Immune Response to U1-C Protein
J. Clin. Invest.
© The American Society for Clinical Investigation, Inc.
Volume 97, Number 11, June 1996, 2619–2626
Distinctive Immune Response Patterns of Human and Murine Autoimmune Sera to
U1 Small Nuclear Ribonucleoprotein C Protein
Minoru Satoh,* Jenifer J. Langdon,* Kimberly J. Hamilton,* Hanno B. Richards,* David Panka,
and Westley H. Reeves*
*Departments of Medicine and Microbiology/Immunology, Thurston Arthritis Research Center and Lineberger Comprehensive Cancer
Center, University of North Carolina, Chapel Hill, North Carolina 27599-7280;
Medicine, Boston, Massachusetts 02118-2394; and
Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Robert A. Eisenberg,
Department of Microbiology, Boston University School of
The U1 small nuclear ribonucleoprotein (snRNP), a com-
plex of nine proteins with U1 RNA, is a frequent target of
autoantibodies in human and murine systemic lupus erythe-
matosus (SLE). Anti-Sm antibodies recognizing the B
E, F, and G proteins of U1 snRNPs are highly specific for
SLE, and are nearly always accompanied by anti-nRNP an-
tibodies recognizing the U1 snRNP-specific 70K, A, and/or
C proteins. Previous studies suggest that human anti-nRNP
antibodies recognize primarily the U1-70K and U1-A pro-
teins, whereas recognition of U1-C is less frequent. We re-
port here that autoantibodies to U1-C are more common in
human autoimmune sera than believed previously. Using a
novel immunoprecipitation technique to detect autoanti-
bodies to native U1-C, 75/78 human sera with anti-nRNP/
Sm antibodies were anti-U1-C (
only 1/65 anti-nRNP/Sm (
) MRL mouse sera of various
Igh allotypes was positive. Two of ten anti-nRNP/Sm (
sera from BALB/c mice with a lupus-like syndrome induced
by pristane recognized U1-C. Thus, lupus in MRL mice was
characterized by a markedly lower frequency of anti-U1-C
antibodies than seen in human SLE or pristane-induced lu-
pus. The results may indicate different pathways of inter-
molecular-intrastructural diversification of autoantibody
responses to the components of U1 snRNPs in human and
murine lupus, possibly mediated by alterations in antigen
processing induced by the autoantibodies themselves. (
1996. 97:2619–2626.) Key words: anti-Sm anti-
temic lupus erythematosus
). In striking contrast,
The U small nuclear ribonucleoproteins (snRNPs)
quent targets of autoantibodies in human and murine systemic
lupus erythematosus (SLE) (1–3). Anti-Sm antibodies recog-
nize the proteins B
/B, D1/D2/D3, E, F, and G, which are
shared by U1, U2, U5, and U4-6 snRNPs (3–5), and are highly
specific for the diagnosis of SLE (1, 2). In contrast, anti-nRNP
antibodies are specific for the unique proteins of U1 snRNPs
(U1-70K, U1-A, and U1-C) (3, 5), although reactivity with a
/B epitope that is unique to U1 snRNPs also has been de-
scribed (6). Anti-nRNP antibodies, unaccompanied by anti-
Sm, reach the highest titers in mixed connective tissue disease
(MCTD) (7), but they also may be seen in SLE and other con-
ditions. The specificities of anti-nRNP antibodies have been
characterized by immunoblotting, and it has been suggested
that autoantibodies to U1-A and U1-70K are most frequent,
whereas anti-U1-C antibodies are detected in only 20–70% of
) sera (5, 8–12). However, in immunoprecipita-
tion assays using 6-min pulse-labeled cell extracts, nearly all
human anti-nRNP (
) sera immunoprecipitate both A and C
(4), suggesting that autoantibodies to native U1-C might be
more common than antibodies to the denatured protein. This
issue was re-examined in the present studies. In agreement
with the pulse-labeling studies, human anti-nRNP sera almost
invariably immunoprecipitated native U1-C. Some sera from
mice with autoantibodies induced by pristane (13) also immu-
noprecipitated U1-C. Unexpectedly, however, sera from MRL/
mice, which contain high titer anti-Sm (2) and anti-nRNP
(14) antibodies, did not recognize U1-C, even though U1
snRNPs are a primary target of autoimmunity in this strain
(14, 15). Thus, markedly different frequencies of anti-U1-C an-
tibodies distinguish human SLE and, to a lesser degree, mu-
rine pristane-induced lupus, from lupus in MRL/
view of recent evidence supporting the role of intermolecular-
intrastructural spreading of autoimmunity to multiple compo-
nents of a particle (14–17), our data raise the possibility that
the spreading of autoimmunity to other components of the U1
snRNP particle may proceed by different pathways in murine
and human SLE. Clarification of the mechanisms of autoanti-
body spreading may shed light on the high degree of disease
specificity of anti-Sm antibodies in contrast to the lower speci-
ficity of anti-nRNP antibodies.
teins, including 2.73 (anti-U1-70 kD) (18), 22G12 (anti-B
/B and D) (20), 2-12 (anti-D) (19), 7-13 (anti-D) (19), and
2G7 (anti-D) (21) were confirmed by immunoblotting with purified
snRNPs (13, 19). mAbs 9A9 (anti-U1-A
) (22) were provided by Dr. W.J. van Venrooij (University of
Nijmegen, The Netherlands).
