Group Title: Arthritis Research & Therapy
Title: Autoantibodies against the replication protein A complex in systemic lupus erythematosus and other autoimmune diseases
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Title: Autoantibodies against the replication protein A complex in systemic lupus erythematosus and other autoimmune diseases
Physical Description: Book
Language: English
Creator: Yamasaki, Yoshioki
Narain, Sonali
Hernandez, Liza
Barker, Tolga
Ikeda, Keigo
Segal, Mark
Richards, Hanno
Chan, Edward
Reeves, Westley
Satoh, Minoru
Publisher: Arthritis Research and Therapy
Publication Date: 2006
 Notes
Abstract: Replication protein A (RPA), a heterotrimer with subunits of molecular masses 70, 32, and 14 kDa, is a single-stranded-DNA-binding factor involved in DNA replication, repair, and recombination. There have been only three reported cases of anti-RPA in systemic lupus erythematosus (SLE) and Sjögren syndrome (SjS). This study sought to clarify the clinical significance of autoantibodies against RPA. Sera from 1,119 patients enrolled during the period 2000 to 2005 were screened by immunoprecipitation (IP) of 35S-labeled K562 cell extract. Antigen-capture ELISA with anti-RPA32 mAb, immunofluorescent antinuclear antibodies (ANA) and western blot analysis with purified RPA were also performed. Our results show that nine sera immunoprecipitated the RPA70–RPA32–RPA14 complex and all were strongly positive by ELISA (titers 1:62,500 to 1:312,500). No additional sera were positive by ELISA and subsequently confirmed by IP or western blotting. All sera showed fine speckled/homogeneous nuclear staining. Anti-RPA was found in 1.4% (4/276) of SLE and 2.5% (1/40) of SjS sera, but not in rheumatoid arthritis (0/35), systemic sclerosis (0/47), or polymyositis/dermatomyositis (0/43). Eight of nine patients were female and there was no racial predilection. Other positive patients had interstitial lung disease, autoimmune thyroiditis/hepatitis C virus/pernicious anemia, or an unknown diagnosis. Autoantibody specificities found in up to 40% of SLE and other diseases, such as anti-nRNP, anti-Sm, anti-Ro, and anti-La, were unusual in anti-RPA-positive sera. Only one of nine had anti-Ro, and zero of nine had anti-nRNP, anti-Sm, anti-La, or anti-ribosomal P antibodies. In summary, high titers of anti-RPA antibodies were found in nine patients (1.4% of SLE and other diseases). Other autoantibodies found in SLE were rare in this subset, suggesting that patients with anti-RPA may form a unique clinical and immunological subset.
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Source Institution: University of Florida
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Research article


Autoantibodies against the replication protein A complex in
systemic lupus erythematosus and other autoimmune diseases
Yoshioki Yamasaki1, Sonali Narain1, Liza Hernandez1, Tolga Barker1, Keigo Ikeda3, Mark S Segal4,
Hanno B Richards1,2, Edward KL Chan3, Westley H Reeves1,2 and Minoru Satohl,2


1Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Florida, PO Box 100221, Gainesville, Florida, 32610, USA
2Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, PO Box 100221, Gainesville, Florida, 32610, USA
3Department of Oral Biology, University of Florida, PO Box 100424, Gainesville, Florida, 32610, USA
4Division of Nephrology, Department of Medicine, University of Florida, PO Box 100221, Gainesville, Florida, 32610, USA

Corresponding author: Minoru Satoh, satohm@medicine.ufl.edu

Received: 19 Apr 2006 Revisions requested: 10 May 2006 Revisions received: 14 Jun 2006 Accepted: 28 Jun 2006 Published: 17 Jul 2006

Arthritis Research & Therapy 2006, 8:R111 (doi:10.1186/ar2000)
This article is online at: http://arthritis-research.com/content/8/4/R111
C 2006 Yamasaki et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract


Replication protein A (RPA), a heterotrimer with subunits of
molecular masses 70, 32, and 14 kDa, is a single-stranded-
DNA-binding factor involved in DNA replication, repair, and
recombination. There have been only three reported cases of
anti-RPA in systemic lupus erythematosus (SLE) and Sj6gren
syndrome (SjS). This study sought to clarify the clinical
significance of autoantibodies against RPA. Sera from 1,119
patients enrolled during the period 2000 to 2005 were
screened by immunoprecipitation (IP) of 35S-labeled K562 cell
extract. Antigen-capture ELISA with anti-RPA32 mAb,
immunofluorescent antinuclear antibodies (ANA) and western
blot analysis with purified RPA were also performed. Our results
show that nine sera immunoprecipitated the RPA70-RPA32-
RPA14 complex and all were strongly positive by ELISA (titers
1:62,500 to 1:312,500). No additional sera were positive by
ELISA and subsequently confirmed by IP or western blotting. All


Introduction
Autoantibodies in systemic autoimmune rheumatic diseases
such as systemic lupus erythematosus (SLE) often recognize
molecules involved in the critical biological functions of cells
such as DNA replication, repair, and recombination, splicing,
transcription, translation, and cell cycle control [1]. These tar-
get antigens are subcellular particles consisting of multipro-
teins often with DNA or RNAs. Furthermore, many of these
autoantibodies are specific for particular diagnoses and have


sera showed fine speckled/homogeneous nuclear staining. Anti-
RPA was found in 1.4% (4/276) of SLE and 2.5% (1/40) of SjS
sera, but not in rheumatoid arthritis (0/35), systemic sclerosis
(0/47), or polymyositis/dermatomyositis (0/43). Eight of nine
patients were female and there was no racial predilection. Other
positive patients had interstitial lung disease, autoimmune
thyroiditis/hepatitis C virus/pernicious anemia, or an unknown
diagnosis. Autoantibody specificities found in up to 40% of SLE
and other diseases, such as anti-nRNP, anti-Sm, anti-Ro, and
anti-La, were unusual in anti-RPA-positive sera. Only one of nine
had anti-Ro, and zero of nine had anti-nRNP, anti-Sm, anti-La, or
anti-ribosomal P antibodies. In summary, high titers of anti-RPA
antibodies were found in nine patients (1.4% of SLE and other
diseases). Other autoantibodies found in SLE were rare in this
subset, suggesting that patients with anti-RPA may form a
unique clinical and immunological subset.


been used as a disease marker [1]. Some of these are also
associated with certain clinical symptoms or subset of disease
and are useful in monitoring certain organ involvement and
predicting outcome.

