autoantibodies recognized conformational epitopes on Spl.
Comparison of EMSA supershift assay, immunoprecipitation, and immunoblotting for detection of antibodies to Spl. The relative sensitivities of the EMSA
supershift assay, immunoprecipitation, and immunoblotting were compared by serial dilution of autoantiserum
7336, the only antiserum that was reactive to Spl by all
3 assays (Figures 5A-D and Table 2). The titer of
anti-Spl antibody in serum 7336 was expressed as the
dilution at which activity was 50% of the activity in
undiluted serum. The anti-Spl titer as measured by
EMSA was 1:407, whereas the titers measured by immunoprecipitation and immunoblotting were 1:4.1 and
1:2.1, respectively. These titers were determined by
interpolation from serial dilutions of antiserum 7336.
Therefore, EMSA was at least 100-fold more sensitive
than immunoprecipitation or immunoblotting in the
detection of antibodies to Spl.
Determination of anti-Spl titer by EMSA supershift assay. The data in Figure 2B show that sera 7336
and 6388 produced a complete supershift of Spl (Figure
1089
A
m
a
Q -
i
a
d
n
-
+ =
a m
m
106-
-Spl
80-
50-
32-
Lane:
2
1
3
4
a - S p l 7336
B
Lane:
i
2
3
4
s
6
7
8
9
10
11
12 13
14
lOl\
Sp1
r
83-
50.6-
35.529.1
-
Figure 4. Immunodetection of Spl by autoimmune sera using different assays. A, Immunoblot studies, in which BJAB cellular extract
(lanes 1 and 3) and Spl (lanes 2 and 4) were resolved, blotted, and
probed with anti-Spl and serum 7336. B, Immunoprecipitation studies,
in which a panel of supershift-positive autoimmune sera (lanes 2-9),
a panel of supershift-negative autoimmune sera (lanes 10-14), and
anti-Spl (lane 1, positive control) were used to immunoprecipitate
purified Spl. Immunoprecipitate was resolved by 8% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Immunoprecipitated Spl was detected by rabbit anti-Spl.
SPAIN ET AL
1090
B
A
Serum: (r-spi
1.1
,
7336
( x - ~
1
106-
Spl+
8050-
Lane:i
2
3
5
4
6
7
8
Y
lmmunoblot
0.01
0.001
0.1
1
Dilution
D
Serum:
-
-
Dilution:
-
-
-
- 3 2 8- 88
Lane:
1
2
3
4
sc
Spl
7336
n - ~
s
6
7
8
8
0
-
0
9 10
-
C
Serum: tx-spi c x - ~
50
7336
-
Lane:
1
2
3
4
5
6
7
lmmunoprecipitation
8
9
P-
EMSA Supershift
Figure 5. Relative sensitivity of the supershift, immunoprecipitation, and immunoblotting assays. Serum 7336 was diluted and used in all 3 assays
with Spl. A, Assay activities were plotted against serum dilutions. W = supershift activities; 0 = immunoprecipitation activities; A = immunoblotting
activities. B, Titration of immunoblotting activity. C, Titration of immunoprecipitation activity. D, Titration of electrophoretic mobility shift assay
(EMSA) supershift activity. Spl = Spl shift complex; see Figure 1 for other definitions. a-R is a nonreactive rabbit antiserum.
Spl, A NOVEL AUTOANTIGEN
1091
Table 2. Comparison of titers of antibody to Spl in serum 7336 using
3 assays
Assay
Titer*
Supershift
Immunoprecipitation
Immunoblot
1:407
1:4.1
1:2.1
* Expressed as the dilution of serum that results in 50% of the activity
of undiluted serum using the indicated assay.
DISCUSSION
In these experiments, we have provided evidence
that the transcription factor, Spl, is one component of a
complex of autoantigenic DNA binding proteins known
as TRBP, which recognize GC-rich sequences in cellular
A
1.1 4
2B, lanes 3 and 4), whereas sera 6770 and 7405 caused
partial supershifts (Figure 2B, lanes 5 and 6). Moreover,
the data in Figure 4B show that sera 7336 and 6388
strongly immunoprecipitated Spl (Figure 4B, lanes 2
and 3), serum 6770 showed an intermediate effect
(Figure 4B, lane 4), and serum 7405 showed weak
immunoprecipation of Spl (Figure 4B, lane 5). To test
the hypothesis that these differences in reactivity with
Spl were due to antibody titer, these 4 antisera were
serially diluted and their reactivity by EMSA was compared (Figure 6 and Table 3, and data not shown).
Serum 7336, which was strongly reactive by both assays,
had the highest titer at 1:407. The titer of anti-Spl
reactivity in serum 7336 was markedly higher than in the
other anti-Sp1 reactive sera. Serum 7405, which was
weakly reactive by both assays, had the lowest titer at
1:2.2, and sera 6388 and 6770 had intermediate titers.