The specificities of murine mAbs specific for U snRNP pro-
/B) (19), Y2
) and 4G3 (anti-
Address correspondence to Westley H. Reeves, Division of Rheuma-
tology and Immunology, University of North Carolina at Chapel Hill,
3330 Thurston Building, CB# 7280, Chapel Hill, NC 27599-7280.
Phone: 919-966-4191; FAX: 919-966-1739; E-mail: reeves.thurston@
Received for publication 13 October 1995 and accepted in revised
form 6 March 1996.
disease; nRNP, nuclear ribonucleoprotein antigen; snRNP, small nu-
clear ribonucleoprotein; SSc, systemic sclerosis.
Abbreviations used in this paper:
MCTD, mixed connective tissue
Satoh et al.
other autoimmune disorders seen at Keio University Hospital, Na-
tional Murayama Hospital or Fussa Hospital (Tokyo, Japan). Pa-
tients were selected on the basis of autoantibodies to nRNP and/or
Sm by double immunodiffusion using rabbit thymus extract (2). Ad-
ditional serum samples were obtained from University of North
Carolina Hospitals (Chapel Hill, NC) and The Rockefeller University
Hospital (New York, NY). The clinical diagnoses of SLE and sclero-
derma (systemic sclerosis, SSc) were made based on ACR criteria
(23, 24). Sjögren’s syndrome and polymyositis/dermatomyositis (PM/
DM) were diagnosed based on the criteria of the Ministry of Health
and Welfare, Japanese government (25) and Bohan’s criteria (26), re-
spectively. Patients with features of two or more of SLE, SSc, and
PM/DM were classified as MCTD/overlap syndrome.
IgH or IgH
(IgH ) mice were maintained in our breeding facility. Sera were col-
lected from the tail vein of 4–10-mo-old mice. MRL/
were generated by backcrossing at Boston University, Boston, MA.
In some experiments, 2–5-mo-old female BALB/c mice were injected
once i.p. with 0.5 ml of pristane, and sera collected 5 mo later were
analyzed for anti-Sm and anti-nRNP antibodies as described (13).
The proteins recognized by human or mu-
rine autoimmune sera were evaluated by immunoprecipitation of ra-
diolabeled K562 (human erythroleukemia) cell extract and SDS-PAGE
as described (13). The cells were labeled with [
cysteine (DuPont-New England Nuclear, Boston, MA), lysed in
NET/NP40 buffer (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH
7.5, 0.3% NP-40), containing 0.5 mM PMSF, 0.3 TIU/ml aprotinin,
and immunoprecipitated on protein A Sepharose beads (Pharmacia LKB
Biotechnology, Inc., Piscataway, NJ) coated with 10
l of mouse sera plus 12
l of rabbit anti–mouse IgG (1 mg/ml).
Immunoprecipitates were washed with 0.5 M NaCl NET/NP40 (0.5 M
NaCl, 2 mM EDTA, 50 mM Tris-HCl, pH 7.5, 0.3% NP-40) or mixed
micelle buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM
EDTA, 0.5% SDS, 2.5% Triton X-100, 0.25 M sucrose). In some ex-
periments, the immunoprecipitates were washed with NET/NP40
buffer containing 0.15, 0.5, or 1.5 M NaCl. In other experiments, the
beads were washed with 0.1, 0.25, 0.5, or 1.0 M MgCl
HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.3%NP40, followed by
NET buffer. L-929 cells (murine fibroblast cell line; American Type
Culture Collection, Rockville, MD) were radiolabeled, and extract
was immunoprecipitated in the same manner. Immunoprecipitated
proteins were analyzed by SDS-PAGE and autoradiography (13).
When the presence or absence of U1-C in the immunoprecipitates
was equivocal, the autoradiograph was scanned and the band densi-
ties (volumes) of the U1-A and U1-C proteins were quantified using
Image Quant software (Molecular Dynamics, Sunnyvale, CA). If the
volume of U1-C was
10% that of U1-A after subtracting local
background, the sample was classified as anti-U1-C negative.
Immunoprecipitation of free U1-C protein.
rived from 3
cells was immunoprecipitated with 300
Y2 culture supernatant absorbed on 20
Sepharose beads as above. Immunoprecipitates were washed three
times with NET/NP40, and then incubated for 2 min at 22
l of either MMB or 0.25 M MgCl
NaCl, 2 mM EDTA, 0.3%NP40. The supernatants were saved, and
absorbed twice for 30 min at 4
C with 20
Sepharose beads coated with a mixture of 300
l of 2.73 ascitic fluid, and 200
l of rabbit anti–mouse IgG, followed by two more absorp-
tions with 20
l of packed protein A Sepharose. The absorbed super-
natants were incubated with 5
l of either human or murine serum,
washed three times with 0.5 M NaCl NET/NP40, and once with NET,
and analyzed by SDS-PAGE.