Among molecules involved in DNA replication, PCNA (prolifer-
ating-cell nuclear antigen) was identified as a target of autoan-
tibodies in SLE more than 20 years ago [2,3]. Later the PCNA
was identified as an auxiliary protein of DNA polymerase delta


Page 1 of 10
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ANA = antinuclear antibodies; DNA-PK = DNA-dependent protein kinase; dsDNA = double-stranded DNA; dsRNA = double-stranded RNA; ELISA
= enzyme-linked immunosorbent assay; IP = immunoprecipitation; mAb = monoclonal antibody; NHS = normal human serum; PCNA = proliferating-
cell nuclear antigen; PM/DM = polymyositis/dermatomyositis; RA= rheumatoid arthritis; RNAP= RNA polymerase; RPA= replication protein A; SjS
= Sjogren syndrome; SLE= systemic lupus erythematosus; snRNP = small nuclear ribonucleoprotein; SSc = systemic sclerosis; ssDNA = sin-
glestranded DNA; UFCAD= University of Florida Center for Autoimmune Diseases.


Open







Arthritis Research & Therapy Vol 8 No 4 Yamasaki et al.



[4]. Anti-PCNA is considered an SLE-specific serological
marker along with anti-Sm, anti-ribosomal P, and anti-dsDNA,
although its frequency in SLE is only about 2% [1,5]. PCNA is
a part of the large complex replication machinery, but little is
known about the autoimmune response in rheumatic diseases
to other components involved in DNA replication. Replication
protein A (RPA), a heterotrimer with subunits of molecular
masses 70, 32, and 14 kDa (RPA70, RPA32, and RPA14,
respectively), is a single-stranded DNA-binding protein with
multiple and essential roles in almost every aspect of DNA
metabolism, including replication, repair, and recombination
[6]. Autoantibodies against RPA in rheumatic diseases have
been described in only three cases of SLE and Sjdgren syn-
drome (SjS) from a screening of about 150 sera [7,8]. No sys-
tematic analysis in the rheumatic diseases or clinical
significance of this specificity in human SLE is available. The
screening in the previous studies was only by western blot
analysis with recombinant RPA70 and RPA32 [7,8]. The reac-
tivity with native RPA has not been evaluated. Autoimmune B-
cell epitopes are often discontinuous [9,10], recognize native
conformational epitopes, and in some cases are poorly reac-
tive in western blot [11,12]. There are also antibodies that rec-
ognize quaternary structure consisting of several protein
components in snRNPs [13] and DNA-dependent protein
kinase (DNA-PK) [14]. On the basis of these observations in
other autoantibody systems, we suspected that the frequency
of anti-RPA might have been underestimated as a result of
their preferential recognition of the native molecule and
because anti-RPA may be associated with a specific clinical
subset of SLE. We performed systematic screening of autoan-
tibodies against the native form of RPA using immunoprecipi-
tation (IP) and antigen-capture ELISA in sera from patients
with rheumatic diseases, and analyzed the clinical significance
of these autoantibodies.

Materials and methods
Patients
A total of 1,119 subjects enrolled at the University of Florida
Center for Autoimmune Diseases (UFCAD) in the period 2000
to 2005 were studied. The subjects included 276 patients
with SLE, 43 with polymyositis/dermatomyositis (PM/DM), 47
with scleroderma (systemic sclerosis (SSc)), 35 with rheuma-
toid arthritis (RA), and 40 with SjS. Diagnosis was established
by American College of Rheumatology criteria (SLE, SSc, and
RA) [15-17], Bohan's criteria (PM/DM) [18], or the European
criteria (SjS) [19]. Clinical information was from the UFCAD
database. The protocol was approved by the University of Flor-
ida's Institutional Review Board. This study meets and is in
compliance with all ethical standards in medicine, and written
informed consent was obtained from all patients in accord-
ance with the Declaration of Helsinki.

Monoclonal antibodies against RPA
mAbs against RPA70 (clone 2H10) and RPA32 (clone 9H8),
obtained by immunization of RPA from HeLa cells, were from


Lab Vision Corp (Fremont, CA, USA). Other mAbs against
RPA32 and RPA14, made by immunization of HiS6-tagged
recombinant human protein, were from QED Bioscience Inc
(San Diego, CA, USA).

Immunoprecipitation and confirmation of anti-RPA
The proteins recognized by human sera were evaluated by IP
of radiolabeled K562 cell extracts and SDS-PAGE as
described [12]. In brief, cells were labeled with [35S]methio-
nine and cysteine, lysed in 0.5 M NaCI, 2 mM EDTA, 50 mM
Tris pH 7.5, 0.3% Nonidet P40 (0.5 M NaCI NET/Nonidet
P40) buffer containing 1 mM phenylmethyl sulfonyl fluoride
and 0.3 TIU (trypsin inhibitor units)/ml aprotinin, and immuno-
precipitated with Protein A-Sepharose beads coated with 8 pl
of human serum. Immunoprecipitates were washed three
times with 0.5 M NaCI NET/Nonidet P40 and once with NET/
Nonidet P40 followed by SDS-PAGE and autoradiography.
The specificity for anti-RPA was confirmed on the basis of IP
of the characteristic set of three proteins of 70, 32, and 14
kDa that co-migrated with the proteins immunoprecipitated by
monoclonal antibodies against RPA. The positive reaction was
also confirmed by strong reactivity in antigen-capture ELISA.

