Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec complex recognized by autoantibodies from patients withtype-1 autoimmune hepatitis

Abstract

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.

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′ 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′) 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. 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% polyacrylamide/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′-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 [, 42]. The TGA stop codon (*) is followed by a long 3′ 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. 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 SDS−10% 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.

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.