Journal of Autoimmune Diseases BioMed Central

Background Antibodies to a cytosolic soluble liver antigen (SLA) are specifically detected in patients with autoimmune hepatitis (AIH). The target of anti-SLA has been identified as a ~50 kDa UGA serine tRNA-associated protein complex (tRNP(Ser)Sec), through the screening of cDNA libraries. A recent report questioned the identity of tRNP(Ser)Sec as the real SLA antigen. The latter study identified α-enolase as a major anti-SLA target, through proteomic analysis. Methods In an attempt to explain the observed discrepancy we have investigated reactivity of SLA positive sera against α-enolase and tRNP(Ser)Sec using rat and primate liver homogenate and the recombinant antigens. Thirty-three serum samples, 11 from SLA-positive patients and 22 from SLA negative controls were investigated. SLA antibodies were detected by an inhibition ELISA and confirmed by immunoblot using human liver homogenate. Autoantibody reactivity was further evaluated using preparations of primate and rat liver homogenates. Anti-α-enolase antibody reactivity has been tested by immunoblot using recombinant α-enolase. An affinity purified goat polyclonal anti-α-enolase IgG antibody was used as reference serum sample. Anti-tRNP(Ser)Sec antibody reactivity was detected by ELISA or dot blot using recombinant tRNP(Ser)Sec antigen. Results and Discussion The affinity purified IgG antibody directed to human α-enolase gave a band of approximately 48 kDa in both human and rat liver homogenates. A high titre anti-tRNP(Ser)Sec antibody serum gave a single band of ~50 kDa in both liver preparations. All but one anti-SLA antibody positive sera reacted with a ~50 kDa but none immunofixed a 48 kDa band. All anti-SLA antibody positive sera reacted strongly with the recombinant full length tRNP(Ser)Sec protein. None of the anti-SLA negative sera reacted with tRNP(Ser)Sec. Anti-SLA positive, and anti-SLA negative sera reacted equally against recombinant α-enolase by immunoblot. Pre-incubation of anti-SLA positive sera with tRNP(Ser)Sec completely abolished the 50 kDa band. The findings of the present study indicate that α-enolase and tRNP(Ser)Sec are both expressed in primate and rat liver and have a respective MW of 48 and 50 kDa. They also show that anti-tRNP(Ser)Sec – but not anti-α-enolase – correlates with anti-SLA antibody reactivity. Conclusion Our findings indicate that tRNP(Ser)Sec is the most likely target of anti-SLA.

Using recombinant tRNP (Ser)Sec antigen as competitor in inhibition experiments it has been found removal of the 50 kDa band immunofixed by SLA positive sera from immunoblots of primate liver homogenate [19]. Though this finding indicates tRNP (Ser)Sec as a major component of SLA, a view apparently shared by Ballot et al, several questions still remain unanswered: 1. Are there any differences in α-enolase expression between rat -used by Ballot et al [11] -and primate liver homogenate -used by our study [19] -that could explain the discrepancy between these studies? 2. Is it true that failure of proteomic analysis to detect tRNP (Ser)Sec is due to its presence in trace amounts in the supernatant of liver homogenate [11]? 3. What is the reactivity of SLA positive and negative sera against recombinant α-enolase?
4. How do we explain the apparent paradox of SLA being identified as α-enolase by proteomic analysis and as tRNP (Ser)Sec by the screening of cDNA libraries? Do α-enolase and tRNP (Ser)Sec cross-react?
In the present study, we have investigated reactivity of SLA positive sera against α-enolase and tRNP (Ser)Sec using rat and primate liver homogenate and the recombinant antigens.