Antigen-capture ELISA for anti-RPA
Antigen-capture ELISA for anti-RPA was performed as
described previously for anti-Ku, anti-nRNP/Sm, anti-Su, and
anti-RNA helicase A, with some modification [20]. In brief,
microtiter plates were coated overnight with 2 uig/ml mAb
against RPA70, RPA32, or RPA14 in 0.1 M Na2HPO4/
NaH2PO4, pH 9.0 at 40C. Plates were washed, blocked with
0.5% BSA NET/Nonidet P40 (50 mM Tris-HCI, pH 7.5, 150
mM NaCI, 2 mM EDTA, 0.3% Nonidet P40) for one hour at
room temperature. K562 cells (108) were sonicated twice in
2.5 ml of 0.5 M NaCI NET/Nonidet P40 for 45 seconds and
the cell extracts were cleared by microcentrifugation at
12,000 r.p.m. 11,269 x g for 30 minutes at 40C. Supernatants
were harvested and the plates were incubated with 50 pl of
cell extract. Wells on half of each of the plates were incubated
with 0.5% BSA in 0.5 M NaCI NET/Nonidet P40 (50 pl per
well) as control. After incubation for 1 hour, plates were
washed three times with TBS Tween 20 (20 mM Tris-HCI, pH
7.5, 150 mM NaCI, 0.1% Tween 20), and incubated with
1:2,500 diluted human sera in 0.5% BSA in 0.5 M NaCI NET/
Nonidet P40 at room temperature for 1 hour. After being
washed, the plates were incubated for 1 hour with alkaline
phosphatase-labeled mouse anti-human IgG (dilution
1:1,000; Sigma, St Louis, MO, USA) and developed; A405 was
then measured. The absorbances of wells without K562 cell
extracts were subtracted from those of wells containing cell
extracts and were converted into units as described [21]. In
some experiments, cell extracts made with 0.15 M NaCI NET/
Nonidet P40 and with 0.5 M NaCI NET/Nonidet P40 buffer
were compared.


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Figure 1

(a) mAb Human anti-RPA
32 1 2 3 4 5 6 7 8 NHS

No4 ...-205s
RPA70 00- 4. f-* 66

-045
-- .iln -s-w r- 45


RPA32> -
29





RPA14 -- -14


RPA70 b-
Ku p70


DDA32 .


---- w
=. .



a n a
4_i (SL) r


Ki (SL) Q
U1-A

a


RPA14 b-
Histone H4


- m S @


Immunoprecipitation of replication protein A (RPA). (a) Immunoprecipi-
tation of RPA by mAbs and human autoimmune sera. 35S-labeled K562
cell extracts were immunoprecipitated with mAbs against RPA32 (lane
32), human sera with anti-RPA (lanes 1 to 4, SLE; lanes 5 to 8, others)
or with normal human serum (NHS). Coexisting anti-Ro (lane 2) and
anti-Su (lanes 3 and 8) are indicated by the open arrowheads. (b)
Immunoprecipitation of lupus autoantigens that co-migrate or overlap
with RPA. [35S]-labeled K562 cell extracts were immunoprecipitated
with sera from patients with SLE (lanes labeled Ki to Histones except
lane Ku) or PM (lane Ku), or mouse mAbs BM6.5 (anti-histones). These
sera or mAbs recognize autoantigens co-migrate with components of
RPA. RPA32 co-migrates with Ki (SL, lanes Ki and rP/Ki/his) and
Ul snRNP-A (U1-A, lanes nRNP and Ku/nRNP), RPA70 co-migrates
with Ku p70 (lanes Ku/nRNP and Ku), and RPA1 4 co-migrates with his-
tone H4 (lanes rP/Ki/his, Histones, and BM6.5). The specificities of
human autoimmune sera are indicated. The numbers at the right are the
molecular masses of protein standards. his, histones; rP, ribosomal P.


In other experiments, inhibition of anti-RPA antibodies binding
to RPA was examined. RPA was affinity purified on a microtiter
plate as described above with the use of anti-RPA70 mAb.
After washing, wells were incubated with singlestranded DNA
(ssDNA; boiled and chilled calf-thymus DNA; Sigma), double-
stranded DNA (dsDNA; S1 nuclease-treated calf thymus


Available online http://arthritis-research.com/content/8/4/R1 11



DNA; Sigma), or synthesized double-stranded RNA (dsRNA;
polyinosinic acid cytidylic acid, poly I:C; Sigma) in TE buffer
(10 mM Tris-HCI, pH 8, 2 mM EDTA) at 0.01 to 100 gg/ml, or
with buffer alone, for 30 minutes. Wells were then incubated
with 1:2,000 diluted anti-RPA-positive sera for 1 hour, fol-
lowed by incubation with ALP mouse anti-human IgG mAb
(1:2,000 dilution) for 1 hour and then developed.