Particularly with regard to the antisera showing high and
low reactivity, these data reveal a general correlation
between the EMSA supershift activity, potency of
immunoprecipitation, and titer of antibody to Spl. However, this correlation was not absolute for the antisera
that had an intermediate titer (see Discussion).
Clinical profile of patients with autoantibodies to
Spl. The 8 patients identified as having autoantibodies
to Spl were white women with a median age of 43 years
(range 25-63 years). The most common symptoms were
fatigue and photosensitivity, which were each present
in one-half of the patients. One-half of the patients had
arthritis, while Raynaud's phenomenon, oral ulcers, malar
rash, and sicca symptoms were less frequent (Table 4).
Only 1 of the patients met the criteria for SLE.
ANA with a speckled pattern were present in the
sera of all 8 patients. The ANA titers ranged from 1:640
to 1:20,480. In general, there was no correlation between
ANA titer and reactivity to Spl by EMSA and immunoprecipitation. However, the most highly reactive serum,
7336, had the highest ANA titer. The speckled pattern of
human autoantibodies was similar to that produced by
monospecific antibodies to Spl (Figure 7).
I
0.001
0.01
0:1
Dilution
D
Figure 6. Relative anti-Spl titers of 4 supershift-positive autoimmune
sera. Autoimmune sera 7336, 6388, 6770, and 7405 were serially
diluted and used to supershift Spl. A, Assay activities, which were
calculated based on quantitation of band intensities by phosphorimagery, were plotted against serum dilutions. W = serum 7336; 0 =
serum 6388; A = serum 6770; = serum 7405. B, Data from titration
of sera 7336 and 7405. Spl = Spl shift complex; see Figure 1 for other
definitions.
+
SPAIN ET AL
1092
Table 3. Comparison of titers of antibody to Spl in 4 sera by
supershift assay*
Serum number
Titer (SS-50)
7336
6388
6770
7405
1:407
1:7.2
1:6.8
1 :2.2
A
* SS-50 is the dilution of serum that results in 50% of the supershift
activity of undiluted serum.
and viral DNA. We obtained the following evidence for
the identity of TRBP complex A and Spl. All autoimmune sera that supershifted TRBP complex A (Figure
2A) also supershifted purified Spl bound to DNA
(Figure 2B). Conversely, sera with ANA that did not
react with TRBP did not supershift Spl. In addition, all
autoantisera that recognized Spl in the EMSA supershift assay were also capable of immunoprecipitating
Spl (Figure 4B). Only a single high-titer autoimmune
serum could recognize Spl on immunoblot. Thus, the
majority of autoantisera were likely to be recognizing a
conformational epitope on Spl.
The initial recognition of Spl as an autoantigen
was made possible by use of the highly sensitive EMSA
supershift assay. In a direct comparison (Figures SA-D),
the EMSA supershift assay was found to be at least
100-fold more sensitive than immunoprecipitation, followed by Western blotting. The EMSA supershift assay
could unambiguously detect antibodies to Spl even
in sera such as 7405, which were weak by immunoprecipitation.
There was not a clear correlation between ANA
titer and EMSA supershift titer, probably because the
Table 4.
Characteristics of patients with autoantibodies to Spl*
Characteristic
General characteristics
White
Female
Age range, years
Symptoms
Fatigue
Photosensitivity
Signs
Arthritis
Raynaud’s phenomenon
Oral ulcers
Malar rash
Sicca
Antinuclear antibodies
Speckled
Titer
818
818
25-63 (median 43)
418
418
418
318
218
218
118
818
1540-1:20,480 (median 1:2,560)
* Except where otherwise indicated, values are the no. of patients.
a-Spl
7336
Figure 7. Indirect antinuclear immunofluorescence with serum 7336
and anti-Spl. A monospecific rabbit serum to A, Spl and B, autoimmune serum 7336 was examined by indirect immunofluorescence on
HEp-2 cells at a dilution of 1:40. Cells were photographed at X1,OOO
magnification.
sera contained antibodies to other autoantigens. However, there was a strong correlation between the titer of
antibody to Spl as measured by EMSA supershift assay
and the capacity of antisera to immunoprecipitate Spl.
Antisera that supershifted Spl completely were highly
efficient at immunoprecipitating Spl, while low-titer
sera immunoprecipitated Spl weakly. However, among
sera of intermediate titer by EMSA supershift assay,
there was more discordance between the 2 assays. Differences in antibody affinities and possible differences in
IgG subclasses may influence reactivity within the 2
assays.