Purification of U snRNPs and immunoblotting.
vol) protein A Sepharose in 20 mM Tris-HCl, pH 8, was incubated
with 1.5 ml of Y2 (anti-Sm B
/B and D) culture supernatant and the
bound antibodies were cross-linked to the beads using dimethyl pime-
Sera were obtained from patients with SLE or
mice (27), and MRL
l of human sera
, in 50 mM Tris-
K562 cell extract de-
l of mAb
l of packed protein A
C with 200
, 50 mM Tris-HCl, pH 7.5, 150 mM
l of packed protein A
l of Y2 culture super-
l 9A9 culture supernatant
l of 50% (vol/
limidate (28). Y2 mAb–coated beads were then incubated with cell
lysate from 10 cells in 2 ml of NET/NP40, and washed three times with
NET/NP40 and once with NET. These conditions allowed the U1-C
protein to remain attached to the beads after washing (see Fig. 1
). The affinity purified proteins were eluted by boiling in SDS
sample buffer, fractionated by SDS-PAGE on 12.5% gels, and trans-
ferred to nitrocellulose filters (13). Nitrocellulose strips 2.5-mm wide
were incubated with 0.8 ml of 1:200 human or murine sera or with mAbs
at appropriate dilutions in 5% non-fat dry milk in PBS at 4
Strips were then washed three times with NET/NP40, incubated with
1:400 alkaline phosphatase-conjugated goat anti–human or mouse
chain specific; Southern Biotechnology, Birmingham, AL) for
3 h at 22
C, washed again with NET/NP40, and developed as de-
scribed (13). The strip incubated with mAb 2G7 (IgG3) was probed
with alkaline phosphatase–labeled goat anti–mouse IgG3 antibodies.
C for 16 h.
Although U1-C is generally considered to be a less important
target of autoantibodies than U1-A and U1-70K (5, 8–12),
pulse labeling studies (4) suggest that autoantibodies to the na-
tive U1-C protein may actually be relatively common. To de-
termine the frequency of autoantibodies to native U1-C, the
possibility of dissociating U1-C from the U1 snRNP under
mild conditions was evaluated as the basis for a screening as-
say. U1-C is associated with the U1 snRNP via protein–protein
interactions (29), and in view of previous observations that
MMB and/or high concentrations of salt disrupt certain pro-
tein–protein interactions with relatively little effect on pro-
tein–nucleic acid interactions (30), we examined whether U1-C
could be released from the U1 snRNP by these treatments.
Dissociation of affinity-purified U1 snRNPs on protein A
K562 cell extract was immunoprecipitated
with the anti-Sm B
/B and D mAb Y2, prototype human anti-
Sm autoimmune serum, or human anti-nRNP serum (positive
in double immunodiffusion) as shown in Fig. 1,
spectively, followed by washing with 0.15 M NaCl NET/NP40
), 0.5 M NaCl NET/NP40 (lane
), or MMB (lane
raphy revealed that when the beads were washed with 0.15 or
0.5 M NaCl NET/ NP40 buffer, all anti-nRNP/Sm sera or mAb
immunoprecipitated the proteins A, B
). In contrast, when the beads were washed with 1.5 M
NaCl NET/NP40 buffer (lane
), U1-C was only weakly visible
in Y2 and anti-Sm immunoprecipitates (Fig. 1,
), whereas it remained associated with the anti-nRNP immu-
noprecipitates with little or no reduction in intensity (Fig. 1
). The intensity of the A, B
modestly in all three cases (Fig. 1,
200-kD doublet was also much fainter after 1.5 M NaCl
washing. When the immunoprecipitates were washed with
), the U1-C protein was lost completely from Y2
and anti-Sm immunoprecipitates (Fig. 1,
from anti-nRNP immunoprecipitates (Fig. 1
ence was seen in the intensity of other U1 snRNP subunits af-
ter MMB washing, nor was there much change in the intensity
of the U5 200-kD doublet, in contrast to what was seen with
1.5 M NaCl NET/NP40 washing. These results suggest that
MMB dissociated U1-C from U1 snRNPs, with minimal effect
on other components. Moreover, anti-U1-C antibodies re-
tained this subunit on the beads under these conditions.
Immunoprecipitation with mAbs recognizing components
of U1 snRNPs also suggested that U1-C was dissociated from
), 1.5 M NaCl NET/
). SDS-PAGE and autoradiog-
/B, C, D, E, F, G (lanes
/B, and D bands decreased
). The U5 spe-
), but not
). Little differ-
Immune Response to U1-C Protein
U1 snRNPs by MMB treatment (Fig. 2). Immunoprecipitates
with a panel of anti-snRNP mAbs were compared after wash-
ing with either 0.5 M NaCl NET/NP40 (Fig. 2
). L-929 cell extract was used because murine cells do
not contain the B
protein, facilitating the identification of au-
toantibodies that immunoprecipitate the U2-B
which has a similar mobility to that of B
immunoprecipitates with all of the anti-nRNP and anti-Sm
mAbs tested if the beads were washed with 0.5 M NaCl NET/
NP40 (Fig. 2
), but was absent if the beads were
washed with MMB (Fig. 2
a weak U1-C signal in the 22G12 lane (Fig. 2
pected, mAb 4G3 (anti-U2-B
) and the anti-Ku mAb 162 did
not immunoprecipitate the U1-C protein regardless of how the
immunoprecipitates were washed (lanes
the p70 and p80 Ku antigens are not visualized due to the lack
of Ku in L-929 cells ). These results further support the in-
), or MMB
. U1-C was visible in
), with the exception of
). As ex-
terpretation that U1-C was dissociated from U1 snRNPs by
washing with MMB, but could be retained on the beads by anti-
U1-C antibodies (Fig. 1). The weak retention of U1-C by mAb
22G12 was probably due to crossreactivity of 22G12 with
U1RNP-C, and was confirmed later by immunoblotting (see
Fig. 5, lane
). However, the possibility that the mAb stabilizes
the interaction of U1-C with B
Immunoprecipitation with human autoimmune sera.