Anti-ssDNA and dsDNA ELISA
A microtiter plate was coated with ssDNA or dsDNA with the
use of Reacti-Bind DNA coating solution (Pierce, Rockford, IL,
USA) in accordance with the manufacturer's instructions. Calf
thymus DNA (Sigma) boiled for 10 minutes and rapidly chilled
on ice for 10 minutes, was used as ssDNA. dsDNA was made
by digesting calf thymus DNA with S1 nuclease [22]. Wells
were coated with ssDNA or dsDNA (3 gig/ml, 100 gtl per well)
for 2 hours at 220C, washed with TBS/Tween 20 and blocked
with 0.5% BSA NET/Nonidet P40. Wells were then incubated
with sera diluted 1:500 in the same buffer, followed by ALP
mouse anti-human IgG mAb, and then washed and developed.
Absorbances greater than the mean plus 3 standard devia-
tions of 20 normal controls were considered positive.

Immunofluorescent antinuclear antibodies
Immunofluorescent antinuclear antibodies (ANA) in the sera
were tested at 1:160 dilution with the use of HEp2 cells and
1:200 diluted Alexa 488 goat anti-human IgG (H and L chain
specific; Molecular Probes, Eugene, OR, USA) as described
[23].

Western blot analysis
RPA was affinity-purified from K562 cell extracts with the use
of mAb against RPA70. Cell extracts from 3 x 108 cells in 0.5
M NaCI NET/Nonidet P40 were immunoprecipitated with 15
pg of mAb against RPA70. Purified proteins were fractionated
by 12% SDS-PAGE and transferred to a nitrocellulose filter
[12]. A strip of the filter 3 mm wide was probed with 1 pg/ml
mouse mAb against RPA or human anti-RPA-positive or con-
trol serum at a dilution of 1:500. Blots were then incubated
with 1:2,000-diluted horseradish peroxidase-labeled goat IgG
anti-mouse IgG (y-chain specific; Southern Biotechnology,
(Birmingham, AL, USA) or goat IgG F(ab')2 anti-human IgG (y-
chain specific, Southern Biotechnology) and developed with
SuperSignal West Pico Chemiluminescent Substrate
(Pierce).

Statistical analysis
All statistical analysis was performed with Prism 4.0c for Mac-
intosh (GraphPad Software, Inc., San Diego, CA, USA).
Fisher's exact test was used for analysis of association of anti-
RPA with other specificities. A relationship between ELISA
with anti-RPA70 versus anti-RPA32 mAb was analyzed by
Spearman correlation. Anti-RPA ELISA between groups was
compared by using Kruskal-Wallis with Dunn's multiple com-
parison test.


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Arthritis Research & Therapy Vol 8 No 4 Yamasaki et al.


Figure 2


(a) Anti-RPA32 Anti-RPA70 (b)
3 I120
00405 3

2 80--
60

1 ;// 40 -- ssDNA
S* -- dsDNA
-. A20-- poly C
o o 0
0.15M 0.5M 0.15M 0.5M 0 0.01 0.1 1 10 100
pg/ml


(c) (d)
4
OD405 1
3.OD405
0 3 Anti-RPA
1 positive
2
*. r 0.9524 V
p= 0.0011 0.5NHS
S4-o.oo NHS



0 1 2 3 4 *1 *^. % '. > '*4
Anti-RPA32 OD0405 4 "0 "o "
Dilution

(e)
0
unit
100 U


10 *0




0.2-
SLE RA SSc PM/DM SjS NHS

Anti-replication protein A (RPA) antigen-capture ELISA. (a) Effects of NaCI concentration of the cell extracts on the reactivity of anti-RPA human
sera. ELISAs were performed as described in the Materials and methods section with mAbs against RPA32 or RPA70 to coat ELISA plates and to
capture RPA from K562 cell extracts, which were prepared in buffer containing either 0.15 M or 0.5 M NaCI. Sera diluted to 1:500 in 0.5 M NaCI
NET/Nonidet P40 were tested. (b) Effects of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), or double-stranded RNA (dsRNA) on
the reactivity of human anti-RPA autoantibodies. Affinity-purified RPA on a microtiter plate was incubated for 30 minutes with ssDNA, dsDNA, or
dsRNA (poly I:C) at concentrations of 0.01 to 100 gg/ml or with buffer alone. Wells were then incubated with 1:2,000 diluted anti-RPA-positive sera
followed by ALP mouse mAb anti-human IgG, and developed. The percentage reactivity compared with RPA incubated with buffer alone (100%) is
shown. ssDNA or dsDNA, but not dsRNA, inhibited the human anti-RPA binding in a dose-dependent manner. (c) Correlation between levels of anti-
RPA by antigen-capture ELISA with mAbs against RPA32 and against RPA70. The reactivity of eight anti-RPA-positive human autoimmune sera in
ELISA with mAbs against RPA32 and against RPA70 was compared. Spearman r= 0.9524, p = 0.0011. (d) Titration curves of anti-RPA-positive
human sera. Titration curves of nine anti-RPA-positive autoimmune sera and four normal human sera (NHS) were created by ELISA with mAb against
RPA32. K562 cell extracts in 0.5 M NaCI NET/Nonidet P40 buffer were used and sera were serially diluted 1:5 starting from 1:500. (e) Screening
of anti-RPA antibodies in sera from patients with various systemic rheumatic diseases by ELISA. Sera from SLE (n = 276), rheumatoid arthritis (RA;
n = 35), SSc (n = 47), PM/DM (n = 43), SjS (n = 40), and normal control (NHS, n = 30) were tested at 1:2,500 dilutions by ELISA with mAb
against RPA32. SLE (p < 0.001 versus RA, p < 0.05 versus SSc, p < 0.01 versus PM/DM, p < 0.001 versus NHS) and SjS (p < 0.001 versus RA
or NHS, p < 0.05 versus SSc, p < 0.01 versus PM/DM) showed high reactivity. RA versus SSc, p < 0.05; SSc versus NHS, p < 0.05; all other pairs
were not significant. All comparisons were made with the Kruskal-Wallis test with Dunn's multiple comparison test. Open symbols, immunoprecipi-
tation negative; filled symbols, immunoprecipitation positive. SjS, Sjogren syndrome; SSc, systemic sclerosis.


