Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec...
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Clin Exp Immunol 2000; 121:364±374
Isolation and characterization of cDNA encoding the antigenic protein of the
human tRNP(Ser)Sec complex recognized by autoantibodies from patients with
type-1 autoimmune hepatitis
M. COSTA, J. L. RODRIÂGUEZ-SAÂ NCHEZ, A. J. CZAJA* & C. GELPIÂ Department of Immunology, Sant Pau Hospital,
Barcelona, Spain, and *Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA
(Accepted for publication 31 March 2000)
SUMMARY
We previously described autoantibodies against a UGA serine tRNA±protein complex (tRNP(Ser)Sec) in
patients with type-1 autoimmune hepatitis [1] and now define the specificity and frequency of this
autoantibody and the DNA sequence encoding the tRNA(Ser)Sec-associated antigenic protein. The
presence of anti-tRNP(Ser)Sec antibodies was highly specific for type-1 autoimmune hepatitis, as 47´5%
of patients were positive compared with none of the control subjects. To characterize the antigenic
protein(s), we immunoscreened a human cDNA library with anti-tRNP(Ser)Sec-positive sera. Two clones
(19 and 13) were isolated. Clone 19 encodes a protein with a predicted molecular mass of 48´8 kD.
Clone 13 is a shorter cDNA, almost identical to clone 19, which encodes a 35´9-kD protein. Expression
of both cDNAs was accomplished in Escherichia coli as His-tagged recombinant proteins. Antibodies
eluted from both purified recombinant proteins were able to immunoprecipitate the tRNA(Ser)Sec from a
HeLa S3 cell extract, demonstrating their cross-reactivity with the mammalian antigenic complex.
Recent cloning data relating to the target antigen(s) of autoantibodies in autoimmune hepatitis patients
that react with a soluble liver antigen (SLA) and a liver-pancreas antigen (LP) have revealed that these
two autoantibodies are identical and that the cloned antigen shows 99% amino acid sequence homology
with tRNP(Ser)Sec.
Keywords tRNP(Ser)Sec UGA suppressor tRNA±protein complex autoantibodies autoantigen
autoimmune hepatitis
INTRODUCTION
Autoimmune hepatitis defines a subgroup of chronic liver diseases
of unknown cause and encompasses a heterogeneous group of
syndromes in which patients appear to lose immunological
tolerance to the liver [2,3]. Many autoantibodies have been
described in autoimmune hepatitis [1,4,5] and some define
patients with distinctive clinical, laboratory and prognostic
features [1,6±9]. Seropositivity for anti-nuclear antibodies
(ANA) and/or smooth muscle antibodies (SMA) characterizes
patients with type 1 autoimmune hepatitis (AIH), whereas
seropositivity for antibodies to liver/kidney microsome type 1
(anti-LKM1) typifies patients with type 2 AIH [10]. As the
presence of SMA and/or ANA has no prognostic value [11], new
markers should be investigated to characterize further these
subtypes of AIH. Patients in each of these subgroups have
mutually exclusive autoantibodies with different clinical mani-
festations, genetic associations [12] and responses to therapy [13].
Antibodies anti-serine tRNA±protein complexes (tRNP(Ser)Sec)
were described in an earlier paper [1] in a subgroup of patients with
type-1 AIH, which is recalcitrant to corticosteroid therapy. These
antibodies precipitate a 90-nucleotide RNA from human whole cell
extracts and recognize a 48-kD polypeptide in immunoblot assays
[1]. The RNA is a UGA suppressor serine tRNA (tRNA(Ser)Sec)
(where Sec is selenocysteine) as shown by sequence analysis,
and it functions in the pathway of selenoprotein synthesis in
human cells. This tRNA is a requisite for the co-translational
incorporation of selenocysteine into growing polypeptide chains
[14]. The insertion of selenocysteine is directed by certain UGA
triplets, which in other contexts act as termination codons. In
brief, a specialized tRNA (tRNA(Ser)Sec) is initially charged with
serine to form seryl-tRNASec, and is then converted to
selenocysteyl-tRNASec by the action of a selenocysteine
synthase, a selenophosphate synthetase, and factors not yet
clearly defined. Moreover, a tRNASec-specific elongation factor,
performing the function executed by the elongation factor Tu
364 q 2000 Blackwell Science
Correspondence: Carmen GelpõÂ, Department of Immunology, Sant Pau
Hospital, Avgda Sant Antoni Ma Claret 167, Barcelona 08025, Spain.