and 2 suggest that anti-U1-C antibodies retain the U1-C pro-
tein on beads after MMB washing. Accordingly, sera from 78
anti-nRNP and/or Sm positive Japanese autoimmune disease
patients were screened for anti-U1-C antibodies by immuno-
precipitation with MMB washing (Table I). All 54 anti-
sera (24 SLE, 25 MCTD/overlap, and 5 SSc),
sera (16 SLE, 4 MCTD/overlap, and
one Sjögren’s) immunoprecipitated U1-C. In contrast, all three
/B (29) could not be excluded
Figure 1. Immunoprecipitation of snRNPs with differ-
ent washing conditions. Radiolabeled K562 cell extract
was immunoprecipitated with mAb Y2 (anti-Sm B?/B
and D), human anti-Sm serum, or human anti-nRNP
serum (A, B, and C, respectively), followed by washing
with buffer containing 0.15, 0.5, or 1.5 M NaCl (lanes
1–3, respectively) or MMB (lane 4). The U1-70K pro-
tein is visualized poorly after labeling cells with
[35S]methionine (45) and was not readily apparent
here. Note that the U5 200K protein doublet was also
dissociated from immunoprecipitates washed with
1.5 M NaCl, but not by MMB washing (cf. lanes 3 and
4, panel A). Positions of the immunoprecipitated A,
B?/B, C, D, E, F, G polypeptides are indicated on the
left, along with a prominent 60K protein immunopre-
cipitated by the sera in panels B and C, which was
identified as the 60K Ro (SS-A) antigen using refer-
ence sera (not shown). Positions of molecular weight
standards are indicated on the right.
Figure 2. Immunoprecipitation with
mAbs to snRNPs. Radiolabeled
mouse L929 cell extract was immuno-
precipitated with murine mAbs 2.73
(anti-U1-70K, lane 1), 9A9 (anti-U1-A
? U2-B??, lane 2), 22G12 (anti-B?/B,
lane 3), Y2 (anti-B?/B and D, lane 4),
2-12 (anti-D, lane 5), 7-13 (anti-D,
lane 6), 2G7 (anti-D, lane 7), 4G3
(anti-U2-B??, lane 8), or 162 (anti-Ku
antigen, lane 9), and washed with
0.5 M NaCl buffer (panel A) or MMB
Satoh et al.
anti-Sm?/nRNP? SLE sera failed to immunoprecipitate U1-C
after MMB washing. In addition to the Japanese patients, we
found that immunoprecipitation of U1-C was common in Cau-
casian (8/8) and Black (25/27) patients with anti-nRNP/Sm an-
tibodies. These results strongly suggest that the immunopre-
cipitation of U1-C after MMB washing was a nearly universal
characteristic of human anti-nRNP/Sm? sera.
Immunoprecipitation with murine autoimmune sera. MRL/
lpr mice produce anti-Sm and anti-nRNP antibodies and have
been studied extensively as a model of autoantibody produc-
tion in SLE. The presence of anti-U1-C antibodies in sera from
MRL/lpr and MRL ?/? mice was evaluated by immunopre-
cipitating human K562 cell lysate and washing immunoprecipi-
tates with 0.5 M NaCl NET/NP40 or MMB as above. As ex-
pected, all sera that were positive for anti-Sm and/or nRNP by
double immunodiffusion immunoprecipitated the A, B?/B, C,
D, E/F, and G proteins after 0.5 M NaCl NET/NP40 washing.
However, unexpectedly, U1-C was absent in all cases when the
immunoprecipitates were washed with MMB (data not shown).
Although most of the human U1 snRNP subunits have nearly
identical sequences to those of the corresponding murine pro-
teins, the sequence of murine U1-C has not been reported.
The possibility that murine anti-U1-C might not recognize hu-
man U1-C was therefore tested by immunoprecipitating ex-
tract from the murine fibroblast cell line L-929, instead of ex-
tract from human K562 cells (Fig. 3, A and B). Human
prototype sera specific for nRNP (lane 1), nRNP plus Sm (lane
2), or Sm alone (lane 3), produced the expected immunopre-
cipitation patterns after 0.5 M NaCl NET/NP40 or MMB
washing (Fig. 3, A and B, respectively). As noted above, mu-
rine U snRNPs contain only a single B polypeptide (31) rather
than the B?/B doublet characteristic of human U snRNPs (cf.