Other assays, including ELISA and immunoprecipitation of radiolabeled cell extracts, may prove to be
more sensitive and time-efficient in future large-scale
seroepidemiologic studies that investigate autoreactivity
Spl, A NOVEL AUTOANTIGEN
to Spl. We did not use ELISA in these experiments
because the purpose of our study was to identify the
TRBP autoantigen and solidify the evidence that it was
Spl. To this end, we used assays with greater degrees of
specificity than ELISA, although they may be less sensitive. Immunoprecipitation of radiolabeled cell extracts
was confounded by the presence of other autoantigens in
the immunoprecipitate, and is therefore also less specific
than EMSA. The high specificity of the EMSA supershift assay is derived from combining a high-affinity
protein-DNA interaction with that of an antigenantibody reaction. EMSA is likely to be more sensitive
than other assays of high specificity, such as immunoprecipitation and immunoblotting, in part because there
is minimal manipulation of the antigen, which might
denature the antigen or disrupt the antigen-antibody
complex. Moreover, the EMSA is a relatively timeefficient and facile method compared with other assays
of high specificity.
The group of patients with antibodies to Spl that
we identified shared several characteristics. They were
all white women with signs and symptoms of connective
tissue diseases. However, only 1 patient met the diagnostic criteria for SLE. Their autoantibodies produced a
speckled pattern by indirect immunofluorescence. These
autoantisera lacked reactivity to dsDNA and also failed
to react with a group of common nuclear autoantigens,
which included RNPs, topoisomerase I, and Ku. A larger
population survey and followup studies are needed to
determine whether antibodies to Spl define a distinct
clinical entity.
Autoantisera have been proven to be powerful
reagents in cell biology. Since autoantisera recognize
conserved epitopes, they have been useful in the identification of analogs that are conserved through evolution.
Proliferating cell nuclear antigen is an example of a
protein originally identified as a human autoantigen that
was later shown to have counterparts in many eukaryotic
cells of different species (24). Anti-Spl autoimmune
sera may be useful to search for Spl analogs in other
species and to identify other human Spl-like proteins
(27-30). Some autoantibodies also recognize epitopes
that are functionally important (24). Several functional
domains on Spl have been well characterized (31-34);
identification of the epitope(s) recognized by the autoimmune sera may elucidate other functionally important
domains or help to further define a functionally important region in an already characterized domain on Spl.
Although several heteroantisera to Spl have been described (27,35,36), autoantisera to Spl may provide
1093
another source of Spl antibodies that may target conserved functional domains.
Other transcription factors have been found to be
autoantigenic. NOR-90, a 90-kd protein localized to the
nucleolus organizer region, was found to be identical to
the RNA polymerase I transcription factor, human
upstream binding factor (37,38). Interestingly, some sera
from patients that recognized human upstream binding
factor also recognized RNA polymerase I and RNA
polymerase 11. It was hypothesized that the target of
autoantibodies in these sera might be the ribosomal
RNA transcription complex (39). Since autoantibodies
are known to recognize subcellular complexes involved
in cellular processes such as RNA splicing, RNA polymerase I11 transcript processing, and DNA replication
(40), it is reasonable to consider that nuclear complexes
involved in RNA polymerase I1 transcription initiation
may also be recognized by autoimmune sera that contain
antibodies to transcriptional activators such as Spl. The
basal transcription factors, RNA polymerase I1 and TFII
B, have been shown to be autoantigens (41,42), which
supports the hypothesis that the RNA polymerase I1
transcription complex is autoimmunogenic.
Spl is a member of a family of zinc-complexing
proteins with highly conserved structures called zinc
fingers that are involved in contacting DNA (12,13). The
52-kd Ro protein, one of the major antigens recognized
by the sera of SLE and Sjogren’s syndrome patients, is
also a zinc-finger protein (43). However, the function of
52-kd Ro is unknown. Our own (unpublished) observations indicate that other members of the Spl family are
also autoantigenic. Furthermore, autoantibodies that
react with Spl also react with other Spl family members.
Since autoantibodies recognize evolutionarily conserved
motifs in a conformation-specific manner (40), it is
possible that the zinc finger itself is an autoantigenic
structural motif. Mapping of the autoantigenic epitopes
on Spl, 52-kd Ro, and other zinc-finger proteins will be
needed to test this hypothesis.
In the present study, we have shown that the
transcription activator, Spl, is a component of the
protein complex, TRBP, that binds to recombinogenic
regions of EBV and cellular DNA. Spl is an autoantigen
and is the first described example of an RNA polymerase I1 transcription activator as an autoantigen. Spl is
recognized by sera from a group of patients with symptoms of connective tissue disease. These sera contain
ANA that do not react with dsDNA or with any commonly tested nuclear autoantigen. As many as 3% of
patients with ANA of unknown specificity may show
reactivity with Spl. EMSA is a highly sensitive and
SPAIN ET AL
1094
specific method for demonstration of antibody to Spl.
Future large-SCale seroepidemiologic studies
provide a more precise definition of the clinical syndrome
associated with autoimmunity to Spl.
ACKNOWLEDGMENTS
we thank D ~ sam
. peng for the immunofluorescence
analyses, and Ms Mary Breitenstein for the ELISA tests of the
autoimmune sera.
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