Antibody Detection
All sera have been tested for conventional antibodies by indirect IFL using rodent liver, kidney, stomach tissues. SLA antibodies were detected by a modified inhibition ELISA [1] and confirmed by immunoblot using human liver homogenate [20]. Autoantibody reactivity was further evaluated using preparations of primate (Euroimmun, Lubeck, Germany) and rat (AID Autoimmun Diagnostika GmbH, Strassberg, Germany) liver homogenates, according to manufacturers' instructions. Anti-αenolase antibody reactivity has been tested by immunoblot using recombinant α-enolase, as described previously [17]. Briefly, the complete complementary DNA (cDNA) encoding human α-enolase was isolated from a cDNA expression library derived from synoviocytes obtained from a patient with rheumatoid arthritis (Stratagene, La Jolla, CA) and immunoscreened with goat anti-enolase antibodies. This cDNA was subcloned in frame in the pSPUTK in vitro translation vector (Stratagene) using the Apa I and Bam HI restriction sites. The translation product was synthesized as a separated biotinylated polypeptide, purified by SoftLink Soft Release Avidin Resin (Promega, Madison, WI), migrated in SDS-PAGE, according to the method of Laemmli, and electrotransferred onto a nitrocellulose membrane [17]. The filters were then incubated with goat anti-enolase antibodies (see below), a monospecific anti-α-enolase antibody positive serum from a patient with rheumatoid arthritis or with individual serum samples, in Tris buffered saline, 0.05% Tween 20, 5% dry milk for 2 hours. After washing, the filters were incubated for 1 hour with 1:15,000-diluted peroxidaseconjugated goat anti-human IgG (Sigma-Aldrich) in 0.05% TBST-milk. The filters were washed and revealed by a chemiluminescence reaction (Supersignal; Pierce, Rockford, IL) [17].
An affinity purified goat polyclonal anti-α-enolase IgG antibody raised against a peptide mapping near the car-boxyl-terminus of human α-enolase, which is common to α, β, and γ isoforms of mouse, rat and human enolase (200 µg/ml; Santa Cruz Biotechnology, Santa Cruz, California, USA) was used as reference serum sample at a dilution of 1:100, according to the manufacturer's instructions.
Anti-tRNP (Ser)Sec antibody reactivity was detected by ELISA or dot blot using recombinant tRNP (Ser)Sec antigen (Euroimmun). A high-titre anti-tRNP (Ser)Sec antibody positive serum was used as a positive control.
Immunoblot patterns produced by anti-α-enolases and anti-tRNP (Ser)Sec antibodies on electrophoretically separated primate and rat liver homogenates and dot blot results with recombinant tRNP (Ser)Sec Figure 1 Immunoblot patterns produced by anti-α-enolases and anti-tRNP (Ser)Sec antibodies on electrophoretically separated primate and rat liver homogenates and dot blot results with recombinant tRNP (Ser)Sec . In both rat and primate liver preparations, a band of ~48 kDa is immunofixed by a polyclonal goat IgG anti-α-enolase specific antibody; a band of ~50 kDa is immunofixed by a serum containing a high-titre anti-tRNP (Ser)Sec antibody. Anti-α-enolase antibody does not recognize tRNP (Ser)Sec by dot blot analysis.

Results and Discussion
The affinity purified IgG antibody directed to human αenolase gave a band of approximately 48 kDa in both human and rat liver homogenates as shown in Figure 1. A high titre anti-tRNP (Ser)Sec antibody serum gave a single band of ~50 kDa in both liver preparations ( Figure 1). All but one anti-SLA antibody positive sera reacted with a ~50 kDa band similar to that obtained by the high titre anti-tRNP (Ser)Sec antibody serum in rat liver preparations (Figure 2) but none immunofixed a 48 kDa band. All anti-SLA antibody positive sera reacted strongly with the recombinant full length tRNP (Ser)Sec protein both in ELISA (mean titre 87 ± 23 RU/ml, cut off: 20 RU/ml) and dot blot. None of the anti-SLA negative sera reacted with tRNP (Ser)Sec . Anti-SLA positive, and anti-SLA negative sera reacted equally against recombinant α-enolase by immunoblot with 5/9 cases in each group giving a strong band ( Figure 3).
Pre-incubation of anti-SLA positive sera with tRNP (Ser)Sec completely abolished the 50 kDa band immunofixed in either rat or human liver preparation. In contrast, a parallel experiment where the anti-α-enolase antiserum was pre-incubated with recombinant tRNP (Ser)Sec left unaltered the reactivity to the 48 kDa band.
However, Ballot et al [11] state that α-enolase is a major SLA antigen since rat α-enolases have MW of 47.4 to 47.5 and Pi values of 5.8 to 6.2. These characteristics do not match the MW (48.8) and Pi (8.6) of tRNP (Ser)Sec . Ballot et al [11], however, show a Coomassie stained 2D-gel over the Pi range 6 to 11 and an immunoblot of this gel with several bands of a Pi above 8, but have not investigated these bands by MALDI-TOF analysis being therefore unable to rule out their possible relation to tRNP (Ser)Sec .

Conclusion
All the above observations indicate that tRNP (Ser)Sec is the most likely target of anti-SLA.

List of Abbreviations
AIH, autoimmune hepatitis; SLA, soluble liver antigen; tRNP (Ser)Sec , tRNA-associated antigenic protein Immunoblot patterns obtained using 9 SLA positive and 9 SLA negative serum samples against recombinant α-enolase Figure 3 Immunoblot patterns obtained using 9 SLA positive and 9 SLA negative serum samples against recombinant α-enolase. A polyclonal goat IgG anti-α-enolase specific antibody has been used as a reference positive serum. Ab, antibody; ag, antigen Anti--enolase ab SLA negative sera SLA positive sera Publish with Bio Med Central and every scientist can read your work free of charge