Fig. 3 A, lane 1 with Fig. 1). Human anti-Sm sera (Fig. 3, A and
B, lanes 2 and 3) immunoprecipitated a prominent polypeptide
migrating just above Sm-B that has been shown to be the U2-
B?? protein (32). In agreement with the immunoprecipitation
studies using K562 extract, sera from four representative
MRL/lpr mice (Fig. 3, A and B, lanes a–d), immunoprecipi-
tated all components of L-929 cell U1 snRNPs, including U1-C,
when washed with 0.5 M NaCl NET/NP40 (Fig. 3 A). How-
ever, after MMB washing, U1-C could not be seen in immuno-
precipitates of L-929 cell extract using either human anti-Sm
prototype serum (Fig. 3 B, lane 3) or MRL/lpr mouse sera
(lanes a–d). Similar results were obtained with an additional 24
anti-Sm (?) sera from MRL ?/? mice, none of which retained
U1-C on the beads after MMB washing (Table I).
Screening of sera from MRL allotype congenic mice for
anti-U1-C antibodies. Since immunoglobulin allotype can in-
fluence autoantibody production in MRL mice (27, 33), the
U1-C reactivity of sera from anti-Sm/nRNP (?) standard
MRL/lpr (IgHj, n ? 28), MRL/lpr IgHb (n ? 12), and MRL/lpr
IgHe (n ? 1) mice was evaluated (Fig. 3 C, Table I). None of
the 28 anti-Sm/nRNP (?) MRL/lpr IgHj or 12 MRL/lpr IgHb
sera retained U1-C on the beads after MMB washing (lanes
8–13 and 1–7, respectively; Table I). The single anti-Sm (?)
MRL/lpr IgHe serum retained U1-C on the beads after MMB
washing (Fig. 3 C, lane 14), indicating that, unlike the sera
from MRL congenic mice of other allotypes, it contained anti-
U1-C antibodies. Unfortunately, samples were not available to
determine whether the production of anti-U1-C antibodies
was a general characteristic of MRL/lpr IgHe mice. None of
the sera from MRL/lpr or MRL ?/? mice, regardless of allo-
type, were positive for anti-nRNP alone, since the U2-B??
protein and the U5 specific 200-kD doublet were always re-
tained on the beads after MMB washing, along with U1-A and
the Sm proteins B, D, E, F, G (Fig. 3). The reactivity with
U2- and U5- snRNPs probably reflects anti-Sm antibodies, or
less likely, autoantibodies to U2 and U5 snRNP-specific pro-
Sera from BALB/c mice with pristane-induced autoimmu-
nity. In contrast to what was seen in MRL mice, anti-nRNP
alone was much more frequent in human autoimmune sera
(Table I) and in sera from BALB/c mice with a lupus-like syn-
drome induced by pristane. Two of ten anti-nRNP/Sm (?)
sera from BALB/c (IgHa) mice injected with pristane exhib-
ited reactivity with U1-C by immunoprecipitation (Table I),
even though reactivity with U1-C by immunoblotting is un-
usual in these mice [(13); see also Fig. 4]. Also, the U5 200-kD
Table I. Frequency of Autoantibodies to U1-C by Immunoprecipitation and Immunoblotting in Sera from Japanese Patients with
anti-nRNP and/or Sm Antibodies
Immunoprecipitation with MMB washingImmunoblotting
*Specificity defined by double immunodiffusion. ‡One serum was completely negative for anti-nRNP precipitin line and did not immunoprecipitate
U1-C. Two sera produced an equivocal anti-nRNP precipitin line (line for anti-nRNP was not observed, but anti-nRNP precipitin line deviated
around the well with patient’s sera). These two sera immunoprecipitated U1-C protein very faintly, but band volumes were ? 10% of the volume of
U1-A, and they were defined as anti-U1-C (?) by immunoprecipitation. n.d., not done. §Defined as anti-Sm and/or nRNP positive by double immu-
Immune Response to U1-C Protein
doublet was immunoprecipitated only weakly, primarily late in
the course of pristane induced autoimmunity (13).
Immunoprecipitation of free U1-C. Although the above stud-
ies suggested that human and murine autoantibodies recognize
the U1 snRNP differently, it was not certain whether retention
of U1-C on beads was due to direct binding of U1-C by the hu-
man, but not murine, autoantibodies, or to a general stabiliza-
tion of the U1 snRNP structure caused by the binding of au-
toantibodies to subunits other than U1-C. The failure of anti-Sm
prototype serum to retain U1-C on the beads argues against
the latter possibility. Nevertheless, the possibility remained
that certain anti-nRNP antibodies might prevent the dissocia-
tion of U1-C by binding to an epitope comprised of U1-C plus
some other subunit, or by an indirect mechanism. This ques-
tion was addressed by immunoprecipitating biochemically pu-
rified U1-C protein.
