From the Departamento de Bioquímica y Biología
Molecular, Facultad de Ciencias, Universidad de Cádiz, 11510 Puerto Real, Cádiz, Spain and the
Area de
Biología Molecular, Facultad de Ciencias Bioquímicas y
Farmaceúticas, Universidad Nacional de Rosario,
2000 Rosario, Argentina
We describe a new human autoimmune antigen in a
patient suffering from scleroderma with high levels of antibodies to
nucleolus and cytoplasmic antigens. Using a Chinese hamster ovary cell
expression library, we have shown that this antigen corresponds to the
autosomal Fragile-X-related gene FXR1. The deduced amino acid sequence
from the hamster cDNA is 97, 98, and 58% homologous to the human,
mouse, and Xenopus laevis FXR1 genes, respectively.
Expression of the hamster cDNA clone in Escherichia
coli and antibody production indicates unequivocally the location
of the FXR1 protein in the cytoplasm of hamster cells. Affinity
chromatography followed by immunofluorescence microscopy analysis and
immunoblots demonstrated the presence of autoimmune IgGs to FXR1 in the
scleroderma patient. Immunolabeling studies in Jurkat cells, induced to
apoptosis by anti-Fas/APO1 serum, indicated that the FXR1 antigens
were clearly displaced from their original cytoplasmic location to
several punctuated foci, resembling the bleb-like membranous structures characteristic of cells at certain stages of apoptosis. This phenomenon could be part of a putative mechanism in which the FXR1 protein is
presented as a target for the autoimmune response in humans.
 |
INTRODUCTION |
Human autoimmune sera have proven to be remarkably powerful
investigative tools for cell and molecular biologists. In addition to
allowing the identification of many cellular proteins, human autoantibodies have also been used to study the details of several intracellular events. One example has been the elucidation of the
mechanisms involved in the processing of heterogenous nuclear RNA to
mRNA (1). Thus, autoantibodies to ribonucleoprotein particles have
been observed in many patients suffering rheumatoid diseases (2-4).
Among others, these include anti-RNP directed against U1RNP (5),
anti-Sm directed against U1, U2, U4, U5, and U6 RNPs (6), anti-Ro/SS-A
directed against Y-RNPs (7), and anti/La/SS-B directed against several
cellular RNPs (8). These autoantigens are particularly prominent in
disorders such as systemic lupus erythematosus (SLE), scleroderma or
systemic sclerosis (SSc),1
and mixed connective tissue disease (MCTD) (9).
Expression of a variety of adhesion molecules is increased on
fibroblasts and endothelial cells in SSc patients, as are circulating levels of the same molecules. In some cases, genetic factors influence the repertory of autoantigens to which the host loses tolerance (10).
When Hep-2 cells are used as substrate, virtually every patient with
scleroderma (95-97%) will have a positive test result for antinuclear
and/or antinucleolar antibody (ANAs) in indirect immunofluorescence
(IF) (11). In particular, nucleolar IF has been suggested as a
diagnostic possibility for SSc since antibodies that target this region
of the cell are unusual in the other connective tissue diseases
(3).
In an attempt to characterize scleroderma autoantigens, we have
screened sera that produced nucleolar staining by immunofluorescence from a large number of patients suffering autoimmune diseases. Here we
describe the identification and molecular characterization of a hamster
cDNA for a novel autoantigen that turned out to be FXR1, an
autosomal homolog of FMR1. Mutations and CGG expansions in the FMR1
gene are associated with Fragile-X syndrome. Immunological studies
presented reveal that the behavior of the FXR1 protein in cells
undergoing apoptosis suggests a putative way for this protein to be
implicated in the human autoimmune response (12).
 |
EXPERIMENTAL PROCEDURES |
Materials--
Restriction enzymes were obtained from Promega
and Amersham Pharmacia Biotech. All radionucleotides were purchased
from Amersham Pharmacia Biotech. All tissue culture reagents were from
Life Technologies, Inc. Oligonucleotides were obtained from Eurogentec, Belgium. Antibodies were purchased from Boehringer Mannheim.