E-mail: [email protected]
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for all other aminoacyl-tRNAs, is required for the synthesis of
selenoproteins [15]. As described in prokaryotes, strong evidence
indicates the existence of a translational elongation factor in
eukaryotes for insertion of selenocysteine into protein [14,16]. The
antigenic 48-kD protein associated with the UGA suppressor tRNA
may be a selenocysteine-specific elongation factor, or an enzyme
involved in the conversion of seryl-tRNA(Ser)Sec to selenocysteyl-
tRNA(Ser)Sec, some other unknown SECIS (selenocysteine-inser-
tion sequence)-binding protein (SBP), or another unknown factor
acting in the selenocysteine insertion pathway. In order to elucidate
the precise nature of this antigenic protein and its relationship with
some of these previously described factors, we cloned, sequenced
and expressed the cDNA of the 48-kD antigenic protein recognized
by autoantibodies from patients with type-1 AIH.
PATIENTS AND METHODS
Sera
Fifty-nine patients who satisfied international criteria for the
diagnosis of autoimmune hepatitis [17] were selected from 303
patients in the chronic hepatitis treatment programme of the Mayo
Clinic because they fulfilled the following additional criteria: (i)
all patients had been screened for the serologic markers of
hepatitis B and C virus infection by second generation assays and
had been found negative [7,8]; (ii) ANA (68%) and/or SMA
(88%) had been demonstrated in each patient at admission, and the
presence of one or both markers had justified their designation as
type-1 AIH [11]; (iii) the observation period ranged from 7 to
348 months (mean 125´6 months). The mean age at diagnosis of
AIH was 39 years (range 16±68 years); (iv) all patients had
received immunosuppressive therapy consisting of prednisone
monotherapy (n � 17) or combination therapy consisting of
azathioprine and prednisone (n � 42) according to previously
published protocols [18]. All patients were participants in a
research programme that had been approved by the Institutional
Review Board of the Mayo Clinic. Patients were evaluated at
presentation and were followed in a uniform fashion in
accordance with a pre-established protocol [18]. Complete
examinations were performed every 6 months during and
immediately after treatment and then at annual intervals if the
clinical condition was stable. During a follow up of approximately
10 years, eight patients died of liver failure. Each treatment had
been shown previously to be equally effective in the management
of severe type-1 AIH and superior to placebo or non-steroidal
regimens [19]. The average duration of treatment was
27 ^ 3 months.
Liver tissue was obtained by needle biopsy in all patients at
the time of presentation. Additional assessments were made as
indicated to document histological remission or to clarify clinical
status. Specimens were interpreted under code and the diagnosis
of cirrhosis required fibrosis and the presence of a complete
regenerative nodule. The histological designations of interface
hepatitis, bridging necrosis and multilobular necrosis required
satisfaction of previously published criteria [20]. Moderate to
severe interface hepatitis was the most advanced histological
pattern at presentation in 25 (42%) patients; bridging necrosis was
present in six (10%) patients; multilobular necrosis in 10 (17%)
patients; and cirrhosis in 18 (30´5%) patients.
As control subjects, we studied 15 patients with type-2 AIH,
10 patients with chronic hepatitis B, 44 patients with chronic
hepatitis C; 20 patients with anti-M2 antibody-positive primary
biliary cirrhosis (PBC), three patients with primary sclerosing
cholangitis who were positive for neutrophil-specific autoanti-
bodies, five patients with alcoholic cirrhosis; 85 patients with
organ-specific autoimmune diseases (60 thyroiditis and 25
diabetes mellitus type 1); 307 patients with non-organ-specific
autoimmune diseases (32 patients with myopathy and/or pulmon-
ary fibrosis, 25 patients with inflammatory bowel diseases, 80
patients with systemic lupus erythematosus (SLE), 75 patients
with SjoÈgren's syndrome (SS), 70 patients with scleroderma, 25
patients with rheumatoid arthritis (RA)) and 20 healthy blood
donors.
Laboratory assessments
Sera were screened for ANA, anti-mitochondrial (AMA) and anti-
smooth muscle antibodies (ASMA) using the indirect immuno-
fluorescence (IIF) technique as described previously [21].
Cryostat sections of rat liver, kidney and stomach were used as
substrates. The fluorescein-labelled anti-human conjugate was
purchased from Dako Labs (Santa Barbara, CA) and used at a
dilution of 1:20.
Anti-LKM and AMA antibodies were also studied by
immunoblot and ELISA tests. Anti-dsDNA antibodies were
studied by Farr technique (Amersham Pharmacia Biotech,
Uppsala, Sweden), and anti-thyroglobulin antibodies were studied
by ELISA (Radim, Angleur, Belgium).
