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Heterogenous nuclear RNP C1 and C2 core proteins are targets for an autoantibody found in the serum of a patient with systemic sclerosis and psoriatic arthritis.

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ARTHRITIS Kr RHEUMATISM
Vol 40. No 12, Deceniher 1907, pp 2172-2177
0 1997. Amerlcan Collegc of Rheumalology
21 72
HETEROGENOUS NUCLEAR RNP C1 AND C2 CORE PROTEINS ARE
TARGETS FOR AN AUTOANTIBODY FOUND IN THE SERUM OF A
PATIENT WITH SYSTEMIC SCLEROSIS AND PSORIATIC ARTHRITIS
DAVID STANEK, JIRi VENCOVSKY, JARMlLA KAFKOVA, and IVAN RASKA
Objective. To determine a target recognized by
anti-Bh autoantibody, found in the serum of a patient
with the unusual coexistence of systemic sclerosis (SSc)
and psoriatic arthritis (PsA).
Methods. Antigens recognized by the anti-Bh serum were characterized by indirect immunofluorescence
on HeLa cells, by conventional immunoblotting using
nuclear extract or partially purified preparation of
heterogenous nuclear RNP (hnRNP) proteins, and by
2-dimensional immunoblotting. For the analysis of
cross-reactivity and immunofluorescence patterns, autoantibodies were affinity-purified by blot elution and
then retested.
Results. Comparison of the reactivity of the antiBh antibody with the monoclonal antibody 4F4 against
both the hnRNP C proteins, together with the determination of biochemical properties of the autoantigens, led
to the identification of C1 and C2 core proteins as the
targets for the anti-Bh autoantibody.
Conclusion. Several essential components of the
spliceosome are targeted by autoantibodies that are
present in the sera of patients with systemic rheumatic
diseases. We also found that the hnRNP core proteins
C1 and C2 are recognized by the autoantibody present
in the serum of a patient with SSc and PsA. C1 and C2
hnRNP proteins should be added to the several intracellular autoantigens recently shown to be cleaved by
~~
~
~~~
Supported by grants from the Ministry of Education, Youth,
and Sports of the Czcch Republic (vS96129), from the ~ntcrnalGrant
Agency of the Czech Ministry of Health (to Dr. Vencovskp and Ms
Kafkovh; 2156-3), and from the Grant Agency of the Czech Republic
(to Mr. StanEk and Dr. RaSka; 304196 K002).
David Stanttk. MSc. Ivan RaSka. PhD. DrSc: Acadcmv of
Sciences of the Czech Republic, and Charles University, Prague,
Czech Republic; Jiii Vencovskp, MD, CSc, Jarrnila Kafkova, MSc:
Institute of ~
l
~
~and Charles
~
~ University,
~
,
Prague.
~
~ Czech
~
l
Re~ublic.
Addrcss reprint requests to Jiii Vencovsky, CSc, Institutc of
Rheumatology, Na Slupi 4, 128 50 Prague 2, Czech Republic.
Submitted for publication April 8, 1997; accepted in revised
form July 15, 1997.
interleukin-1P-converting enzyme-like enzymes during
apoptosis.
Nascent transcripts of RNA polymerase I1
(termed heterogenous nuclear RNA [hnRNA]) associate with hnRNP proteins and small nuclear RNP
(snRNP) to form hnRNA-hnRNP-snRNP complexes.
Both snRNP and hnRNP proteins are directly or indirectly involved in the maturation and transport of
hnRNA. Immunopurification of hnRNP complexes and
2-dimensional electrophoresis demonstrated that
hnRNA exists in the nucleus in association with more
than 20 proteins, designated A1 through U. The most
abundant proteins A l , A2, B1, B2, C1, and C2 are
referred to as “core” hnRNP proteins (1). All of the
hnRNP A/B proteins that have been sequenced to date
have similar structures. The sequence of human C2 is
identical to that of C1 except for an insert of 13 amino
acids near the middle of the C2 protein (2). Even though
the function of the hnRNP proteins in hnRNA maturation is not fully underytood, it is becoming apparent that
they are involved in RNA splicing. The A3 protein
regulates the choice of the 5‘ splice site, while the C
proteins are involved in cleavage at the 5’ splice site and
in the formation of the spliceosome (1). Whereas C1 and
C2 proteins are restricted to the nucleus, and probably
serve to retain the incompletely processed premessenger RNA (mRNA), hnRNP A1 protein also
shuttles between the nucleus and the Cytoplasm and may
play an important role in mRNA export (3).
