Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec...

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]

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

Cloning of the tRNP(Ser)Sec/protein antigen 365

q 2000 Blackwell Science Ltd, Clinical and Experimental Immunology, 121:364±374

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.

366 M. Costa et al:


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.



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.

368 M. Costa et al:


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.



Fig. 3. (See previous page for caption)

370 M. Costa et al:


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.



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:


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).

REFERENCES

1 GelpõÂ C, Sontheimer EJ, Rodriguez-Sanchez JL. Autoantibodies

against a serine tRNA±protein complex implicated in cotranslational

selenocysteine insertion. Proc Natl Acad Sci USA 1992; 89:9737±43.

2 Maddrey WC. Subdivisions of idiopathic autoimmune chronic active

hepatitis. Hepatology 1987; 7:1372±5.

3 Maddrey WC, Willis C. Chronic hepatitis. In: Bone RC, ed. Disease-a-

month, Vol XXXIX, No. 2. 1993:53±126.

4 Manns M. Autoantibodies and antigens in liver diseases-updated. J

Hepatol 1989; 9:272±80.

5 Czaja AJ. Autoantibodies. Bailliere's Clin Gastroenterol 1995; 9:723±

44.

6 Homberg J-C, Abuaf N, Bernard O et al. Chronic active hepatitis

associated with anti-liver/kidney microsome antibody type 1: a second

type of `autoimmune' hepatitis. Hepatology 1987; 7:1333±9.

7 Czaja AJ, Pfeifer K, Decker RH, Vallari A. Frequency and significance

of antibodies to asialoglycoprotein receptor in type 1 autoimmune

hepatitis. Dig Dis Sci 1996; 41:1733±40.

8 Czaja AJ, Cassani F, Cataleta M, Valentini P, Bianchi FB. Frequency

and significance of antibodies to actin in type 1 autoimmune hepatitis.

Hepatology 1996; 24:1068±73.

9 Manns MP. Autoantibodies in chronic hepatitis: diagnostic reagents

and scientific tools to study aetiology, pathogenesis, and cell biology.

Prog Liver Dis 1994; 12:137±56.

10 Czaja AJ, Manns MP, Homburger HA. Frequency and significance of

antibodies to liver/kidney microsome type 1 in adults with chronic

active hepatitis. Gastroenterology 1992; 103:1290±5.

11 Czaja AJ. Behaviour and significance of autoantibodies in type 1

autoimmune hepatitis. J Hepatol 1999; 30:394±401.

12 Czaja AJ, Manns MP. The validity and importance of subtypes of: a

point of view. Am J Gastroenterol 1995; 90:1206±11.

13 Czaja AJ. Autoimmune hepatitis: evolving concepts and treatment

strategies. Dig Dis Sci 1995; 40:435±56.

14 Jung J-E, Karoor V, Sandbaken MG et al. Utilisation of selenocysteyl-

tRNA(Ser)Sec and seryl-tRNA(Ser)Sec in protein synthesis. J Biol Chem

1994; 269:29739±45.

15 Bock A, Forchhammer K, Heider J, Baron C. Selenoprotein synthesis:

an expansion of the genetic code. Trends Biochem Sci 1991; 16:463±

7.

16 Yamada K. A new translational elongation factor for selenocysteyl-

tRNA in eucaryotes. FEBS Letters 1995; 377:313±7.

17 Johnson PJ, McFarlane IG, Alvarez F et al. Meeting Report.

International Autoimmune Hepatitis Group. Hepatology 1993;

18:998±1005.

18 Czaja AJ. Diagnosis, prognosis, and treatment of classical autoimmune

chronic active hepatitis. In: Krawitt EL, Wiesner RH, eds. Auto-

immune liver disease. New York: Raven Press, 1991:143±66.

19 Summerskill WHJ, Korman MG, Ammon HV, Baggentoss AH.

Prednisone for chronic active liver disease: dose titration, standard

dose, and combination with azathioprine compared. Gut 1975; 16:876±

83.

20 Czaja AJ, Carpenter HA. Sensitivity, specificity and predictability of

biopsy interpretations in chronic hepatitis. Gastroenterology 1993;

105:1824±32.