Analysis of immunoprecipitated ribonucleoproteins (RNPs)
To identify autoantibodies capable of binding specific small
nuclear/cytoplasmic ribonucleoproteins (sn/scRNPs), sera or
affinity-purified antibodies were tested for their ability to
immunoprecipitate subsets of small RNAs from extracts of human
HeLa S3 cells. The standard assay method was used [22]. Briefly,
HeLa S3 cells growing in log phase at 4 � 105 cells/ml were
labelled in vivo with 32P-orthophosphate (Amersham) as de-
scribed [23]. Whole cell extracts were prepared as described [24]
and precleared with 1/20th volume of 20% (w/v) suspension of
Sepharose-protein A (Pharmacia) in NET-2 buffer (50 mm Tris
pH 7´4, 0´15 m NaCl, 0´05% Nonidet P-40) plus 1 mg/ml bovine
serum albumin (BSA); immunoprecipitations were performed as
described by Lerner & Steitz [22] for 32P-labelled extracts.
Deproteinized extracts were prepared by PCA extraction and by
incubating 32P-labelled extract in 0´1 mg/ml proteinase K and
0´2% SDS for 2 h at 378C, followed by extraction with phenol/
chloroform/isoamyl alcohol (PCA) (50/49/1) with 0´1% 8-
hydroxiquinoleine and ethanol precipitation.
Immunoprecipitated 32P-labelled RNAs were PCA extracted,
ethanol precipitated, electrophoresed on 10% polyacrylamide
denaturing gels in 0´1 m boric acid/0´1 m Tris base/2 mm EDTA
(1� TBE) pH 8´3, and subjected to autoradiography.
Purification of tRNP(Ser)Sec antigen from HeLa S3 human cells
Affinity-purified protein±tRNA(Ser)Sec antigen complexes were
prepared as described [1] with modifications, using the anti-
tRNP(Ser)Sec pattern serum immobilized on Sepharose-protein A.
Briefly, Sepharose-protein A was incubated with pattern serum for
2 h at room temperature. After washing three times with buffer
IPP (10 mm Tris pH 8, 0´5 m NaCl, 0´1% NP-40) and one with
Tris buffer saline (TBS) (40 mm Tris pH 7´4, 0´13 m NaCl), IgG
coupled to Sepharose was treated with glutaraldehyde at 10% for
30 min at room temperature. The beads were then blocked with a
solution of 1 mg/ml BSA in TBS for 2 h at 48C. After washing
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with NET-2 buffer, the beads were ready for incubation with a
NET-2 extract from HeLa cells prepared in the usual manner [24].
The antigen was eluted with glycine±HCl 0´2 m pH 2´5. After
being neutralized the eluted antigen was dialysed with TBS and
used for separation of proteins in a 10% SDS±PAGE as described
[25] and transferred to nitrocellulose sheets in 25 mm Tris,
192 mm glycine pH 8´3, without SDS [26].
Western blot analysis
Crude extracts from total HeLa S3, CEM, and HL-60 cells, and rat
liver, as well as bacterial lysates and purified recombinant
proteins, were separated on 10% (w/v) polyacrylamide gels and
transferred to nitrocellulose as described by Towbin et al. [26]
with modifications. Immunoblots using cell and tissue extracts
were performed as described previously [1] using 3% casein as
blocking solution and 125I-protein A (Amersham Pharmacia
Biotech) for detection of bound immunoglobulins. When bacterial
lysates were used for immunoblotting, nitrocellulose was
incubated with anti-tRNP(Ser)Sec-positive sera, normal human sera
and with anti-Xpress antibody (Invitrogen, San Diego, CA)
diluted 1:1000. Membranes were washed and consecutively
incubated with alkaline phosphatase-conjugated anti-human rabbit
immunoglobulins (Dako, Glostrup, Denmark). Positive reactions
were performed using 5-bromo-4-chloro-3-indolyl phosphate
(BCIP) with nitro blue tetrazolium (NBT) (Promega, Madison,
WI) as a substrate.
Affinity purification of antibodies
Antibodies were affinity-purified as described in [21] and were
used without dilution in Western blot assays and immunopreci-
pitation experiments.
Statistical analysis
Quantitative variables were compared by the Fisher test or x2 test.
Since the variables for comparison had been formulated a priori
and then assessed systematically in each group, an unadjusted P
value of 0´05 was used to determine statistical significance.
Cloning
Screening of a human liver cDNA library in Uni-ZAP XR
(Stratagene, La Jolla, CA) was performed [27] using anti-
tRNP(Ser)Sec-positive sera. Detection of antigen±antibody com-
plexes was performed using alkaline phosphatase-conjugated
anti-human rabbit immunoglobulins (Dako). Positive clones were
purified by repeated screening until all progeny plaques were
positive. In a final screening, each filter was divided into eight
pieces that were incubated with four different anti-tRNP(Ser)Sec-
positive sera, and with anti-M2, anti-Ro, anti-NOR 90 and normal
human sera, as unrelated and negative controls. Two positive
clones (13 and 19) were isolated, and pBluescript plasmids
containing cDNA inserts were obtained by in vivo Exassist/SOLR
excision according to the protocol provided by Stratagene.