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Figure 3


(a) (i)




(iii)




(v)




(vii)


(b)


RPA70


RPA32



RPA14


ELISA (+)
mAb Anti-RPA (+) IP (-) NHS
a b cd 1 2 3 4 5 6 7 8 9 1011 12 1314




S w. -.


Immumofluorescent ANA and western blot with anti-RPA positive sera
(a) Immunofluorescent ANA testing with anti-RPA-positive sera. HEp-2
cells were stained with mAb against RPA32 (i), RPA70 (ii), human
autoimmune sera with anti-RPA (1:160 dilution, iii-vii), or normal con-
trol (viii). All anti-RPA-positive sera showed nuclear fine speckled/
homogeneous staining, similar to the staining by anti-RPA32 or anti-
RPA70 mAb. Some sera had an additional immunofluorescent pattern
from the other coexisting specificities; mitochondria (vi) and centro-
mere (vii). (b) Western blot analysis of anti-RPA antibodies. RPA was
immunoprecipitated from K562 cell extract, fractionated by 12% SDS-
PAGE, and transferred to a nitrocellulose filter. Strips of the filter were
probed with mAbs against RPA (lanes a to d: a, RPA1 4; b, RPA32; c,
RPA32; d, RPA70), anti-RPA immunoprecipitation-positive sera (lanes
1 to 9), anti-RPA ELISA-positive immunoprecipitation-negative sera
(lanes 10 to 12), or control sera (normal human serum (NHS), lanes 13
and 14). H, mouse IgG heavy chain.

Results
Screening and identification of anti-RPA antibodies
Autoantibodies against RPA were screened on the basis of
the IP of the characteristic set of 70, 32, and 14 kDa proteins
that co-migrated with those immunoprecipitated by anti-


Available online http://arthritis-research.com/content/8/4/R1 11



RPA32 mAb (Figure la, lane 32) from [35S]methionine-
labeled K562 cell extracts. A representative IP by human anti-
RPA sera and mAb against RPA32 is shown (Figure 1 a). Nine
human autoimmune sera (eight are shown in Figure la, lanes
1 to 8) clearly immunoprecipitated all three RPA proteins that
co-migrated with those immunoprecipitated by mAb against
RPA32 (Figure la, lane 32). During the screening, it was
noted that each component of RPA co-migrated or overlapped
with known lupus-related autoantigens on SDS-PAGE. Exam-
ples of lupus-related autoantigens that co-migrate with RPA
are shown in Figure 1b. RPA32 co-migrates with the
U1snRNPs-A protein (U1-A, immunoprecipitated by anti-
nRNP and anti-Sm antibodies), that can be found in about
40% of SLE sera [5], and Ki (SL) antigen, which is recognized
by about 10% of SLE sera [24,25]. RPA70 co-migrates with
the p70 subunit of the Ku/DNA-PK antigen, which is recog-
nized by about 6% of sera from SLE and other diseases [20].
RPA14 co-migrates with the histone H4 of the core histone
complex [20,21] immunoprecipitated by certain autoimmune
sera. If the molecular masses of proteins are not carefully com-
pared, the pattern by anti-ribosomal P can also appear similar
to that of RPA; in particular the coexistence of Ki or U1 snRNPs
in the same serum sample can be confusing. From the routine
screening of autoantibodies by IP, nine sera were found to
immunoprecipitate RPA; however, the co-migration of compo-
nents of RPA with other lupus autoantigens shown in Figure
1 b suggests that some anti-RPA sera might have been over-
looked when other specificities coexisted. Thus, screening of
sera by antigen-capture ELISA using mAbs to RPA was per-
formed to find additional anti-RPA-positive sera that might
have been overlooked by screening with IP.

mAbs established by immunization of recombinant histidine-
tagged RPA32 or RPA14 proteins (QED Bioscience) did not
efficiently immunoprecipitate RPA from K562 cell extracts (not
shown) though they were positive by western blot (Figure 3b
see below). In contrast, mAbs against RPA70 (clone 2H10)
and RPA32 (clone 9H8) made by immunization of RPA from
HeLa cells (Lab Vision) immunoprecipitated RPA from K562
cell extracts (Figure 1) and worked well in an antigen-capture
ELISA after establishing appropriate conditions (see below).
Anti-RPA32 mAb clearly immunoprecipitated all three compo-
nents from K562 (Figure la, lane 32), HEp-2, and HeLa cells
(not shown). Anti-RPA70 mAb efficiently immunoprecipitated
all three components of RPA from HEp-2 cells but the IP of
RPA32 and RPA14 from K562 and HeLa cells was very weak
(not shown).

The reactivity of anti-RPA IP-positive sera in antigen-capture
ELISA with the use of cell extracts made with 0.15 M NaCI and
with 0.5 M NaCI in otherwise identical lysis buffer (50 mM Tris-
HCI, 2 mM EDTA, 0.3% Nonidet P40) was compared (Figure
2a). All eight sera tested reacted weakly when cell extracts
were made with 0.15 M NaCI buffer; however, the absorbance
increased markedly when the 0.5 M NaCI cell extracts were


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Arthritis Research & Therapy Vol 8 No 4 Yamasaki et al.