21 GelpõÂ C, AlgueroÂ A, Martinez MA, Vidal S, Juarez C, Rodriguez-

Sanchez JL. Identification of protein components reactive with anti-

PM/Scl autoantibodies. Clin Exp Immunol 1990; 81:59±64.

22 Lerner MR, Steitz JA. Antibodies to small nuclear RNAs complex with

proteins are produced by patients with systemic lupus erythematosus.

Proc Natl Acad Sci USA 1979; 76:5495±9.

23 Mimori T, Hinterberger M, Pettersson I, Steitz JA. Autoantibodies to

the U2 small nuclear ribonucleoprotein in a patient with scleroderma±

polymyositis overlap syndrome. J Biol Chem 1984; 259:560±5.

24 Forman MS, Nakamura M, Mimori T, GelpõÂ C, Hardin JA. Detection

of antibodies to small nuclear ribonucleoproteins and small cytoplas-

mic ribonucleoproteins using unlabeled extracts. Arthritis Rheum

1985; 28:1356±61.

25 Laemmli UK. Cleavage of structural proteins during the assembly of

the head of bacteriophage T4. Nature 1979; 227:680±5.

26 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins

from polyacrylamide gels to nitrocellulose sheets: procedure and some

applications. Proc Natl Acad Sci USA 1979; 76:4350±4.

27 Sambrook J, Fritsch E, Maniatis T. Molecular cloning, 2nd edn. New

York: Cold Spring Harbor Laboratory Press, 1989:12.16±12.20.

28 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-

terminating inhibitors. Proc Natl Acad Sci USA 1977; 74:5463±7.

29 Altschul SF, Madden TL, SchaÈffer AA, Zhang J, Zhang Z, Miller W,

Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of

protein database search programs. Nucl Acids Res 1997; 25:3389±402.

30 Kozak M. Compilation and analysis of sequences upstream from the

translational start site in eukaryotic mRNAs. Nucl Acids Res 1984;

12:857±72.

31 Hanas JS, Hazuda DJ, Bogenhagen DF, Wu FY-H, Wu C-W. Xenopus

transcription factor A requires zinc for binding to the 5S RNA gene. J

Biol Chem 1983; 258:14120±5.

32 Draper DE. Protein-RNA recognition. Ann Rev Biochem 1995;

64:593±620.

33 Vincent C, Leberman R, Hartlein M. EMBL/GENBANK/DDBJ Data

Banks Accession: P49591.

34 Mizutani T, Kurata H, Yamada K, Totsuka T. Some properties of

murine selenocysteine synthase. Biochem J 1992; 284:827±34.

35 Yamada K, Mizutani T, Ejiri S, Totsuka T. A factor protecting

mammalian [75Se]SeCys-tRNA is different from EF-1a. FEBS Letters

1994; 347:137±42.

36 Burk RF, Hill KE. Regulation of selenoproteins. Ann Rev Nutr 1993;

13:65±81.



37 Keene JD. RNA surfaces as functional mimetics of proteins. Chem

Biol 1996; 3:505±13.

38 Manns M, Gerken G, Kyriatsoulis A, Staritz M, Meyer zum

BuÈschenfelde KH. Characterization of a new subgroup of autoimmune

chronic active hepatitis by autoantibodies against a soluble liver

antigen. Lancet 1987; 7:292±4.

39 Stechemesser E, Klein R, Berg PA. Characterization and clinical

relevance of liver-pancreas antibodies in autoimmune hepatitis.

Hepatology 1993; 18:1±9.

40 WaÈchter B, Kyriatsoulis A, Lohse AW, Gerken G, Meyer zum

BuÈschenfelde KH, Manns M. Characterization of liver cytokeratin as a

major target antigen of anti-SLA antibodies. J Hepatol 1990; 11:232±

9.

41 Wesierska-Gadek J, Grimm R, Hitchman E, Penner E. Members of the

glutathione-S-transferase gene family are antigens in autoimmune

hepatitis. Gastroenterology 1998; 114:329±35.

374 M. Costa et al:


Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec...

Documents

Transcript of Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec...