From the Departamento de Microbiología,
Facultad de Biología, Universidad de Sevilla, Apdo. 1095, 41080 Sevilla, Spain and the ** Institut Curie, Section Recherche, UMR
144 du CNRS, 26 rue d'Ulm, 75248 Paris, Cedex 05, France
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ABSTRACT |
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The serum from a patient with Sjögren's
syndrome (RM serum) was used to screen a human testis cDNA
expression library. A cDNA of 865 base pairs containing the entire
coding sequence for a novel protein was isolated. The 14-kDa predicted
protein contains an acidic domain (amino acids 6-80) with a high
frequency of heptad repeats characteristic of -helices that form
dimeric coiled-coil structures and an alkaline carboxyl-terminal domain
(amino acids 81-119). It seems to be widely expressed, but its
expression level varies depending on tissues. A protein of apparent
molecular mass of 14 kDa was immunoprecipitated from cell lysates by
the autoimmune serum, and it was recognized by rabbit antibodies raised
to a recombinant bacterial fusion protein generated from the cDNA
clone. Conventional and confocal immunofluorescence microscopy on HeLa and 3T3 cells transiently transfected with a tagged form of the protein
showed numerous punctate structures scattered throughout the nucleus.
This novel protein has been termed NA14 for Nuclear Autoantigen of 14 kDa.
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INTRODUCTION |
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Human autoantibodies are valuable reagents for identification and study of novel cellular constituents since they usually recognize highly conserved components, which otherwise are difficult to detect due to their low immunogenicity. In addition, the autoantibodies usually react with the active sites of the antigen, and therefore, they are often capable of inhibiting its functional activity (1).
In a number of rheumatic diseases, the presence of the so-called antinuclear antibodies is a dominant feature. These diseases include systemic lupus erythematosus, mixed connective disease, polymyositis, scleroderma, and Sjögren's syndrome (2).
Human autoimmune sera have been used to characterize and identify several nuclear proteins including La antigen (3), centromere protein B (CENP-B) (4), Ku70 and Ku86 (5, 6), Sp-100 (7), PM-Scl (8), Mi-2 (9), and Ngp-1 (10).
RM serum, from a patient with Sjögren's syndrome, contains high titer autoantibodies to the Golgi apparatus and low titer autoantibodies to the nucleus (11). Western blotting and immunoprecipitation experiments demonstrated that this serum shows multiple autoreactivities and recognizes three major bands of 210, 130, and 45 kDa (p210, p130, and p45, respectively) in HeLa cell extracts. p210 was shown to be localized at the Golgi complex (11). We have recently cloned and characterized the cDNA encoding p210, now called GMAP-210, by immunoscreening of a HeLa cell cDNA library.1
In the present study, we used RM serum to screen a human testis oligo(dT) cDNA library, and we isolated a clone encoding a novel protein of 14 kDa, termed NA14, that localizes to the nucleus. The analysis of its amino acid sequence revealed the presence of a domain probably involved in the formation of a coiled-coil structure and sequence similarity with a number of proteins with coiled-coil motifs.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Lysis-- Jurkat cells were grown in RPMI 1640 medium (Bio-Whittaker) supplemented with 10% heat-inactivated FCS.2 HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS. Mouse NIH3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum. 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin were included in all culture media. All cells were maintained in a 5% CO2 humidified atmosphere at 37 °C.
For cell lysis, cells were washed and harvested in phosphate-buffered saline. 2 × 107 to 108 cells per ml were lysed at 4 °C in Nonidet P-40 buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml pepstatin, leupeptin, and aprotinin) for 20 min. The extract was centrifuged at 20,000 × g for 20 min, and the supernatant was stored atAntibodies--
RM serum of the patient suffering from
Sjögren's syndrome (12, 13) was aliquoted, had sodium azide
added, and then stored at 70 °C. IgG fraction (10 mg/ml final
concentration) was purified from whole serum on protein A-Sepharose
columns and stored in 50% glycerol at
70 °C. Specific antibodies
from RM serum were affinity purified on nitrocellulose strips using
immunoreactive proteins from total cell extracts as described (14).
cDNA Cloning and Sequence Analysis--
RM serum diluted
1:1500 in TBST (10 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 0.1% Tween 20) containing 5% non-fat dry milk was used for
immunoscreening of 5 × 105 recombinants from a human
testis oligo(dT)-primed cDNA library in gt11. Positive clones
were rescreened and plaque-purified. Two positive clones (
5 and
10) were isolated and subcloned into pTZ19R plasmid. The entire
10 EcoRI insert was used as a probe to hybridize against
a HeLa random primed cDNA
ZAPII library. The clones obtained
were isolated and subcloned in vivo into pBluescript plasmid
using R408 helper phage as described in the manufacturer instructions
(Stratagene). The nucleotide sequence was determined by using an
automated sequencer from Pharmacia Biotech Inc.
Preparation of GST Fusion Proteins and Rabbit Anti-autoantigen
Fusion Protein Antibodies--
A fusion protein containing the
complete EcoRI insert from 10 was constructed at the
carboxyl terminus of GST using the prokaryotic expression vector
pGEX-4T-1. Plasmids were transformed in Escherichia coli
strain DH5
. Expression of the GST fusion proteins was induced by the
addition of 1 mM
isopropyl-
-D-thiogalactoside, and the fusion proteins
were isolated from bacterial lysates by affinity chromatography with
glutathione-agarose beads (Sigma). NA14 was released from the fusion
protein by treatment with 1% (w/w) thrombin in cleavage buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2.5 mM CaCl2) for 2 h at 25 °C. A
polyclonal antibody was generated in rabbit using the entire GST-NA14
fusion protein. The antiserum obtained after the fourth immunization,
named anti-NA14, was used in the experiments described.
Northern Blotting--
A poly(A)+ RNA blot from
CLONTECH was hybridized with the
32P-labeled cDNA insert from clone 10 and washed as
described in the manufacturer instructions.
Electrophoresis and Immunoblot Analyses-- Proteins were separated by SDS-PAGE on 15% acrylamide gels (21) and stained with Coomassie Brilliant Blue. Gels were electrophoretically transferred to nitrocellulose filters (22), and immunoblot analyses were carried out as described (11).
Immunoprecipitation-- HeLa and Jurkat cell lysates (1.5 × 107 cells) were incubated with 5 µl of RM serum, normal human serum, or an irrelevant human autoimmune serum for 2 h at 4 °C, with agitation. 50 µl of a 50% solution of protein A-Sepharose was added, and incubation was continued for 1 h. Then the protein A-Sepharose beads were collected and washed five times with Nonidet P-40-buffer. Proteins were released from the beads by boiling in SDS-PAGE sample buffer and analyzed by immunoblotting.
Transient Transfection of HeLa and 3T3 Cells--
The
full-length open reading frame of na14 (EcoRI
insert from 10) was cloned in-frame with the HA epitope (23) into
the eukaryotic expression vector pECE (24) to obtain an HA
epitope-tagged NA14. The resulting plasmid, pECE-HA-NA14, was purified
with a plasmids kit from Qiagen followed by phenol extraction and
ethanol precipitation. HeLa or 3T3 cells were split 24-48 h before
transfection so that they were 60-80% confluent on the day of
transfection. 5 × 106 cells per assay were
resuspended in 200 µl of 15 mM HEPES-buffered serum-containing medium, mixed with 50 µl of 210 mM NaCl
containing 20-40 µg of plasmid DNA, and electroporated at 240 V and
950 microfarads using a Bio-Rad Gene Pulser. 12 h after
electroporation, medium was replaced by fresh medium, and cells were
processed for Western blotting or immunofluorescence staining after
24 h.
Immunofluorescence Microscopy--
Cells were grown on
culture-treated slides for 24 or 48 h before an experiment. Cells
were rinsed twice with phosphate-buffered saline and incubated in
methanol at 20 °C for 6 min to simultaneously fix and permeabilize
the cells. After methanol treatment, cells were processed as described
(11). Conventional immunofluorescence analysis was performed on a Leica
epifluorescence microscope. Confocal laser scanning microscopy was
performed using a Leica CLSM instrument based on a Leitz Diaplan
microscope with a plan-Neofluar objective × 100. A series of
optical sections at 0.25 µm intervals were recorded.
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RESULTS AND DISCUSSION |
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Cloning of na14 cDNA--
When serum RM was used to screen
5 × 105 plaques from a human testis gt11
oligo(dT)-primed cDNA expression library, two clones (
5 and
10) were isolated. The cDNA inserts were subcloned into pTZ19R
and sequenced. The two clones contained the poly(A) tail as expected
and gave identical sequences in the overlapping regions, clone
5
being 14 bp longer than
10 and containing an 865-bp insert. Analysis
of the sequence showed that it was not part of GMAP-210
cDNA.1 In addition, affinity purified antibodies from
p130 and p45 did not show staining of the phage plaques. From these
data, we concluded that the clones obtained did not correspond to any
of the three major proteins recognized by the autoimmune RM serum (11).