DNA sequencing and sequence analysis
Sequencing of double-stranded DNA templates was achieved by a
modified dideoxy chain-termination method [28] using the
ALFexpress Autoread Sequencing kit and the ALFexpress DNA
Sequencer (Amersham Pharmacia Biotech). For priming, 5 0-cyanine-labelled primers were used. Specific internal oligonucleo-
tides were designed as the sequencing progressed. Sequences of
the recombinant clones were compared with the non-redundant
databases at the National Center for Biotechnology Information
(NCBI) using the BLAST program [29].
Expression and purification of His-tagged recombinant proteins
The cDNA insert from clone 19 (3378 bp) was SacI/KpnI digested
and gel-purified using GeneClean II kit (p
Bio 101; Vista, CA).
The DNA fragment was ligated in frame to SacI/KpnI digested
and gel-purified (p
Bio 101; Vista) expression vector pTrcHisA
(Invitrogen, San Diego, CA), allowing the translation of a fusion
protein bearing a 6-histidine tail at the NH2-terminus. The insert
from clone 13 (2911 bp) was subcloned into pTrcHisC (Invitro-
gen) at BamHI and KpnI sites. In order to express His-tagged
recombinant proteins, the constructs were transformed into the
Escherichia coli strain TOP10 and selected on ampicillin-
containing plates. Each recombinant clone was verified by
sequence analysis. The pTrcHis vector with no insert was used
as negative expression control, and the pTrcHisBCAT construct
(Invitrogen) as positive expression control. Cells were grown in
LB at 378C to an OD600 � 0´6, isopropyl-b-d-thiogalactopyrano-
side was added to a final concentration of 1 mm and incubated for
a further 3 h. Preparation of cell lysates and purification of
recombinant proteins were performed under native conditions as
specified in the manufacturer's instructions (Invitrogen). Briefly,
TOP10 bacteria containing each recombinant protein were
harvested and resuspended in buffer A (20 mm sodium phosphate,
500 mm NaCl, pH 7´8), treated with white lysozyme (100 mg/ml)
and lysed by four rapid freeze±thaw and sonication cycles. Debris
was spun down, and supernatants were loaded onto pre-equilibrated
nickel-charged ProBond columns (Invitrogen). Washes were
performed with buffer A at several pH: 6´3, 6´0 and 5´5. Elution
was performed with a pH gradient from 4´5 to 2´5 in buffer A.
RESULTS
AIH sera immunoprecipitate an opal suppressor tRNA(Ser)Sec
Sera randomly collected during follow up from 59 selected
patients with type-1 AIH were studied by immunoprecipitation.
Of the 59 analysed sera, 28 had antibodies which immunopreci-
pitated RNA species migrating at 4´5S region in a denaturing 10%
urea±PAGE, between hY4 and hY5. This RNA had earlier been
identified as the UGA suppressor serine tRNA (tRNA(Ser)Sec).
Moreover, and as shown in Fig. 1, some sera immunoprecipitated
other sn/scRNAs. Figure 1(a±c) shows three representative
experiments of the analysis of the RNAs immunoprecipitated by
patients with severe AIH.
Since most small RNAs precipitated by autoimmune sera are
associated with antigenic proteins, we tested the ability of these
sera to immunoprecipitate the tRNA(Ser)Sec from deproteinized
extracts. Approximately 5% of all the patients studied had
antibodies in their sera that recognized the structure of the
RNA. Figure 1a shows a representative result of the immunopre-
cipitation assay performed with the same sera shown in Fig. 1a
and deproteinized a HeLa cell extract. Serum 8 precipitated RNA
about 50% as efficiently from deproteinized extracts. The other
five sera as well as a prototype anti-tRNP(Ser)Sec control serum
(DPP) showed no detectable immunoprecipitation of tRNA(Ser)Sec.
No patients from the control groups studied (patients with
organ-specific autoimmune diseases, patients with non-organ-
specific autoimmune diseases and normal blood donor volunteers)
were found positive in the immunoprecipitation assay for anti-
tRNP(Ser)Sec antibodies.