Many systemic rheumatic diseases are associated
with the occurrence of autoantibodies that react with
nuclear, nucleolar, and cytoplasmic proteins that are
~often~ involved
,
in vital cellular processes. These targeted
Droteins are often comdexed with nucleic acids (4).
Ribonucleoprotein particles that form the spljceosome
are Some Of the most
targeted
Of
the autoimmune response. Autoantibodies to snRNP,
\
I
AUTOANTIBODIES TO hnRNP PROTEINS C1 AND C2
anti-Ul R N P , and anti-Sm, are well characterized a n d
serve as diagnostic markers for several r h e u m a t i c diseases (5). In t h e last few years, autoantibodies against all
A/B hnRNP proteins have also been f o u n d in t h e sera of
patients with rheumatoid arthritis ( R A ) , systemic lupus
erythematosus (SLE), and mixed connective tissue disease (MCTD) (for review, see ref. 6). Recently, antibodies to h n R N P I protein were associated with t h e clinical
features of systemic sclerosis (SSc) (7).
H e r e i n we characterize t h e a u t o i m m u n e serum of
a patient with t h e unusual coexistence of SSc and
psoriatic arthritis (PsA) t h a t reacts strongly with both of
the hnRNP C proteins, b u t not with t h e other “core”
proteins A/B.
PATIENTS AND METHODS
The patient. Serum anti-Bh was obtained from a
70-year-old man who had had psoriasis since the age of 15.
Psoriatic arthritis was diagnosed at age 66, when he began to
complain of swollen joints and severe pain in the small joints of
his hands, radiocarpal joints, and talocrural joints. Radiographic investigation revealed typical psoriatic erosions in the
small joints of the hands and ankylosis of both sacroiliac joints,
together with paravertebral parasyndesmophytes. At about the
same time, cutaneous signs of diffuse sclerodcrma appcared.
Skin biopsy was compatible with a diagnosis of SSc. Fibrotic
changes in the lungs on chest radiographs, inflammatory
alveolitis in bronchoalveolar lavage fluid, and severe impairment of diffusing capacity of the lungs established a diagnosis
of fibrosing alveolitis. There was also mild pericardial effusion
on echocardiography. Because of intermittent proteinuria, a
kidney biopsy was performed. This showed vascular nephrosclerosis possibly associated with SSc.
Laboratory investigation showed positive antinuclear
antibodies (ANA; titer 1:640) and positive latex agglutination
test results for rheumatoid factor (titer 1:1,280). Attempts to
determine the specificity of the ANA revealed an unidentified
precipitin line on countcrimmunoelectrophoresis using calf
thymus extract and unidentified strong bands on immunoblotting. Therefore, we made a further attempt to characterize the
target antigen. The antibody activity of this serum was named
anti-Bh, according to the initials of the patient’s name.
Cells, sera, and antibodies. HeLa cells were cultured in
Dulbecco’s modified Eagle’s medium supplemented with 10%
fetal calf serum and gentamicin. A monoclonal antibody 4F4 to
hnRNP proteins C1 and C2 (8) was kindly provided by
Professor G. Dreyfuss and Dr. S. Pinol-Roma (Department of
Biochemistry and Biophysics, University of Pennsylvania, Philadelphia). Anti-k’B hnRNP serum was obtained from a patient with RA. This serum showed an identical pattern to a
standard anti-hnRNP A2/RA33 serum on immunoblots with
semipurified hnRNP (9).
Preparation of cell extracts. Nuclear and cytoplasmic
extracts were prepared according to the method described by
Kevin et a1 (10). For 2-dimensional (2-D) electrophoresis, cells
were washed with cold phosphate buffered saline (PBS),
2173
harvested, resuspended in lysis buffer (9.Xil.I urea, 25% Nonidet
P40, 100 mA4 dithiothreitol, and 2% ampholytes), and incubated for 1 0 minutes at room temperature. Before loading, the
cell extract was centrifuged for 60 minutes at 24,OOOg at 4°C.
Purification of’ hnKNP. Strips of nitrocellulose mcmbrane with blotted semipurified hnRNP wcrc a generous gift
from Dr. G. Steiner (Department of Rheumatology, University
of Vienna, Vienna, Austria). The hnRNP proteins were purified from HeLa cells by affinity chromatography on heparinSepharose, as previously dcscri5ed (Y).