Table 1

Cases with anti-RPA autoantibodies

Case Age Sex Race


1 27 F Black
2 45 F Latin


3 35


Mixed


Diagnosis


SLE criteria ELISA (unit) Western blot


RPA70 RPA32 RPA14

189.0 ++ ++ +
11.5 ++ ++ +


SLE
SLE
SLE


19.6 +


4 23 M Black


5 41
6 61


White
White


Possible SLE


SiS, PBC


++ +


27.0
149.1 + ++
125 ++ ++ +


7 56 F Black


8 48


White


1 >625 +


Thyroiditis, HCV, pernicious anemia 1


81.8


9 51 F Black


Unknown


1 34.4


SLE, systemic lupus erythematosus; SjS, Sj6gren syndrome; PBC, primary biliary cirrhosis; ILD, interstitial lung disease; HCV, hepatitis C virus
infection; ELISA, enzyme-linked immunosorbent assay; RPA, replication protein A.


used with either anti-RPA32 or anti-RPA70 mAb (Figure 2a).
These results suggest that RPA can be extracted more effi-
ciently in high NaCI, and/or that the dissociation of interacting
proteins and DNA by high NaCI and possibly secondary con-
formational changes help the binding of anti-RPA antibodies in
autoimmune sera.

We examined whether the binding of DNA to RPA can inter-
fere the reactivity of anti-RPA autoantibodies by incubating
affinity-purified RPA with ssDNA, dsDNA, or dsRNA before
antibody binding. When the RPA was incubated with ssDNA
or dsDNA before the reaction with autoimmune sera, the bind-
ing of all eight sera tested was inhibited by about 50% by
ssDNA (47.2 11.2% (mean SD), range 34.0 to 66.9%) or
dsDNA (52.0 11.8%, range 36.3 to 63.2%), but not by
dsRNA (Figure 2b) at 100 uig/ml. dsDNA showed stronger
inhibition than ssDNA in all eight cases, inhibiting anti-RPA
binding in a dose-dependent manner up to 0.1 to 1 uig/ml. At
1 uig/ml, ssDNA inhibited by more than 10% in zero of eight
cases, whereas dsDNA showed the same effects in seven of
eight cases. These data are consistent with Figure 2a and sug-
gest that the binding of DNA to RPA in 0.15 M NaCI was at
least partly responsible for the poor binding of anti-RPA anti-
bodies against RPA when cell extracts were made in 0.15 M
NaCI buffer (Figure 2a).

Anti-ssDNA and anti-dsDNA antibodies were positive in four
of nine and one of nine cases by ELISA, respectively (not
shown). In cases with high anti-ssDNA antibodies, reactivity
with RPA after incubation with ssDNA increased at a moder-
ate concentration of ssDNA. This is consistent with the reac-
tivity of anti-ssDNA antibodies against ssDNA that binds to
RPA. In the presence of a high concentration of ssDNA, inhib-
itory effects on anti-RPA-RPA binding seemed to be dominant


compared with enhanced reactivity via anti-ssDNA antibody
binding to ssDNA on RPA. However, at a low concentration of
ssDNA, inhibition on anti-RPA binding by ssDNA was minimal,
whereas anti-ssDNA antibodies caused a false high binding
via ssDNA on RPA.

When the reactivity of ELISA with anti-RPA70 mAb was com-
pared with that of anti-RPA32 mAb, there was a nearly perfect
correlation (Figure 2c; Spearman r= 0.9524, p= 0.0011). On
the basis of these data, the screening of sera for anti-RPA anti-
bodies was performed with anti-RPA32 mAb and cell extracts
with 0.5 M NaCI NET/Nonidet P40. Considering the DNA-
binding capability of RPA, sera were diluted in 0.5 M NaCI
buffer to minimize the false-positive reactions caused by the
binding of the DNA-anti-DNA immune complex to RPA.

Titration curves of the nine anti-RPA IP positive sera and four
controls by ELISA with anti-RPA32 mAb are shown in Figure
2d. Sera were serially diluted 1:5, starting from 1:500 dilu-
tions. All sera were clearly positive on ELISA and their titers
were 1:12,500 in one case, 1:62,500 in six, and 1:312,500 in
two, indicating that the titers of anti-RPA were as high as those
of other high-affinity IgG autoantibodies in SLE.

Sera from patients with SLE and other systemic rheumatic dis-
eases were screened by antigen-capture ELISA (Figure 2e).
As a group, sera from patients with SLE (p < 0.001 versus RA,
p < 0.05 versus SSc, p < 0.01 versus PM/DM, p < 0.001 ver-
sus normal human serum (NHS)) and SjS (p < 0.001 versus
RA or NHS, p < 0.05 versus SSc, p < 0.01 versus PM/DM)
showed high reactivity. In addition to the five sera (four SLE,
one SjS) that were confirmed for anti-RPA by IP (filled circles),
there were sera that showed comparable or higher reactivity
on ELISA. However, after careful evaluation of proteins immu-


Page 6 of 10
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++ +
++ +












noprecipitated by these sera and by western blotting, none of
the additional ELISA positive sera were considered positive
(Figure 3b, and data not shown). Thus, anti-RPA was found in
nine cases by IP and no additional cases were found from
ELISA screening. The false-positive reactivity of many SLE and
SjS sera in this ELISA is probably due to their reactivity with
DNA (see Figure 2a,b) and other proteins co-purified with RPA
(see Figure 3b), similar to their false-positive reaction in anti-
Ku ELISA [20]. Although all nine IP-positive sera showed high
reactivity, the ELISA was not useful because of the poor sig-
nal:noise ratios and the high frequency of false positives.