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Immunoblotting assays; anti-tRNP(Ser)Sec autoantibodies
recognize a 48/52-kD protein
Autoantibodies against tRNA(Ser)Sec-associated protein(s) were
studied by immunoblot using affinity-purified antigen from HeLa
cell extracts prepared as described earlier. Fifteen of the 59 AIH
sera were positive. Of these, 12 immunoprecipitated the
tRNA(Ser)Sec. Figure 2 shows a representative number of sera
with a positive immunoblot reaction. These antibodies recognized
protein(s) of 48 and/or 52 kD. None of the sera from the control
groups was positive (data not shown).
Sensitivity and specificity of anti-tRNA(Ser)Sec antibodies and AIH
Patients with and without anti-tRNP(Ser)Sec antibodies had similar
clinical, laboratory, histological, and immunological features at
presentation. There were no significant differences in the
conventional laboratory indices of inflammatory activity or
Fig. 1. (See next page) Immunoprecipitation of small RNAs by sera from patients with autoimmune hepatitis (AIH). 32P-labelled HeLa cell
sonicates were immunoprecipitated and gel fractionated. Numbers at the top of each lane correspond to the number given to each patient. DPP
and DOD are the identification names of two prototype anti-tRNP(Ser)Sec sera. Known RNAs are indicated on the side; tRNA(Ser)Sec (shown by
an arrow) denotes the tRNA immunoprecipitated by AIH sera. (a,b,c) Three autoradiograms of the RNAs immunoprecipitated by sera from a
representative number of AIH patients studied. Panel A 0 shows the RNAs immunoprecipitated by the same sera described in panel A, from a
deproteinized HeLa cell extract. Lanes Total RNA show the total RNAs extracted from the whole (a,b,c) and from the deproteinized (panel
A 0) HeLa cell sonicates prior to immunoprecipitation; lanes Normal serum and NHS show the immunoprecipitated RNAs by sera from
healthy non-autoimmune donors; lanes DPP and DOD show the RNAs immunoprecipitated by two anti-tRNP(Ser)Sec reference sera.
Cloning of the tRNP(Ser)Sec/protein antigen 367
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Fig. 1. (See previous page for caption)
Table 1 Clinical and serological features in patients with type-1 autoimmune hepatitis (AIH)
AIH patients*
Anti-tRNA(Ser)Sec-positive Anti-tRNA(Ser)Sec-negative P
Number 28 31
Age (years) 36 ^ 2 42 ^ 3 0´107
Female:male 22:6 24:7 0´915
Concurrent autoimmune diseases 11 (39) 15 (48) 0´953
SMA $ 1:40 24 (86) 28 (90) 0´630
ANA $ 1:40 19 (68) 21 (68) 0´449
Duration of follow up (months) 121 ^ 14´5 130 ^ 17 0´709
²Fatal outcome 7 (25) 1 (3) 0´040
*Two groups of type-1 AIH patients with or without antibodies to tRNA(Ser)Sec in their sera.
²Liver-related death.
Numbers in parentheses are percentages. Significantly different from each other at levels of P. Differences
between groups (, 0´05) are in bold.
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immunoreactivity in the patients with type-1 AIH who did and
those who did not have anti-tRNP(Ser)Sec antibodies (data not
shown). The frequencies of anti-tRNP(Ser)Sec were statistically
different between AIH and control groups. Moreover, the patients
who died of liver failure were more commonly seropositive for
anti-tRNP(Ser)Sec than patients who did not have these antibodies
(25% versus 3%, P � 0´04). Both groups of patients were
followed for a similar period of time (121 versus 130 months)
(Table 1) and received similar treatment.
Cloning and sequence analysis
Four anti-tRNPSer(Sec)-positive sera from type-1 AIH patients (all of
them reacting with the 48/52-kD tRNA(Ser)Sec-associated protein
from HeLa cells) were used to screen a human liver cDNA
expression library to isolate and characterize the cDNA encoding
the antigenic protein associated with the tRNA(Ser)Sec. A total of
5 � 105 plaques was screened and two positive clones (number 19
and number 13) recognized by all anti-tRNP(Ser)Sec (4/4) and by
none of the control sera (one anti-Ro, one anti-M2, one anti-NOR 90
control sera and four normal human sera) were isolated. Both clones
were converted into plasmids and used for further analysis.
Nucleotide sequences and deduced amino acid sequences are
shown in Fig. 3. Clone 19 had an insert of 3378 bp including 174 bp
of 5 0-untranslated region, a putative initiating ATG codon at
nucleotide 175 followed by an open reading frame of 1326 bp, and
1878 bp of 3 0-untranslated region. The open reading frame was
predicted to encode a protein of 441 amino acids. The theoretical
molecular mass was 48´8 kD, close to the estimated molecular mass
from SDS±PAGE of the antigenic protein associated with
tRNA(Ser)Sec (48 kD) [1]. The putative polypeptide showed a high
content of basic amino acids, resulting in a theoretical pI of 9´40.