To obtain an additional sequence, a screening of a
ZAPII HeLa random primed cDNA library was performed using the insert from clone
10
as a probe. Two clones (
Z2 and
Z20) were obtained, and the cDNA inserts were subcloned into pBluescript and sequenced. Both inserts were contained in the sequence present in
5, and no new sequence was obtained (Fig.
1A).
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Sequence Analysis--
As shown in Fig. 1B, the entire
sequence comprises 865 bp and contains an open reading frame of 360 bp.
The first ATG, at positions 47-49, is in excellent agreement with
Kozak's sequence for eukaryotic translation initiation (25). The open
reading frame is terminated by a stop codon at positions 404-406. A
polyadenylation signal AATAAA (26) is found at positions 837-842 in
the 3-untranslated region, and a poly(A) tail is present at the end of
the sequence. These data confirm that the complete coding sequence was
present in
5 and
10.
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Tissue Distribution of NA14--
To investigate the expression of
na14, Northern blot analysis of mRNAs from different
human tissues was performed with 10 insert as a probe. As seen in
Fig. 2, the highest level of expression is observed in testes although
na14 can be detected in all the tissues assayed. This result
agrees with the data summarized in Table I.
Polyclonal Antiserum Production and Analysis of Expression of NA14
by Western Blotting and Immunoprecipitation--
The entire insert
from 10 clone was subcloned into a pGEX-4T-1 bacterial expression
vector. A GST fusion protein of the expected molecular mass of 41 kDa
was produced. This protein was recognized by RM autoimmune serum (Fig.
3A, left panel). To show the
specificity of the recognition, the fusion protein was cut with
thrombin to generate GST and NA14. RM serum only recognized the NA14
moiety (Fig. 3A, left panel).
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Transfection and Subcellular Localization-- Subcellular localization of endogenous NA14 was examined in several cell lines by indirect immunofluorescence with anti-NA14 serum, but no staining pattern could be easily recognized (not shown), consistent with the low level of the protein shown by immunoblot analysis (Fig. 3). This prompted us to investigate the subcellular location of NA14 in transfected cells. An HA-tagged form of NA14 was subcloned into the eukaryotic expression vector pECE. Plasmid pECE-HA-NA14 was introduced into human HeLa cells and mouse 3T3 cells by electroporation. Expression of the protein was assessed by Western blotting (Fig. 4, a and f). HeLa and 3T3 cells transiently transfected with HA-NA14 were examined by indirect immunofluorescence microscopy with anti-HA antibodies, anti-NA14 polyclonal antibodies, and RM serum (Fig. 4). All the antibodies, in both HeLa (Fig. 4, b-e) and 3T3 cells (Fig. 4, g-j), revealed the same dotted pattern that localized exclusively to the nucleus. This stain was completely removed by treatment with 0.5% Triton X-100 for 1 min at 37 °C (not shown).
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ACKNOWLEDGEMENTS |
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We are indebted to Drs.
Carré-Eusèbe and Picard (Paris, France) for the generous
gift of oligo(dT)-primed gt11 library and Dr. Chambon (Strasbourg,
France) for random primed
ZAPII library.
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FOOTNOTES |
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* This work was supported by the Junta de Andalucía, by the Ministerio de Educación y Ciencia of Spain (SAF96-0275), and by Institut Curie, France.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Z96932.
§ Contributed equally to this work.
¶ Supported by a return grant of the TMR program from the European Union.
Supported by a Fundación Cámara fellowship from
the University of Seville.
Recipient of a Poste Rouge from the CNRS (UMR 144) and to whom
correspondence should be addressed. Tel.: 34-5-4557120; Fax: 34-5-4557830; E-mail: rmrios{at}cica.es.
1 C. Infante, F. Ramos-Morales, C. Fedriani, M. Bornens, and R. M. Rios, submitted for publication.
2 The abbreviations used are: FCS, fetal calf serum; ECL, enhanced chemiluminescence; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; PKC, protein kinase C; bp, base pair(s).
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REFERENCES |
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