Since the previous experiments had suggested that U1-C
could be released from the U1 snRNP by MMB treatment, the
supernatant after MMB washing was immunoprecipitated with
human or murine autoimmune sera after extensive immunode-
pletions to remove all traces of other snRNP polypeptides or
intact particles. Most sera that retained U1-C on beads after
MMB washing failed to recognize the free U1-C protein after
it was released into the supernatant by MMB treatment of U1
snRNPs (data not shown). Since partial denaturation of U1-C
by MMB (which contains SDS) might occur, an alternative
scheme for purifying U1-C protein was developed, based on
treatment of the immunoprecipitates with MgCl2.
When mAb Y2 (anti-Sm B?/B and D) immunoprecipitates
of K562 cell extract were washed with 0.15 M NaCl NET/NP40
buffer containing 0–1.0 M MgCl2, and U1-C was dissociated
from U1 snRNPs in the presence of 0.25 M MgCl2 (Fig. 4 A).
Accordingly, U1-C was eluted from affinity purified U1 snRNPs
with 0.25 M MgCl2, and the purified protein was immunopre-
cipitated with human or murine autoimmune sera (Fig. 4 B).
All five human sera containing anti-nRNP antibodies clearly
immunoprecipitated U1-C (Fig. 4 B, lanes 1–5), whereas hu-
man anti-Sm serum (lane 6), normal human serum (lane 7),
and anti-Sm/nRNP (?) MRL/lpr mouse sera (lanes a–f) did
not. These results strongly suggest that the retention of U1-C
Figure 3. Immunoprecipitation of snRNPs with human and murine
autoimmune sera. (A and B) Radiolabeled L929 cell extract was
immunoprecipitated with human anti-nRNP (lane 1), anti-nRNP
and Sm (lane 2), or anti-Sm sera (lane 3) or with MRL/lpr (IgHj)
sera (lanes a–d), and washed with 0.5 M NaCl buffer (A) or MMB
(B). (C) MRL allotype congenic mice were screened for anti-U1-C
antibodies. Radiolabeled L929 cell extract was immunoprecipitated
with MRL/lpr IgHb (lanes 1–7), MRL/lpr IgHj (lanes 8–13), or
MRL/lpr IgHe (lane 14) sera, and washed with MMB.
Figure 4. Immunoprecipitation of
purified U1-C protein. (A) Immuno-
precipitation of snRNPs by Y2
mAbs washed with MgCl2. Radio-
labeled K562 cell extract was immu-
noprecipitated with Y2 mAbs and
washed with NET/NP40 containing
0, 0.1, 0.25, 0.5, or 1 M MgCl2 (lanes
1–5, respectively). The U1-C protein
was dissociated from U1 snRNPs in
the presence of 0.25 M or higher
MgCl2 (lanes 3–5). (B) Immunopre-
cipitation of free U1-C dissociated
by MgCl2 buffer. Radiolabeled U1-C
was purified from Y2 immunopre-
cipitates with 0.25 M MgCl2 as de-
scribed in the Methods and immuno-
precipitated with human anti-nRNP
(lanes 1 and 2), anti-nRNP plus Sm
(lanes 3–5), or anti-Sm (lane 6) sera,
or with normal human serum (lane
7), or with MRL/lpr sera (lanes a–f).
Satoh et al.
after MMB washing is at least partially due to antibodies rec-
ognizing the U1-C protein itself. In addition, it is likely that the
binding of autoantibodies to U1-C partially stabilizes the na-
tive conformation in the presence of MMB. It is uncertain
whether the epitope(s) involved is (are) dependent on the qua-
ternary structure of the U1 snRNP or the tertiary structure of
U1-C. Finally, the inability of human anti-Sm or MRL/lpr sera
to immunoprecipitate U1-C after its release from the U1 snRNP
by mild MgCl2 treatment suggests that these sera did not rec-
ognize determinants of U1-C that were buried within the U1
Immunoblot analysis for autoantibodies to U1-C. The present
demonstration of a high frequency of autoantibodies to native
U1-C in human anti-nRNP (?) sera contrasts with previous
data suggesting that autoantibodies to U1-C are relatively un-
common by immunoblotting. Therefore, the sera used in the
current study also were tested by immunoblotting (Fig. 5, Ta-
ble I). To avoid the loss of U1-C during affinity purification,
the antigens were affinity-purified using anti-Sm mAb Y2 and
washed with NET/NP40 (cf. Fig. 1 A, lane 1). In agreement
with previous reports, antibodies to U1-70K, U1-A, and Sm-B?/B
were detected in 88% (69/78), 79% (62/78), and 97% (76/78),
respectively, of human anti-nRNP/Sm (?) sera. Antibodies to
Sm-D were found in 50% (12/24) of anti-Sm (?) patients.