Electrophoresis and immunoblotting. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
run either on a large gel (14 cm X 10 cm x 1.5 mm) or a
minigel (Mini Protean 11; Bio-Rad, Hertfordshire, London,
UK). A mixture of Pharmalyte 3-10 and Servalyte 5-7 was used
for 2-D electrophoresis (isoelectric focusing/SDS-PAGE). Proteins were transferred onto nitroccllulose membranes and
immunodetected oy systems based on 1-chloro-4-naphthol or
enhanced chemiluminescence (ECL) (Amersham, Little Chalfont, UK).
To compare the reactivity of the anti-Bh autoantibody
with the monoclonal antibody 4F4, immunodetection with both
antibodies was performed on thc same membrane. The proteins resolved by 2-D electrophoresis were blotted onto nitrocellulose membranes and sub,jected to immunodetection with
anti-Bh serum (dilution 152.5). Aftcr detection by ECL, antibodies were washed according to the manufacturer’s instructions (Amersham ECL Western blotting protocol). Brietly, the
membrane was submerged into stripping buffer (62.5 mM Tris
HCI, pH 6.7, 100 mM 2-mercaptoethanol. 2% SDS) for 30
minutes at 56“C, washed with PBS containing 0.1% Tween
(PBS-Tween), and blocked for I hour in a solution of 5%
nonfat dried milk in PBS. T o control for residual binding, the
membrane was incubated only with the anti-human antibody
conjugated with horseradish pcroxidase (HRP) and subjected
again to the ECI., detection. Aftcr elution in stripping buffer,
washing in PBS-Tween, and blocking as described above,
immunodetection was performed with the monoclonal antibody 4F4 (dilution 1:3,000).
Affinity purification of autoantibodies. Autoantibodies
from the anti-Bh serum were affinity-purified by blot clution
(1 I). Briefly, a nuclear extract was separated by large 5 1 5 %
SDS-PAGE and blotted onto nitrocellulose membranes. After
blocking, the membrane was incubated for 2 hours with serum
diluted 1:lOO. Strips from both sides of the membrane werc
cut, and proteins were immunodetected as described ‘1 b ove.
The regions corresponding to the desired bauds werc then
excised and incubated with 0.1M glycine, p H 2.5, for 10
minutes at room temperature. ‘Thc eluatc was immediately
neutralized with a 1/10 volumc of 1M Tris HCI. p H 8.0,
containing bovine serum albumin (1 mgiml), washed, and
concentrated.
Indirect immunofluorescence. HcI,a cells were grown
on covcrslips, rinsed in PBS, and fixed in 2% paraformaldehydc for 10 minutes at room temperature. After permeabilization with 0.2% Triton X-100 in PBS for 5 minutes and
washing in PBS, the cells were incubated for 30 minutes at
room temperature with the anti-Bh serum (dilution I :1 OO),
undiluted affinity-purified antibody, 4F4 antibody (1:100), and
control serum (1 :I OO), respectively. Cells were washed 3 times
in PBS and incubated with the appropriate anti-human or
2174
STANEK ET AL
antimouse antibody, conjugated with Texas Red (Vector,
Burlingame, CA) for 30 minutes at room temperature.
RESULTS
Cell localization and immunologic crossreactivity of 39-kd and 41-kd autoantigens of the antiBh serum. Immunoblotting analysis revealed that the
anti-Bh serum reacted with a unique doublet of 39-kd
and 41-kd proteins, as determined by SDS-PAGE. Immunoblotting on nuclear and cytoplasmic cell extracts
showed that both proteins were largely enriched in the
soluble nuclear fraction (Figure 1A). To determine the
immunochemical relationship between the proteins of
the 39/41-kd doublet, autoantibodies were purified by
blot elution from each band separately, and immunoblotting analysis was performed with these purified
autoantibodies. Both proteins are highly related and
have similar immunologic properties, because antibodies
eluted from each band reacted with both proteins (Figure 2B).
Comparison of the reactivity of the anti-Bh serum with a monoclonal antibody to hnRNP C proteins.