Immunofluorescent ANA test
All nine anti-RPA IP-positive sera showed a fine speckled/
homogeneous nuclear staining (the staining of five cases is
shown in Figure 3a, panels iii-vii) similar to that by anti-RPA32
mAb (Fig. 3a, panel i) or anti-RPA70 (Fig. 3a, panel ii). Some
sera seem to have additional cytoplasmic staining, which is
consistent with previous observations with affinity-purified
anti-RPA antibodies [7]. One serum each among anti-RPA
positive sera also had anti-mitochondria antibodies (Fig. 3a,
panel vi) or anti-centromere antibodies (Fig. 3a, panel vii).

Western blot analysis
Six of nine anti-RPA-positive sera reacted with all three com-
ponents of RPA; of the remainder, one reacted with RPA70
and RPA32, one reacted with RPA14 only, and one was neg-

Table 2

Systemic lupus erythematosus criteria of cases with anti-RPA


Available online http://arthritis-research.com/content/8/4/R1 11



ative (Figure 3b, lanes 1 to 9; Table 1). Generally, the sera with
higher levels of anti-RPA by ELISA showed strong reactivity in
western blotting. Most sera reacted strongly with RPA32, fol-
lowed by RPA70. Reactivity with RPA14 was generally weak.
There was no relationship between reactivity with different
components of RPA and diagnosis. Anti-RPA ELISA-positive
IP-negative sera did not react with RPA but some reacted with
other proteins co-purified by anti-RPA mAb (Figure 3b, lanes
10 to 12). These data explain the false positive results in
ELISA given by some sera.

Clinical and immunologic features of anti-RPA-positive
patients
Nine patients with anti-RPA were identified out of total of
1,119 patients. Anti-RPA was found in 1.4% (4 of 276) in SLE
(includes SLE-overlap syndrome) but not in other systemic
autoimmune rheumatic diseases such as SSc (n = 47), PM/
DM (n = 43), and RA (n = 35). However, anti-RPA was also
found in five cases that do not fulfill SLE criteria including 1 of
40 (2.5%) with SjS. All except one case were female and there
was no race predilection (Table 1). SLE criteria of the positive
cases were not particularly characteristic except for that none
of five cases (including one possible case that had three SLE
criteria) had discoid rash or neurological symptoms (Table 2).
One additional case possibly had SLE leucopeniaa, lymphope-
nia, anti-dsDNA antibodies, ANA, and possible arthritis;
included in Table 2). One case of each had SjS plus primary


Criterion
Malar rash
Discoid rash
Photosensitivity
Oral ulcers
Arthritis
Serositis
Renal disorder
Neurologic
Hematologic
Positive serology
Anti-Sm


Case 1


Case 2


Case 3


Case 4


Case 5


Possible


Possible


Possible


Anti-dsDNA + +
Anti-phospholipid +
Antinuclear + +
antibodies
Number of criteria 7 5
(out of 11 SLE
diagnostic criteria)
dsDNA, double-stranded DNA; RPA, replication protein A.


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Arthritis Research & Therapy Vol 8 No 4 Yamasaki et al.


Table 3

Frequency of autoantibodies (percentages) in patients with anti-RPA


Autoantibody


RPA(+), all (n = 9)


RPA(+), SLE (n= 4)


RPA(-), SLE (n= 272)


Anti-nRNP 0a 0 39a
Anti-Sm 0 0 15
Anti-Ro 11 25 33
Anti-La 0 0 11
Anti-Ku 0 0 2
Rib-P 0 0 8
Anti-RNA helicase A 0 0 6
Anti-Su 22 0 10


RPA, replication protein A; SLE, systemic lupus erythematosus; a, p = 0.00122 by Fisher's exact test.


biliary cirrhosis, interstitial lung disease, autoimmune thyroidi-
tis plus hepatitis C virus infection plus pernicious anemia, and
one case without clinical information.

Frequency of coexisting other autoantibodies found in anti-
RPA-positive versus anti-RPA-negative SLE patients was
compared (Table 3). Interestingly, autoantibodies that can be
found in up to 30 to 40% of SLE patients such as anti-snRNPs
or anti-Ro were rare among anti-RPA-positive sera. None of
the anti-RPA sera were positive for anti-nRNP, anti-Sm, and
anti-La, and only one case was positive for anti-Ro (Figure la,
lane 2, open arrowhead). Two cases had anti-Su (Figure la,
lanes 3 and 8, open arrowhead). Anti-nRNP was significantly
less common in anti-RPA-positive sera than in anti-RPA-nega-
tive SLE (p = 0.00122 by Fisher's exact test).

About 200 patients are enrolled to UFCAD every year. One
case of anti-RPA was found in the year 2000, no cases in
2001 and 2002, four cases in 2003, three cases in 2004, and
one case in 2005. It is possible that there is a year-to-year dif-
ference in the prevalence of anti-RPA (2001 to 2002 versus
2003 to 2004, p < 0.05 by Fisher's exact text) but the number
is too small to be conclusive.

Discussion
In the previous studies in rheumatic diseases, only three cases
of SLE and SjS with anti-RPA have been described [7,8]. The
first report described two cases with anti-RPA from the
screening of 55 autoimmune sera by western blotting with
RPA70 and RPA32 recombinant proteins [7]. One case was
of SjS whose serum reacted with RPA70 and RPA32; the
other was of SLE-SjS complicated with gastric lymphoma
treated with radiotherapy [8], whose serum reacted with
RPA32. A subsequent study from the same authors described
2 of 108 SLE sera that were positive in a western blot; these
were a case reported previously and another case whose
serum reacted with both RPA32 and RPA70 [8]. The fre-
quency of anti-RPA in SLE was 1.9%, similar to that in the


present study. Because certain autoantibodies preferentially
recognize the native molecules, antibodies against native RPA
were screened by IP in this study. Two sera (cases 4 and 9),
which were negative for RPA70 and RPA32 in western blot,
would have been missed if IP had not been used, although this
was a relatively minor population (two of nine; 22% of anti-
RPA-positive sera). ELISA may be helpful in identifying addi-
tional anti-RPA-positive sera in theory; however, false positives
via the reactivity of antibodies against DNA and proteins that
interact with RPA seem to be quite common among patients
with systemic rheumatic diseases, in particular in SLE and SjS
(Figures 2b,e and 3b). The 'true' reactivity of anti-RPA antibod-
ies was significantly inhibited by DNA (Figure 2b), consistent
with the idea that autoantibodies recognize the functional site
of the target molecule [1].

