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Antibodies to soluble liver antigen and α-enolase in patients with autoimmune hepatitis

Antibodies to soluble liver antigen and α-enolase in patients with autoimmune hepatitis 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 (Ser)Sec protein complex (tRNP ), through the screening of cDNA libraries. A recent report questioned the identity (Ser)Sec of tRNP 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 (Ser)Sec against α-enolase and tRNP 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 (Ser)Sec polyclonal anti-α-enolase IgG antibody was used as reference serum sample. Anti-tRNP antibody reactivity (Ser)Sec was detected by ELISA or dot blot using recombinant tRNP antigen. Results and Discussion: The affinity purified IgG antibody directed to human α-enolase gave a band of (Ser)Sec approximately 48 kDa in both human and rat liver homogenates. A high titre anti-tRNP 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 (Ser)Sec (Ser)Sec recombinant full length tRNP protein. None of the anti-SLA negative sera reacted with tRNP . Anti- SLA positive, and anti-SLA negative sera reacted equally against recombinant α-enolase by immunoblot. Pre- (Ser)Sec incubation of anti-SLA positive sera with tRNP completely abolished the 50 kDa band. The findings of the (Ser)Sec present study indicate that α-enolase and tRNP are both expressed in primate and rat liver and have a (Ser)Sec respective MW of 48 and 50 kDa. They also show that anti-tRNP – but not anti-α-enolase – correlates with anti-SLA antibody reactivity. (Ser)Sec Conclusion: Our findings indicate that tRNP is the most likely target of anti-SLA. Page 1 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:4 http://www.jautoimdis.com/content/1/1/4 3. What is the reactivity of SLA positive and negative sera Background Antibodies to a cytosolic soluble liver antigen (SLA), against recombinant α-enolase? detected originally by an inhibition ELISA using cytosolic liver fractions in a sub-group of patients with autoim- 4. How do we explain the apparent paradox of SLA being mune hepatitis (AIH) negative for other autoantibodies, identified as α-enolase by proteomic analysis and as (Ser)Sec have recently been also reported in adult patients with tRNP by the screening of cDNA libraries? Do α-eno- (Ser)Sec anti-nuclear and/or smooth muscle antibody (ANA/SMA) lase and tRNP cross-react? positive type 1 AIH and in seronegative patients with a form of cryptogenic hepatitis resembling type 1 AIH [1-6]. In the present study, we have investigated reactivity of SLA (Ser)Sec In pediatric patients, anti-SLA has been described not only positive sera against α-enolase and tRNP using rat in type 1 AIH but also in anti-liver kidney microsomal-1 and primate liver homogenate and the recombinant antibody positive type 2 AIH and autoimmune sclerosing antigens. cholangitis [7-10]. Anti-SLA is specific for these autoim- mune liver diseases, where it is associated with a more Methods Patients severe course and is virtually absent in non-hepatic autoimmune disorders [1-9]. The target of anti-SLA has Thirty-three serum samples, 11 from SLA-positive patients been identified by several groups as a ~50 kDa UGA serine and 22 from SLA negative controls were investigated. SLA- (Ser)Sec tRNA-associated protein complex (tRNP ), through positive patients included 8 paediatric patients with AIH1 (Ser)Sec the screening of cDNA libraries [2-4,7]. Anti-tRNP [7 female, median age 12, range 5–17 years, all ANA pos- antibodies have been detected in up to 90% of serum itive, median immunofluorescence (IFL) titre: 1/320, samples positive for SLA by the original inhibition ELISA range 1/80–1/1280] and 3 adults with AIH/PBC overlap [1-8]. syndrome, (2 female, median age 56, range 47–65), all but one AMA positive (1/5120), and ANA positive Using anti-SLA positive sera against rat liver cytosolic frac- (median tire 1/320, range 1/80–1/640). Eleven case- tion in one and two-dimensional immunoblotting analy- matched SLA negative patients were tested as pathological ses and through peptide mass fingerprint analysis, controls including 8 with AIH1 and 3 with AIH/PBC over- following MALDI-TOF mass spectrometry, Ballot et al. lap syndrome. Eleven demographically matched healthy [11] identified four isoforms of α-enolase, – a cytosolic subjects including 8 children (7 female, median age 11, antigen of 48–50 kDa –, as the major target of anti-SLA range 