Using 2-D gel electrophoresis, it was determined that
both proteins had the same isoelectric point (PI 4.9)
(data not shown). After a search through the Celis
Database of 2-D gels (http:\\biobase.dk\cgi\bin\celis) we
found that properties of the 2 antigens recognized by the
anti-Bh serum (M, and PI) best matched the C1 and C2
hnRNP proteins. To confirm this finding, we compared
the anti-Bh serum with a monoclonal antibody to
hnRNP C proteins (4F4) on immunoblots with nuclear
extract (data not shown), with total cell extract by 2-D
electrophoresis (Figures 2A and B), and with partially
purified hnRNP proteins (Figure 2C).
To eliminate the diversity between different 2-D
gels, we performed immunodetection with anti-Bh and
4F4 antibodies on the same nitrocellulose membrane.
After removal of the bound anti-Bh antibody from the
membrane and before immunodetection with antibody
4F4, we incubated the membrane with HRP-conjugated
anti-human antibody alone in order to exclude the
possibility that any antibody from the immunodetection
with anti-Bh serum remained on the membrane. No
positive signal was observed after 15 minutes of exposure (data not shown); normal exposure time is -10
seconds. We established that anti-Bh autoantibody and
monoclonal antibody 4F4 recognized the same doublet
of proteins on SDS-PAGE and 2-D electrophoresis.
To confirm that the 39-kd and 41-kd antigens are
indeed hnRNP proteins C1 and C2, the immunoblotting
94 67 43 30 -
A
B
Figure 1. Immunoblotting using anti-Bh autoantibodies. A, The same
amount of proteins from nuclear and cytoplasmic (cytoplasm.) extracts
were separated by 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and immunodetected with crude anti-Bh
serum. Protein markers are shown at the left. B, Nuclcar proteins were
separated on large 5-15% SDS-PAGE gels and subjected to immunodetection with autoantibodies affinity-purified by blot elution from the
39141-kd doublet. Autoantibodies were separately purified from either
41-kd protein (upper band) or 39-kd protein (lower band).
was performed with semipurified hnRNP proteins as the
source of antigens (9). Although the C proteins are not
the major antigens in this preparation, they copurify
together with the AIB proteins and can be immunodetected as shown in Figure 2C. We also compared the
anti-Bh serum with an autoimmune serum reacting with
the other core proteins of the hnRNP complex. While
the anti-Bh serum and antibody 4F4 recognized identical
antigens, anti-A/B protein antibodies reacted with a
different set of proteins with lower molecular mass than
the hnRNP C proteins.
Immunofluorescence of anti-Bh autoantibody,
afinity-purified anti-Bh autoantibodies, and monoclonal antibody to hnRNP C proteins. Finally, we compared the immunofluorescence patterns of the anti-Bh
serum and antibody 4F4.Anti-Bh serum showed the
same extranucleolar signal as antibody 4F4 (Figure 3).
To determine if autoantibodies reactive with the 39/
41-kd doublet are indeed responsible for the nucleoplasmic signal, we performed immunofluorescence with autoantibodies purified from these bands by blot elution.
Immunofluorescence with the purified autoantibodies
showed the same pattern of labeling as crude serum and
antibody 4F4, but with lower intensity. This diminution
of intensity is probably inherent to the purification
method (11). We were not able to detect any positive
immunofluorescence signal with autoantibodies eluted
AUTOANTIBODIES TO hnRNP PROTEINS C1 AND C2
anti-Bh
2175
-
-
IEF
IEF
I‘
94 -
43 -
67
I
67 94
43 -
30 -
30 -
20 -
20 -
A
c2c1-
cn
b
cn
B
- BllB2
-A2
- A1
Figure 2. Comparison of thc anti-Bh scrum with a monoclonal antibody to heterogenous nuclear RNP (hnRNP) C proteins (4F4). A and
B, The proteins from total cell extracts were s e p r a t e d by 2dimensional gel electrophoresis (isoclectric focusing [IEF]/sodium
dodecyl sulfate [SDSI-polyacrylamide gel electrophoresis), blotted
onto nitrocellulose mcmbranes, and immunodetected by the anti-Bh
serum (A) and antibody 4F4 (B). Protein markers are shown at the left.
C, Partially purified hnRNP proteins were electrophoresed as dcscribed by Steiner et a1 (9). Immunodetection was performed with the
anti-Bh serum, monoclonal antibody 4F4, and anti-A/B protein autoantibodies. The positions of the core hnRNP proteins Al; A2, BlIB2,
C1, and C2 are indicated.
C
from other weak bands with which the anti-Bh serum
reacted (data not shown).