ZAP II
hamster expression library was obtained from Stratagene (La Jolla, CA).
CNBr-activated Sepharose was obtained from Amersham Pharmacia Biotech.
All other chemicals were of the highest grade available and obtained
from Sigma.
Immunofluorescence Microscopy--
Immunofluorescence was
carried out on CHO, HeLa, and Jurkat cells using formaldehyde fixation
and a brief Triton X-100 lysis as described (13). Rabbit anti-FXR1 sera
was generated as indicated below and used at a dilution of 1:500 in
PBS. In some cases, affinity purified human autoimmune IgGs was used as
a primary serum. Detection of the primary antibodies was with
fluorescein isothiocyanate-conjugated secondary antibodies made in goat
and used at 1:100 dilution in PBS. Coverslips were counterstained with
Hoechst 33528 for DNA, mounted in PBS:glycerol 1:9 (v/v), and observed
in a Zeiss Axiophot fluorescence microscope equipped with a 1:63
immersion oil objective.
Isolation of cDNA and Sequencing--
3 × 105 plaques of
ZAP hamster cDNA library were
screened to obtain FXR1 cDNAs using a human autoimmune serum named
JC. Three positives clones sequenced were shown to be highly homologous to the human FXR1 gene (14). Since no initial clones contained the
entire open reading frame encoding FXR1, a 5'-RACE approach was used as
described. All plasmids and cDNA fragments were manipulated using
standard techniques (15). The nucleotide sequence of overlapping clones
was verified by DNA sequencing (16).
Expression of Hamster FXR1 cDNA and Antisera
Production--
An N-terminal region (residues 1-222) of the hamster
FXR1 cDNA was expressed by the pET-3a system in Escherichia
coli using an NdeI-site at the 5' end and a
BamHI-site at the 3' end included in the polymerase chain
reaction primers used for cloning. The expressed protein (34 kDa) was
analyzed by SDS gel electrophoresis, isolated from inclusion bodies
after cell sonication, and used as immunogen for polyclonal antibodies
production in rabbits. Western blots were performed with recombinant
truncated FXR1 and CHO whole cell extracts with both human autoimmune
serum JC (diluted 1:200) and rabbit anti-FXR1 sera (diluted 1:500).
Detection of the primary antibodies was with peroxidase-conjugated
secondary antibodies used at 1:2000 dilution in PBS. Development of the blot membranes was with 4-chloro-1-naphthol.
Affinity Chromatography of Human Autoimmune Anti-FXR
Immunoglobulins--
The FXR1 expressed polypeptide was bound to
CNBr-activated Sepharose according to the instructions of the
manufacturer. The resin was washed with PBS and equilibrated with PBS
containing 0.5 mM NaCl (final concentration). 3 ml of human
autoimmune serum JC containing anti FXR1 IgGs were loaded into the
affinity column (resin volume was 1.5 ml), washed extensively with
PBS-NaCl buffer, and eluted with 0.2 M glycine, pH 2.5. Aliquots of 250 µl were collected and equilibrated immediately with 1 M Tris, pH 8.0. IgGs were confirmed in the eluate by
SDS-gel electrophoresis and used for immunofluorescence microscopy and
Western blot analysis without further treatment.
Northern Blot Analysis--
Total RNA from CHO culture cells was
extracted using the acid guanidinium thiocyanate/phenol-chloroform
extraction method (17). RNA samples were separated by electrophoresis
in formaldehyde, 1% agarose in MOPS buffer. Northern blotting (15) was
carried out using a polymerase chain reaction fragment obtained from
the largest clone of the hamster FXR1 cDNA, representing
nucleotides 1-666.
 |
RESULTS |
Cloning and Expression of Hamster FXR1--
We have used a human
autoantibody from a scleroderma patient to screen a CHO cDNA
expression library and have isolated clones corresponding to the
hamster FXR1 gene. The largest CHO FXR1 cDNA characterized is 2141 nucleotides long, encoding a 621 amino acid chain (Fig.