This protein showed one zinc finger motif (amino acids 156±161)
which has been associated with nucleic acid and protein interaction.
Clone 13 had an insert of 2911 bp including 16 bp of 5 0-untranslated region, a potential ATG start codon at nucleotide 17,
followed by a 972-bp open reading frame coding for a putative
protein with a predicted molecular mass of 35´9 kD, and a long 3 0-untranslated region of 1923 bp.
Comparative analyses of nucleotide sequences of both clones
demonstrated that the nucleotide sequence of clone 19 from
nucleotide 513 was identical to the complete nucleotide sequence
of clone 13 with minor exceptions. Minor differences were: five
nucleotides of the 5 0-untranslated region of clone 13, a single
nucleotide in the 3 0-untranslated region of both clones (indicated
with bold letters in Fig. 3), and the presence of 72 nucleotides
before the poly(A) tail of clone 13 which did not exist in clone 19.
The alignment of the two nucleotide sequences showed that the
ATG start codon of the cDNA number 13 perfectly matched a
second in frame ATG codon at position 529 of the cDNA number
19. These data support the finding that clone 13 encoded for a
shorter protein which lacked the first 118 amino acids of the
protein encoded by clone 19.
As the sequences of the two clones were not previously
recorded in the GenBank, EMBL, DDBJ and SwissProt databases,
sequence of the cDNA number 19 was submitted to the EMBL
Data library (accession number: AJ238617).
A BLASTN search in non-redundant databases revealed an
identity of cDNA number 19 from nucleotide 629 to nucleotide
3331 (just before the poly(A) tail) with a region of human
chromosome 4 (GenBank accession number AC007073). With
regard to cDNA number 13, the identity region included
Fig. 2. Immunoblotting of the affinity-purified tRNP(Ser)Sec antigen from
HeLa cell sonicate, with autoimmune hepatitis (AIH) sera. The
immunopurified HeLa cell extract was fractionated on 10% polyacryl-
amide/SDS gels, blotted onto nitrocellulose, and probed with antisera as
indicated. The positions of molecular weight markers (in kD) are shown on
the right. Numbers at the top of each lane correspond to the number given
to each patient, lanes DOD and DPP show the immunoblot reaction of anti-
tRNP(Ser)Sec reference sera, and lane Normal serum shows the immunoblot
reaction of a serum from a healthy blood donor volunteer.
Fig. 3. (See next page) Nucleotide and deduced amino acid sequence of clone 19. The amino acid sequence was predicted from the
nucleotide sequence of clone 19 cDNA. A 5 0-untranslated region of 174 bp precedes the open reading frame. The initiator methionine codon
is surrounded by the sequence GCAATCATGG at nucleotides 169±178, which closely approximates to the ideal ribosomal binding
sequence GCCACCATGG [40] and one zinc finger motif (amino acids 156±161), which has been associated with nucleic acid and protein
interactions. Similar sequence has been observed in RNA binding proteins [41, 42]. The TGA stop codon (*) is followed by a long 3 0 non-
coding region. This region includes putative polyadenylation signals AATAAA at nucleotides 3309±3314. First nucleotide of cDNA number
13 is underlined. Bold letters indicate the location of minor differences with clone 19. The symbol arrowhead indicates the site where the
open reading frame of clone 13 starts.
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Fig. 3. (See previous page for caption)
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nucleotide 180 to nucleotide 2891, just before the poly(A) signal
of this cDNA.
The alignment of the predicted amino acid sequences with
databases demonstrated 44% identity (clone 19) and 41% (clone
13), with a hypothetical 53´4-kD protein (481 amino acids) of
Caenorhabditis elegans, whose function has not yet been
described (SWALL accession number: Q18953).
Expression and purification of His-tagged recombinant proteins
In order to demonstrate that we had isolated the cDNA encoding
the antigenic protein, which associates with the tRNA(Ser)Sec, we
proceeded with the expression, purification and analysis of both
recombinant proteins. The expressions of a specific 62-kD protein
(clone 19) and a specific 41-kD protein (clone 13) were
demonstrated in Western blots using anti-tRNP(Ser)Sec-positive
serum (Fig. 4a). Each protein correlated with a band obtained with
anti-Xpress antibodies raised against an NH2-terminal X-press
epitope of pTrcHis vectors (Fig. 4a). As His-tagged recombinant
protein translation started at the ATG of pTrcHis vector, there was
an increment in molecular weight of the recombinant proteins in
comparison with the theoretical molecular mass estimated from
the cDNA. This increment in the molecular weight was due to a
fragment of pTrcHis vector, a fragment of pBluescript vector and
the 5 0-untranslated region of cDNA included in the expressed His-
tagged recombinant protein.