However, despite the nearly universal existence of autoanti-
bodies to native U1-C in these samples, by immunoblotting an-
tibodies to U1-C were detected only in 33% (25/75) of human
sera with anti-nRNP antibodies (Fig. 5, Table I), in agreement
with previous studies. In addition, the reactivity with U1-C was
generally much weaker than that with U1-70K, U1-A or
Sm-B?/B. As observed previously (13–15), anti-U1-A and/or
U1-70K autoantibodies were detectable by immunoblotting in
many MRL mouse sera (Fig. 5, murine, lanes 1–8), but none of
sera were reactive with U1-C. Sera from BALB/c mice in-
jected with pristane contained antibodies mainly to U1-70K
and U1-A, however, and were unreactive with U1-C, including
the two anti-U1-C immunoprecipitation positive sera (Fig. 5,
These studies show that nearly all human sera recognizing U1
snRNPs contain autoantibodies to the native U1-C protein. In
contrast, such autoantibodies are absent in the sera of MRL/
lpr mice, suggesting that the patterns of autoimmune response
to U1 snRNPs are different in human and murine lupus. The
epitope(s) recognized by human anti-U1-C antibodies were
sensitive to MMB, but were stabilized by prior autoantibody
binding, consistent with recent observations that autoantibody
binding stabilizes the structure of other antigens (30). The sta-
bilization of antigen structure after antibody binding could
have consequences for antigen processing (34) and/or the pattern
of intermolecular-intrastructural spreading of autoimmunity.
Selective dissociation of U1-C from U1 snRNPs. Unlike the
nRNP-specific U1-70K and U1-A proteins, U1-C lacks an
RNA recognition motif (35, 36). Consequently, although the
association of U1-C with the U1 snRNP is dependent on U1
RNA, it does not bind directly to the RNA (29, 37). Instead,
U1-C associates with the U1 snRNP particle by interacting
with B?/B (29). MMB or 0.25 M MgCl2 selectively disrupted
this protein–protein interaction, releasing U1-C. The dissocia-
tion of U1-C from the U1 snRNP by MMB or MgCl2 allowed
us to detect autoantibodies to native U1-C (Fig. 4). This tech-
nique made it possible to identify an important difference in
the recognition of U1 snRNPs by human and murine autoim-
Anti-U1-C antibodies are characteristic of human, but not
murine, SLE. Immunoprecipitation of U1-C after MMB wash-
ing was restricted largely to human autoimmune sera and sera
from BALB/c mice with pristane-induced autoimmunity (Ta-
ble I). Out of 78 anti-nRNP/Sm (?) human sera, 75 were anti-
U1-C (?), in agreement with previous observations that anti-Sm
Figure 5. Immunoblot analysis of hu-
man and murine autoimmune sera.
U snRNPs from K562 cells were af-
finity purified and transferred to ni-
trocellulose membrane. Strips of the
membrane were probed with mAbs
2.73 (anti-U1-70K, lane a), 9A9 (anti-
U1-A and B??, lane b), 22G12 (anti-
Sm B?/B, lane c), Y2 (anti-Sm B?/B
and D, lane d), 2G7 (anti-Sm D, lane
e), or control mAb 162 (anti-Ku, lane
f), or human autoimmune sera with
anti-nRNP (human, lanes 1–6), anti-
nRNP plus anti-Sm (lanes 7–10), or
anti-Sm (lane 11) antibodies, or with
normal human serum (lane 12). Ad-
ditional strips were probed with
MRL/lpr mouse sera (murine, lanes
1–8), sera from pristane-treated
BALB/c mice (lanes 9–13), or anti-
nRNP/Sm negative MRL/lpr mouse
serum (lane 14). Positions of the
U1-70K, U1-A, U1-C, Sm-B?/B, and
Sm-D proteins are indicated on the
left. H, residual heavy chain from the
mAb used for affinity purification.
Immune Response to U1-C Protein
antibodies are usually accompanied by high levels of anti-
nRNP antibodies (38, 39). In contrast, only 1 of 65 sera from
mice with spontaneous lupus was anti-U1-C (?). Lack of reac-
tivity of murine sera with U1-C was confirmed by immunopre-
cipitation of biochemically purified U1-C (Fig. 4), arguing
strongly against the possibility that the murine sera recognize
epitopes of U1-C buried within the interior of the U1 snRNP.
However, the crossreactivity of mAb 22G12 (anti-B?/B, estab-
lished from an MRL/lpr mouse), with U1-C, along with the
equivocal immunoprecipitation of U1-C by some MRL/lpr
sera (data not shown), suggest that anti-U1-C reactivity may
be present at very low titer in some MRL/lpr sera. This is also
consistent with the previously reported crossreactivity of hu-
man autoantibodies to Sm B?/B with U1-C (40). The fact that
U1-C previously bound by autoantibodies was not released by
MMB, whereas U1-C generally could not be immunoprecipi-
tated efficiently after its release by MMB, suggests that the anti-
U1-C antibodies in human autoimmune sera stabilize the con-
formation of U1-C. However, the variable intensity of the U1-C
band in immunoprecipitates using the purified protein (Fig. 4
B) suggests that certain epitopes are partially dependent on its
association with the U1 snRNP particle.
Although autoantibodies to U1-C have been recognized
for some time in humans, their reported frequencies have been
inconsistent. In vitro translation and pulse labeling experiments
with a small number of sera suggest that anti-U1-C antibodies
are common in anti-nRNP (?) sera (4, 36), in agreement with
the present results. However, the frequency of antibodies to
U1-C by immunoblotting has been as low as 19% among anti-
nRNP/Sm (?) sera (9). The immunoblot data in the present
report (Fig. 5 and Table I) suggest that the low frequency of
anti-U1-C antibodies in previous publications is not a conse-
quence of the use of relatively U1-C-deficient substrates. The
low frequency of anti-U1-C antibodies in immunoblot assays is
more likely to reflect SDS-sensitivity of U1-C epitopes.