One study reported the frequent detection of anti-RPA autoan-
tibodies in cancer patients [26]. HeLa cDNA expression library
was screened with a high-titer ANA-positive serum from a
breast cancer patient, and RPA32 was cloned. Sera from can-
cer patients were screened by ELISA with the recombinant
RPA32 protein. Antibodies against RPA32 were found in
10.9% (87 of 801) in breast cancer patients and in 10.3% (4
of 39) in intraductal in situ carcinoma patients, in contrast with
non-cancer controls (0 of 65) [26]. Various autoantibodies
have been described in patients with cancer [27,28] and it is
possible that anti-RPA is found in diseases other than sys-
temic rheumatic diseases. However, because the previous
study was based on ELISA alone, which is prone to false pos-
itives as shown in the present study, this finding will need to
be verified in future studies with other methods. None of the
anti-RPA-positive patients in this study had cancer.

In SSc and PM/DM, classifying patients into subsets based on
their autoantibodies has been studied extensively [29,30]. Dis-
ease-associated autoantibodies rarely coexist in SSc and PM/
DM. SSc-related autoantibodies against topoisomerase I,
RNA polymerase 1/Ill, fibrillarin, Th (7-2RNP), centromere, or


Page 8 of 10
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PM-Scl seldom coexist and thus about 80% of SSc has one
of these autoantibodies [30]. In PM/DM, patients with anti-
aminoacyl tRNA synthetase antibodies have antibodies
against only one of the synthetases, and other patients have
anti-SRP, anti-PM-Scl, anti-Ku, and anti-nRNP [31]. Several
autoantibodies including anti-Sm, anti-ribosomal P, anti-
PCNA, and anti-dsDNA have been known to be specific for
SLE [1,5]. However, in contrast with the finding in SSc or PM/
DM, frequent coexistence of disease-specific autoantibodies
has been reported in SLE [32]; many patients have more than
one of anti-Sm, anti-dsDNA, and anti-ribosomal P. The present
study suggests that anti-RPA-positive patients may form a
unique group of SLE without other autoantibodies commonly
found in SLE.

Many of the known autoantigens recognized by sera from
patients with SLE are phosphoproteins including snRNPs, La,
ribosomal P, DNA-PK, RNA polymerase II, histones, and nucle-
olin [5]. We have previously identified autoantibodies against
RNA polymerase (RNAP) II in SLE sera that preferentially rec-
ognize the phosphorylated form of RNAP II but are unreactive
with the unphosphorylated form of RNAP II [33]. In patients
with SSc, autoantibodies specific for the phosphorylated form
of RNAP II always coexisted with autoantibodies against
another phosphoprotein, topoisomerase I, suggesting the role
of phosphoamino acids in the autoimmune B-cell epitope [34].
In contrast, it has been shown that phophorylation is not nec-
essary for ribosomal P antigens to be recognized by autoanti-
bodies in SLE [35].

Both RNAP II and RPA are phosphorylated by exposure to
ultraviolet [36,37] or chemicals such as hydroxyurea and
camptothecin [38]. RPA has a role in sensing damaged DNA,
and ultraviolet or certain chemicals induces the phosphoryla-
tion of RPA32 by DNA-PK, another target of autoimmune
response in SLE, and cdc2 kinase [39]. The phosphorylated
RPA32 becomes unstable, is dissociated from the RPA com-
plex [40] and prevents RPA association with replication cent-
ers [41]. Although anti-RPA described in the present study
recognizes the unphosphorylated form of RPA32, in contrast
with the preferential recognition of the phosphorylated form of
RNAP II by certain SLE sera [33], it is tempting to speculate
that abnormal phosphorylation, disassembly of RPA, and deg-
radation triggered by ultraviolet or chemicals are associated
with the initiation of an autoimmune response to RPA.
Whether anti-RPA is associated with photosensitivity or skin
lesion in SLE, as described for anti-Ro antibodies [42], will be
another point of interest that needs to be examined in future
studies.

Conclusion
High titers of anti-RPA antibodies were found in nine patients
(1.4% of those with SLE and other diseases). Although anti-
RPA seems to be a rare autoantibody specificity, it may repre-
sent a unique clinical and immunological subset of autoim-


Available online http://arthritis-research.com/content/8/4/R1 11



mune disease that does not produce common lupus-related
autoantibodies.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
YY carried out the immunoassays, participated in the data
analysis and in the design of the study, and drafted the manu-
script. SN, LH, TB, KI, MSS, HBR, and WHR helped with data
collection. TB also helped in editing the manuscript. EKLC
provided technical help and advice for immunoassays, took
immunofluorescent ANA pictures, and also helped edit the
manuscript. MS designed and coordinated the study, per-
formed the immunoassays and the data analysis, and also
edited the manuscript. All authors read and approved the final
manuscript.

Acknowledgements
We thank Ms Lisa Oppel and Mr Anthony Chin Loy for technical assist-
ance. This work was supported by NIH Grants A147859, A139645,
AR40391, AR050661, and M01 R00082, and State of Florida funds to
the Center for Autoimmune Diseases.

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