Lysates from bacterial clones containing the recombinant
proteins were loaded onto nickel-charged agarose resin columns.
Western blots of the eluted fractions demonstrated that both
recombinant proteins were highly purified at pH 2´5 eluted
fraction (Fig. 4b).
Immunoprecipitation and Western blot analysis of antibodies
eluted from His-tagged recombinant proteins
Using the purified fraction of the 62-kD recombinant protein
(clone 19), a Western blot with an anti-tRNP(Ser)Sec-positive serum
was performed, and antibodies eluted from the 62-kD band were
assayed by immunoprecipitation of a 32P-labelled HeLa S3 cell
extract. These affinity-purified antibodies clearly precipitated the
tRNA(Ser)Sec (Fig. 5, lane 5). The same experiment was performed
with antibodies eluted from the 41-kD protein encoded by clone
13, showing that these antibodies were also capable of
immunoprecipitating tRNA(Ser)Sec (Fig. 5, lane 7). Control
experiments using eluates from unrelated regions of the
nitrocellulose did not precipitate the tRNA(Ser)Sec (Fig. 5, lanes
Fig. 4. Antigenicity of fusion proteins characterized by Western blot. (a) Escherichia coli TOP10 lysates with the cDNA sequences
expressed from the bacterial vector pTrcHis were fractionated by SDS210% PAGE, transferred to nitrocellulose membrane, and allowed to
react with: normal human serum (NHS), type-1 autoimmune hepatitis (AIH) patient serum containing anti-48-kD associated tRNA(Ser)Sec
antibodies (a-tRNP), and anti-Xpress antibodies that recognize the amino acid sequence -Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys- found in the
NH2-terminus of the vector pTrcHis (a-Xpress). CAT, E. coli TOP10-pTrcHisBCAT extract; 13, E. coli TOP10-pTrcHisC cDNA number 13
extract; 19, E. coli TOP10-pTrcHisA cDNA number 19 extract. Molecular weight markers are indicated on the left. (b) Purified His-tagged
proteins from bacterial lysates were assayed by Western blot with a NHS and a type-1 AIH patient serum with anti-tRNA(Ser)Sec antibodies
(a-tRNP). Arrows indicate the molecular weight of the recombinant proteins reactive with anti-tRNP(Ser)Sec antibodies.
Cloning of the tRNP(Ser)Sec/protein antigen 371
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6 and 8). When these affinity-purified antibodies were used to re-
test a Western blot, they detected the 62- and 41-kD proteins,
respectively (data not shown).
Moreover, the sera that immunoprecipitated the tRNA(Ser)Sec
and strongly reacted in immunoblot with a 48/52-kD protein from
partially purified HeLa cell extracts, recognized the 62-kD His-
tagged recombinant protein.
DISCUSSION
We previously identified anti-tRNP(Ser)Sec autoantibodies, specific
for a subgroup of AIH [1]. These antibodies recognized
tRNA(Ser)Sec-associated protein(s) and/or directly reacted with
the tRNA(Ser)Sec itself. In this study we extend our previous report
to 59 patients with AIH, include a more extensive group of
controls, and demonstrate the high specificity and frequency
(47´5%) of the anti-tRNP(Ser)Sec autoantibodies for severe forms of
type-1 AIH. The complete nucleotide sequence and expression of
an antigenic protein is reported.
The clinical relevance of these autoantibodies arises from the
high specificity of the response of AIH patients, with a higher
frequency in patients who died of liver disease. Autoantibodies
against this antigen are not found in patients with other
autoimmune diseases or in normal human blood donor volunteers.
The biological relevance of this antigen arises from its
association with the human UGA suppressor tRNA(Ser)Sec.
In the present study we cloned and expressed a cDNA
encoding a human protein, with a predicted molecular mass of
48´8 kD, which is specifically recognized by sera from type-1
AIH patients. Affinity-purified antibodies from this protein
specifically immunoprecipitated the tRNA(Ser)Sec from HeLa S3
cells.
The deduced amino acid sequence, although novel, has a 44%
homology with a hypothetical 53´4-kD protein of C. elegans,
described as part of the C. elegans genome study, and whose
function is not yet known.
Data in our previous study [1] and in the present paper
strongly suggest that tRNP(Ser)Sec complex is involved in the
selenocysteine insertion pathway in eukaryotes. However, the
cloning and sequencing of the 48´8-kD protein did not completely
elucidate its precise molecular nature. No relevant similarity was
found with any synthetase [30], synthase [31], or elongation
factors when compared with nucleotide and protein Data/Banks.