3) autoantibodies purified by blot elution from each
band of the doublet reacted with both proteins, and this
is compatible with a high degree of sequence homology
DISCUSSION
We have analyzed the autoantibody reactivity
found in the serum from a patient with the unusual
coexistence of SSc and PsA. This serum reacted with a
previously uncharacterized 39-kd and 41 -kd protein doublet. The hnRNP proteins C1 and C2 were determined
to be the target autoantigens of the anti-Bh serum.
Evidence for this is based on the following observations:
1) the anti-Bh serum and a monoclonal antibody 4F4 to
hnRNP C proteins reacted with the same protein doublet On l-D and 2-D immunoblots’ 2, the
and isoelectric points of the recognized antigens corresponded to the C1 and C2 hnRNP proteins;
A
B
c
Figure 3. Immunofluorewmcc labeling of HeLd ccllc, by A, monoclo-
nal antibody 4F4, B, crude anti-Bh \erum, and C, autonntibodie5
affinity-purified by blot elution from the 39141-kd doublet
2 176
between the C proteins; 4) the aut,lantigens copurified
with the other “core” hnRNP proteins on heparinSepharose chromatography; and 5) the immunofluorescence pattern of the anti-Bh antibodies was indistinguishable from that obtained with the monoclonal
antibody 4F4.
As far as we know, this is the first description of
human anti-hnRNP C autoantibody reactivity. Autoantibodies directed toward other hnRNP proteins have bcen
described. In patients with SLE, these autoantibodies,
particularly anti-hnRNP A l , are significantly associated
with autoantibodies to U1 RNP and/or Sm (5). AntihnRNP A2IRA33 antibodies were found in patients with
several systemic diseases, such as RA, MCTD, and SLE.
These autoantibodies may appear very early in the
courye of RA, and in the absence of anti-snRNP antibodies, they are useful diagnostic markers (6). Autoantibodies to the I protein of the hnRNP complex were
recently identified in 22 of 40 patients with SSc and in 3
of 32 patients with RA, but not in patients with SLE or
MCTD (7).
We have found highly enriched anti-Bh antibody
in a patient who had SSc with lung, kidney, and heart
visceral involvement, as well as PsA that involved the
peripheral and sacroiliac joints. To determine whether
this activity was associated with SSc or with other
connective tissue diseases, we screened by immunoblotting serum samples from 71 patients with SSc, 53 with
SLE, and 139 with RA. In n o other serum $ample from
these patient groups could we confirm strong antihnRNP C reactivity (unpublished observations) However, we cannot exclude low-titer antibodies, because a
few very faint bands of a molecular mass similar to that
recognized by the anti-Bh serum were present. It seems,
therefore, that this is a rare activity for which the
diagnostic and symptom-related relevance will have to
be addressed in future studies. Patients with PsA, and
particularly patients with overlapping features of PsA
and systemic rheumatic diseases, will be of interest. In
fact, 2 patients with coexisting psoriasis and isolated
Raynaud’s phenomenon were found to have a unique
pattern of autoantibodies to A, A‘, and C proteins of U1
and U2 snRNP (12).
The hnRNP proteins C l and C2 have the same
amino acid sequence, except for a short 13-amino acid
insert in the protein C2. The data presented here show
that at least one autoantibody in the serum of patient Bh
was directed against a common region of the hnRNP C
proteins and was responsible for the described crossreactivity between the C l and C2 proteins. On the other
hand, no immunologic cross-reactivity was observed
STANEK ET AL
between ClIC2 and A/B proteins, despite the fact that
all these “core” proteins belong to the family of RNAbinding proteins containing the consensus sequcncetype RNA-binding domain (1,2).
In summary, wc have dcscribed a novel autoantibody that recognizes abundant components of the
hnRNP complex: C1 and C2 proteins. This finding is
important because both the C1 and C2 hnRNP proteins
have been shown to be cleaved by 2 enzymes (CPP32
and Mch3a) from interleukin- 1p-converting enzyme
(ICE)-like protease family (13). The ICE-like enzymes
are activated during apoptosis, and several substrates
of CPP32 protease have been shown to be the autoantigens (refs. 14 and 15 and the present study). It has
been suggested that cleavage of certain autoantigens
during apoptosis may reveal immunocryptic epitopes
that could potentially induce autoantibody responses
(15). The identified autoantibodies to hnRNP proteins C1/C2 thus provide further support for such a
hypothesis and could help to explain a possible role of
apoptotic processes in the induction of the autoantibody response.
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