By double immunodiffusion, anti-Sm, rather than anti-
nRNP, is the main specificity seen in MRL/lpr mouse sera (2).
However, by immunoblotting, the frequencies of autoantibod-
ies to U1 snRNP specific subunits 70K (43%) and A (100%)
are as high as, or higher than, those of antibodies to the Sm
subunits B?/B and D (14, 15). Although the frequency of anti-
U1-C was not addressed in previous studies of the murine im-
mune response to U1 snRNPs, MRL/lpr sera did not exhibit
reactivity with U1-C in immunoblotting here (Table I) or in
other studies (14, 15), suggesting that autoantibodies to native
U1-C may be an important component of precipitating anti-
bodies to nRNP. It has been suggested that the initial autoim-
mune response to U1 snRNPs in MRL/lpr mice is focused on
U1-70K and U1-A, with subsequent intermolecular-intrastruc-
tural diversification leading to the production of anti-Sm B?/B
and D antibodies (14, 15). The present data indicate that, in
contrast to humans, antibodies to U1-C are extremely rare or
absent in MRL/lpr mice, despite the fact that the nRNP anti-
gen is a major target of the autoimmune response in both hu-
mans (38, 39) and mice (14, 15). The reason that the autoim-
mune response to U1 snRNPs “skips” U1-C in MRL/lpr mice
is not known. However, it is likely that anti-U1-C antibodies
appear early, since pristane-treated BALB/c mice were posi-
tive from the onset of anti-snRNP antibody production, and in
the rare case of a patient in whom the onset of anti-snRNP an-
tibodies was observed, anti-U1-C antibodies were also present
The striking difference in U1-C recognition between mice
and humans might reflect interspecies differences in the struc-
ture of U1 snRNPs that alter its immunogenicity. The primary
structure of murine U1-C is not known, and could differ from
that of the human homologue. Moreover, the murine U1
snRNP contains a single B protein, and the absence of B? might
alter its structure sufficiently to reduce the antigenicity of U1-C.
For instance, in the murine U1 snRNP, U1-C might be inacces-
sible to membrane bound Ig on antigen-specific B cells. Fi-
nally, differences in the posttranslational modification of U1-C
might reduce antigenicity of the murine protein. Pulse-chase
studies indicate that the mobility of human (K562 cell) U1-C
on SDS gels is reduced gradually over a period of ? 8 h (42).
In contrast, this mobility shift occurs very rapidly (? 1 h) in the
murine myeloma cell line SP2/0 (M. Satoh, unpublished data).
This difference between the human and murine U1 snRNP
may also affect antigenicity. However, it remains to be con-
firmed that the different rates of posttranslational modifica-
tion of U1-C reflect a general difference between human and
murine cells, or a property of the individual cell lines tested.
It is also possible that differences in genetic loci controlling
immune responsiveness (e.g., MHC antigens or allotypes), or
the effects of exogenous or endogenous infections or other en-
vironmental agents, explain the different patterns of U1-C rec-
ognition in human and murine lupus. The significance of im-
munoprecipitation of U1-C by a single MRL/lpr IgHe serum
and 2/10 sera from pristane-primed BALB/c mice is uncertain
at present. Previous studies suggest that Igh allotype influences
the production of anti-chromatin and anti-Sm antibodies in
MRL/lpr mice (27, 33). Thus, the IgHe allotype (or IgHa of
BALB/c mice), or products of genes in linkage disequilibrium
with the Igh locus, might be associated with enhanced anti-U1-C
antibody production. Further studies are needed to address this
question. Also, the importance of MHC in autoantibody pro-
duction has been suggested. For example, production of anti-
fibrillarin antibodies in mice treated with HgCl2 is restricted to
H-2s (43, 44). Therefore, the possibility that anti-U1-C antibody
production is associated with H-2d must also be considered.
In summary, the present study shows that autoantibodies to
the native U1-C protein are nearly universal in human anti-
nRNP/Sm (?) sera. This contrasts with the low frequency of
antibodies to U1-C reported previously, and reemphasizes the
importance of testing for autoantibodies to native, rather than
denatured, proteins. Analysis of the mechanisms responsible
for the different autoantibody diversification patterns to com-
ponents of snRNPs between human, MRL/lpr, and pristane-
treated BALB/c mice, may offer clues to understand the gen-
eration of immune responses to multiprotein complexes and
basis for the disease specificity of anti-Sm antibodies.
We thank Dr. Walther J. van Venrooij for providing mAbs, Ms.
Sylvia Craven for double immunodiffusion analysis, and Dr. Takashi
Ogasawara (Keio University, Tokyo, Japan) for helpful discussions.
This work was supported by grants AR42573, AR40391,
AR30701, AR40620, AR26574, AR34156, AR33887, and RR00046
from the United States Public Health Service.
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