However, recent data suggest that the 48´8-kD protein could be
the Selenium-tRNA protecting factor (SePF) earlier described as a
50-kD tRNA(Ser)Sec-binding protein protecting mammalian 75Se-
tRNA(Ser)Sec from alkaline hydrolysis [16,32]. Even though the
sequence of SePF is not known, our data suggest that the 48´8-kD
protein could be this selenocysteine-specific factor: it has a similar
molecular weight, its sequence contains a zinc finger motif which
has been associated with nucleic acid and protein binding region,
and data not yet reported have demonstrated that the tRNP(Ser)Sec
is more resistant to alkaline hydrolysis than the tRNA(Ser)Sec itself.
Moreover, it has a putative ribosomal binding sequence.
Knowledge of the biological nature of the antigen may help to
elucidate the aetiopathogenesis of the anti-tRNP(Ser)Sec antibodies
and their possible role in type-1 AIH.
In eukaryotes the tissue where the highest number of
selenoproteins is found is the liver, and factors involved in
selenoprotein synthesis may also possibly be predominant in this
organ [33]. Anti-tRNP(Ser)Sec antibodies may arise following a
breakdown in tolerance to `self-proteins' in the liver, perhaps as a
molecular mimicry mechanism, or to the `self modified'. Anti-
tRNP(Ser)Sec antibodies could result from the direct interaction of
the 48´8-kD protein with a virus RNA, thereby rendering it
`foreign' to the host immune system [34].
Other autoantibodies related to AIH have been described
although their nature has not yet been elucidated. These include
the soluble liver antigen (SLA) [35], the liver pancreas antigen
(LP) [36], cytokeratins 8 and 18 [37] and the glutathione
transferase (GTS) [38].
Almost simultaneously the 48-kD tRNP(Ser)Sec clone was
Fig. 5. Immunoprecipitation of tRNA(Ser)Sec by affinity-purified anti-62-
and anti-41-kD recombinant protein antibodies. A non-immune serum from
a healthy blood donor (lane 2), whole sera with anti-tRNP(Ser)Sec antibodies
(lanes 3 and 4), anti-62-kD recombinant protein antibodies eluted from a
Western blot of purified expressed protein from clone 19 (lane 5), anti-41-kD
recombinant protein antibodies eluted from a Western blot of purified
expressed protein from clone 13 (lane 7), a control eluate from an unrelated
region of the Western blot from expressed clone 19 (lane 6), and a control
eluate from an unrelated region of the Western blot from expressed clone 13
(lane 8) were used to immunoprecipitate a 32P-labelled HeLa S3 cell sonicate,
and the RNAs were analysed. The mobility of known RNAs is given on the
left, and tRNA(Ser)Sec precipitated by type-1 autoimmune hepatitis (AIH)
sera are indicated on the right. Total RNA (lane 1) is RNA from the HeLa S3
cell sonicate prior to immunoprecipitation.
372 M. Costa et al:
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registered (accession number AJ238617), the LP antigen also
related to AIH was cloned (accession number AF146396). The
reported sequence of the latter shows 99% homology with the 48-
kD tRNP(Ser)Sec cloned protein. Moreover, it has recently been
reported that the LP antigen is identical to the SLA antigen [39].
We studied eight sera from AIH patients with anti-SLA antibodies
and found that all of them precipitated the tRNA(Ser)Sec and
reacted with the recombinant 48´8-kD tRNP(Ser)Sec protein. We
concluded that the tRNP(Ser)Sec antigen is the same as the LP
antigen [36], is different from cytokeratins [37] and glutathione
transpherase [38], and is included in the complex SLA [35].
This study demonstrated that the screening of anti-tRNP(Ser)Sec
antibodies may be a very useful marker for type-1 AIH. Moreover,
the improvement of an ELISA test with the recombinant protein
expressed in eukaryote cells to screen the anti-tRNA-associated
protein antibodies would increase both the sensitivity and utility
of the assay. Antibodies that immunoprecipitated the tRNA(Ser)Sec
included at least two subsets of autoantibodies, those that reacted
with a 48´8-kD antigenic protein and those that recognized the
tRNA itself or another tRNA-associated protein different from the
48´8-kD protein. At present, the screening of AIH patients by
immunoprecipitation and analysis of RNAs is the most sensitive
and specific test for anti-tRNP(Ser)Sec antibodies.
ACKNOWLEDGMENTS
We thank Ms Carolina Newey for her assistance with the preparation of
the manuscript. This work was supported by a grant from the `Fondo de
Investigaciones Sanitarias de la Seguridad Social' (94/0751), a grant from
the `Ministerio de EducacioÂn y Ciencia' (SAF 97/0123) and a grant from
the `Generalitat de Catalunya' (1997/SGR/00352) (Spain).
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