1). A 5'-untranslated sequence region CGG
repeat was not found in the sequenced clone. This characteristic CGG
repeat was described before as part of the human FMR1 cDNA (18),
but it is not known if it is present in the human FXR1 cDNA (19). Overall, we found a 97, 98, and 58% homology of amino acid sequence between hamster FXR1 and human, mouse, and frog FXR1 genes,
respectively (Fig. 2).

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 1.
Sequence of the hamster cDNA for
FXR1. The nucleotide sequence of the FXR1 with deduced amino acid
sequence underneath is shown. A putative stop codon at position
1880-1882 is indicated by asterisk. DNA and deduced protein sequence
are in the GenBankTM/EBI Data Bank (accession number
Y12387).
|
|

View larger version (73K):
[in this window]
[in a new window]
|
Fig. 2.
Alignment of hamster (CG) FXR1
amino acid sequence with human (HS), mouse
(MM), and Xenopus laevis (XL) FXR1
proteins. Identical amino acids are shaded. The frog
protein contains 29 extra residues not found in the three mammalian
species (gap). Major amino acid residue variants are found
in three of the proteins at the C-terminal tail. Note that the sequence
of the mouse FXR1 gene represented is a shorter form of the gene
(21).
|
|
The highly conserved N-terminal region contains two KH domains that
represent RNA binding elements (18). The hamster FXR1 gene also
contains an arginine-rich sequence similar to arginine-rich motifs
found in several proteins including HIV Rev (20) as described previously for other FXR1 genes. Major differences in amino acid sequences between the hamster and FXR1 genes from other species are
found at the very end of the C-terminal region (residues 590-618). It
has to be mentioned that the sequence of the mouse FXR1 gene available
represents a shorter form of this gene (21).
The hamster cDNA was used in a Northern blot analysis with
total RNA from CHO cells (Fig. 3). A
prominent transcript of about 2.3 kilobases was detected, which
correlates well with that described in HeLa cells (14). However, our
blot result does not rule out the possibility of additional FXR1
transcripts with close sizes in CHO cells, as was shown previously for
human and mouse tissues, where two FXR1 messengers of 2.4 and 2.2 kilobases were identified as a result of an alternative splicing (14,
21). The hamster FXR1 cDNA was further used to express the N
terminus of the molecule in E. coli, and the expressed
polypeptide was purified to a high concentration from inclusion bodies.
Two polyclonal sera were generated against recombinant FXR1 protein and
used in apoptosis studies (see below).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3.
Expression of FXR1 in CHO cells. Shown
is the total hamster RNA hybridized in Northern blots with FXR1
cDNA showing a prominent 2.3-kilobase mRNA signal.
|
|
FXR1 Is a Human Autoantigen--
We present immunological evidence
that the human autoimmune serum JC used in the screening of the
ZAP
library reacts with the expressed hamster FXR1 antigen (Fig.
4A, center panel).
Data included in the same figure also show the blotting with a
polyclonal serum generated against the recombinant N-terminal region of
expressed hamster FXR1 (Fig. 4B, right panel).
Furthermore, this recombinant truncated FXR1 protein was used to make
an affinity column to isolate human specific anti-FXR1 immunoglobulins.
It is important to state that FXR1 does not appear to be the major
autoantigen found in the serum JC. This was demonstrated by immunoblots
in CHO whole cell extracts using human serum JC (Fig. 4B,
center panel, lane 1), anti-FXR1 affinity
purified human immunoglobulins (Fig. 4B, center panel,
lane 2), and polyclonal serum to hamster FXR1 (Fig. 4B,
right panel, lane 1). These results clearly indicate that:
(i) a 70-kDa protein corresponding to the hamster FXR1 antigen is
recognized by the human autoantibody, and ii) it is not the major
autoantigen of the human serum JC. Another protein of 55 kDa is the
autoantigen most probably responsible for the predominant nucleolar
staining observed by IF with the whole human autoantibody JC (data not
shown).