6–16) and 3 adults (2 female, median age 53, range positive sera. These findings challenge the notion that 42–63) negative for SLA were also tested as controls. (Ser)Sec tRNP is the sole target of anti-SLA antibodies [2-8]. Critically, no absorption studies were performed with Antibody Detection purified α-enolase to confirm this proposal [11]. Moreo- All sera have been tested for conventional antibodies by ver, α-enolase has been described as an antigen in several indirect IFL using rodent liver, kidney, stomach tissues. autoimmune disorders totally unrelated to autoimmune hepatitis [12-18]. SLA antibodies were detected by a modified inhibition ELISA [1] and confirmed by immunoblot using human (Ser)Sec Using recombinant tRNP antigen as competitor in liver homogenate [20]. Autoantibody reactivity was fur- inhibition experiments it has been found removal of the ther evaluated using preparations of primate (Euroim- 50 kDa band immunofixed by SLA positive sera from mun, Lubeck, Germany) and rat (AID Autoimmun immunoblots of primate liver homogenate [19]. Though Diagnostika GmbH, Strassberg, Germany) liver homoge- (Ser)Sec this finding indicates tRNP as a major component of nates, according to manufacturers' instructions. Anti-α- SLA, a view apparently shared by Ballot et al, several ques- enolase antibody reactivity has been tested by immunob- tions still remain unanswered: lot using recombinant α-enolase, as described previously [17]. Briefly, the complete complementary DNA (cDNA) 1. Are there any differences in α-enolase expression encoding human α-enolase was isolated from a cDNA between rat – used by Ballot et al [11] – and primate liver expression library derived from synoviocytes obtained homogenate – used by our study [19] – that could explain from a patient with rheumatoid arthritis (Stratagene, La the discrepancy between these studies? Jolla, CA) and immunoscreened with goat anti-enolase antibodies. This cDNA was subcloned in frame in the 2. Is it true that failure of proteomic analysis to detect pSPUTK in vitro translation vector (Stratagene) using the (Ser)Sec tRNP is due to its presence in trace amounts in the Apa I and Bam HI restriction sites. The translation product supernatant of liver homogenate [11]? was synthesized as a separated biotinylated polypeptide, purified by SoftLink Soft Release Avidin Resin (Promega, Madison, WI), migrated in SDS-PAGE, according to the Page 2 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:4 http://www.jautoimdis.com/content/1/1/4 1 2 50 kDa Human liver ~ 48 kDa Rat liver (Ser)Sec tRNP ag (Ser)Sec Goat polyclonal anti-Į enolase ab 1 2 Anti-tRNP ab positive serum (Ser)Sec(S er)Sec and rat liv Figure 1 Immunoblot p er homogenates and dot b atterns produced by an lot results ti-α-enolawith recombin ses and anti-tRN ant tRNP P antibodies on electrophoretically separated primate (Ser)Sec Immunoblot patterns produced by anti-α-enolases and anti-tRNP antibodies on electrophoretically separated primate (Ser)Sec and rat liver homogenates and dot blot results with recombinant tRNP . 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 (Ser)Sec (Ser)Sec serum containing a high-titre anti-tRNP antibody. Anti-α-enolase antibody does not recognize tRNP by dot blot analysis. method of Laemmli, and electrotransferred onto a nitro- boxyl-terminus of human α-enolase, which is common to cellulose membrane [17]. The filters were then incubated α, β, and γ isoforms of mouse, rat and human enolase with goat anti-enolase antibodies (see below), a mono- (200 µg/ml; Santa Cruz Biotechnology, Santa Cruz, Cali- specific anti-α-enolase antibody positive serum from a fornia, USA) was used as reference serum sample at a dilu- patient with rheumatoid arthritis or with individual tion of 1:100, according to the manufacturer's serum samples, in Tris buffered saline, 0.05% Tween 20, instructions. 