View larger version (91K):
[in this window]
[in a new window]
|
Fig. 4.
Expression of recombinant FXR1 and polyclonal
serum production. A, left, Coomassie Blue
gel staining of expression of hamster FXR1 from uninduced cells
(lane 1),
isopropyl-1-thio- -D-galactopyranoside-induced cells
(lane 2), and renaturated FXR1 polypeptide from inclusion
bodies (lane 3). A, center, Western
blot of the gel on the left with human autoimmune serum JC.
This serum reacts with the expressed 34-kDa truncated FXR1 polypeptide
(arrowhead). A, right, immunoblot of
proteins shown on the left, with rabbit polyclonal serum
generated against the recombinant 34 kDa FXR1 polypeptide
(arrowhead). B, left, Coomassie blue
staining of a CHO cell extract (lane C). B,
center, Western blot of hamster protein extract with human
autoimmune serum JC (lane 1) and anti-FXR1-affinity purified
immunoglobulins from human JC autoimmune serum (lane 2).
Note that the major autoantigen recognized by this serum is a 55-kDa
antigen (arrow), but other autoantigens are also blotted,
including a CHO 70-kDa protein (arrowhead). B,
right, immunoblot of the CHO protein extract of the gel on
the left with rabbit polyclonal anti-FXR1 serum. A similar 70-kDa
protein corresponding to the hamster FXR1 is recognized by this serum
(arrowhead). M, indicates molecular markers
corresponding to 200, 96, 68, 50, 36, 30, 14, and 6 kDa.
|
|
Furthermore, immunofluorescence microscopy of culture cells with
affinity-purified human anti-FXR1 IgGs revealed a punctate cytoplasm
staining in CHO (Fig. 5b) and
HeLa cells (Fig. 5f). This IF staining was similar to that
observed using rabbit antibodies to the recombinant FXR1 hamster
protein generated in vitro (Fig. 5, d and
h). Eventually, some weak nuclear staining was observed with
the rabbit anti-FXR1 sera. Double IF staining demonstrated unambiguously that the serum from patient JC contains antibodies that
co-localized with the serum anti-FXR1 generated in rabbits (data not
shown).

View larger version (72K):
[in this window]
[in a new window]
|
Fig. 5.
Immunofluorescence location of human
autoantigen JC. Affinity purified human autoimmune IgGs against
FXR1 show cytoplasm staining on CHO (b) and HeLa cells
(f). Note that some weak staining was occasionally detected
in a restricted area of some HeLa nuclei. A similar IF location was
seen in CHO (d) and HeLa cells (h) with a rabbit
polyclonal serum generated in vitro against expressed FXR1.
Hoechst staining of the same cells is also shown (a,
c, e, and g). HeLa cells in
g and h are shown at higher magnification for
better detail. Original magnification: × 40 (a-d), × 63 (e, f), and × 100 (g, h).
|
|
FXR1 Is a Autoantigen Redistributed in Cells Undergoing
Apoptosis--
It has been suggested that some mechanistic correlation
may exist between the autoimmune phenomenon and apoptosis (12). It was
proposed that the selective protein cleavage associated with apoptosis
may enhance the immunogenicity of autoantigens by revealing
immunocryptic epitopes that are not efficiently generated during
antigen processing (22). In order to determine the behavior of the FXR1
protein in cells undergoing apoptosis in vitro, we did
immunofluorescence studies of FXR1 localization in Jurkat cells treated
with anti-Fas/APO-1 serum (23). The IF results in Fig.
6 with anti-FXR1 serum showed the
re-distribution of the FXR1 cytoplasmic antigen to certain specific
foci on apoptotic cells. These foci resembled those bleb-like
structures observed during the apoptosis phenomenon (12, 22).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 6.
Immunofluorescence analysis of FXR1 in Jurkat
cells induced to apoptosis. FXR1 distribution on uninduced Jurkat
cells shows a cytoplasm staining (b). Induction with
anti-Fas/APO-1 serum to apoptosis clearly caused a redistribution of
the FXR1 antigen to discrete foci in treated cells (d,
f, h), as shown by IF with human autoimmune IgGs
to FXR1 (d) and two rabbit polyclonal sera to recombinant
FXR1 (f, h). In some apoptotic cells (detail in
h), the FXR1 was distributed to the periphery in bleb-like
structures, characteristic of membrane changes observed during the
process of apoptosis (arrows). Magnification, × 63.
|
|
 |
DISCUSSION |
In this report, we have identified and described a new human
autoantigen in a patient with scleroderma, by cloning the gene from a
CHO cDNA expression library. The cDNA revealed an amino acid
sequence homologous to the human autosomal gene FXR1, a gene related to
the FMR1 protein responsible for the Fragile-X syndrome in humans
(24-26). This syndrome is the most common inherited form of mental
retardation. The human, mouse, and frog cDNAs for FXR1 have been
previously sequenced (14, 21, 27). The major differences in amino acid
sequences we have found between hamster and the other FXR1 cDNAs
isolated are in the final 30 amino acid residues of the C-terminal
region. This region as described for the FMR and FXR protein family is
produced by alternative splicing and introduces potential functional
differences between the gene products. These proteins have similar RNA
binding activities in vitro, and both are located in the
cytoplasm being the RNA 60 S, the putative binding site for FXR1, as
demonstrated by biochemical fractionation in vitro (28).
However, some previous evidence from other groups, and our own IF
analysis, do not rule out an additional nuclear binding site for the
FXR1 antigen during the cell cycle.
By expressing the hamster FXR1 cDNA and the production of specific
anti-FXR1 antibodies, we demonstrated the autoimmune nature of the FXR1
gene product in a patient suffering from scleroderma. As one of the
group of related systemic rheumatic diseases, scleroderma is
characterized by a high level of circulating autoantibodies, especially
to nuclear antigens (4, 29). The observation that FXR1 protein is a
human autoantigen raises questions of possible association between FXR1
(and possibly FMR1) and autoimmune responses in humans. Activation of
the immune system is a consistent early event in SSc, and some
autoantibodies may be generated in SSc patients through the antigen
processing of complexes involved in gene transcription. The generation
of autoantibodies and subsequent tissue deposition of immune complexes
is thought to trigger the pathogenic consequences of systemic
autoimmune disease.
The origin of autoimmune diseases is not yet known but several
hypotheses have been put forward and clues have been found (30). Our IF
staining results indicated that the FXR1 antigen behaves like other
human nuclear autoantigens analyzed (12). It moves from its original
cytoplasmic binding site associated with ribosomes to more punctuated
foci. These new locations resemble the typical bleb-like membrane
organelles found in apoptotic cells. Although the significance of this
change of location and the putative cleavage of FXR1 during apoptosis
requires more specific studies, the re-distribution of the FXR1 antigen
in apoptosis follows a pattern that could contribute to the nature of
the autoimmune response (12, 22). If this is the case for FXR1, it
would be interesting to pursue more extensive studies to gain a better understanding of the nature of the autoimmune response to a gene related to a genetic disorder such as the Fragile-X syndrome. With that
in mind, studies to identify the human autoepitope(s) in the FXR1
protein are underway.
We thank the members of Servicio de
Inmunología of Hospital Puerta del Mar of Cádiz for
assistance with the apoptosis studies and Royston Snart for language
advice during elaboration of the manuscript.