5% dry milk for 2 hours. After washing, the filters were (Ser)Sec incubated for 1 hour with 1:15,000-diluted peroxidase- Anti-tRNP antibody reactivity was detected by ELISA (Ser)Sec conjugated goat anti-human IgG (Sigma-Aldrich) in or dot blot using recombinant tRNP antigen (Ser)Sec 0.05% TBST-milk. The filters were washed and revealed by (Euroimmun). A high-titre anti-tRNP antibody pos- a chemiluminescence reaction (Supersignal; Pierce, Rock- itive serum was used as a positive control. ford, IL) [17]. An affinity purified goat polyclonal anti-α-enolase IgG antibody raised against a peptide mapping near the car- Page 3 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:4 http://www.jautoimdis.com/content/1/1/4 Rat liver homogenate 12 12 335446 (Ser)Sec an (la Immunoblot p Figure 2 ti nes 2–5) four -tRNP atterns an representative anti-solubl tiboproduced on dy rat liver hom e liver o antigen (SLA) posit genate by (lane 1) ia polyclon ve sera; (laal g ne o 6) a at Ig reference serum G anti-α-enolaseconta speciifning h ic antibo igh-titr dy; e Immunoblot patterns produced on rat liver homogenate by (lane 1) a polyclonal goat IgG anti-α-enolase specific antibody; (lanes 2–5) four representative anti-soluble liver antigen (SLA) positive sera; (lane 6) a reference serum containing high-titre (Ser)Sec anti-tRNP antibody. Page 4 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:4 http://www.jautoimdis.com/content/1/1/4 Anti-Į -enolase ab SLA positive sera SLA negative sera Immunoblot p Figure 3 atterns obtained using 9 SLA positive and 9 SLA negative serum samples against recombinant α-enolase Immunoblot patterns obtained using 9 SLA positive and 9 SLA negative serum samples against recombinant α-enolase. A poly- clonal goat IgG anti-α-enolase specific antibody has been used as a reference positive serum. Ab, antibody; ag, antigen Inhibition Studies However, Ballot et al [11] state that α-enolase is a major To investigate whether the 50 kDa band immunofixed by SLA antigen since rat α-enolases have MW of 47.4 to 47.5 (Ser)Sec anti-SLA is tRNP , inhibition experiments were per- and Pi values of 5.8 to 6.2. These characteristics do not (Ser)Sec formed using 3 anti-SLA positive serum samples, diluted match the MW (48.8) and Pi (8.6) of tRNP . Ballot et at 1/1000, and pre-incubated with solid phase recom- al [11], however, show a Coomassie stained 2D-gel over (Ser)Sec binant tRNP (Euroimmun, UK), as previously the Pi range 6 to 11 and an immunoblot of this gel with described [19]. several bands of a Pi above 8, but have not investigated these bands by MALDI-TOF analysis being therefore una- (Ser)Sec Results and Discussion ble to rule out their possible relation to tRNP . The affinity purified IgG antibody directed to human α- enolase gave a band of approximately 48 kDa in both Conclusion (Ser)Sec human and rat liver homogenates as shown in Figure 1. A All the above observations indicate that tRNP is the (Ser)Sec antibody serum gave a single most likely target of anti-SLA. high titre anti-tRNP band of ~50 kDa in both liver preparations (Figure 1). All but one anti-SLA antibody positive sera reacted with a ~50 List of Abbreviations kDa band similar to that obtained by the high titre anti- AIH, autoimmune hepatitis; SLA, soluble liver antigen; (Ser)Sec (Ser)Sec tRNP antibody serum in rat liver preparations (Fig- tRNP , tRNA-associated antigenic protein ure 2) but none immunofixed a 48 kDa band. All anti-SLA antibody positive sera reacted strongly with the recom- Competing Interests (Ser)Sec binant full length tRNP protein both in ELISA The author(s) declare that they have no competing (mean titre 87 ± 23 RU/ml, cut off: 20 RU/ml) and dot interests. blot. None of the anti-SLA negative sera reacted with (Ser)Sec tRNP . Anti-SLA positive, and anti-SLA negative sera Authors' Contributions reacted equally against recombinant α-enolase by immu- DPB designed the study, performed the ELISA and inhibi- tion experiments and contributed in writing the report. noblot with 5/9 cases in each group giving a strong band (Figure 3). DG performed the immunoblot testing of anti-enolase antibodies. IB, SL & YM performed the immunoblot (Ser)Sec Pre-incubation of anti-SLA positive sera with tRNP experiments involving liver preparations. RRM helped completely abolished the 50 kDa band immunofixed in with the artwork. GMV provided clinical material and either rat or human liver preparation. In contrast, a paral- contributed in writing the report. DV supervised the study lel experiment where the anti-α-enolase antiserum was and wrote the report. (Ser)Sec pre-incubated with recombinant tRNP left unaltered the reactivity to the 48 kDa band. Acknowledgements We thank Professor Huub J.M. Op den Camp (Department of Microbiol- ogy, University of Nijmegen, The Netherlands) for helpful comments. DP The findings of the present study indicate that α-enolase Bogdanos and G Mieli-Vergani are supported by the Children's Liver Dis- (Ser)Sec and tRNP are both expressed in primate and rat liver ease Foundation (Birmingham, U.K.). G Mieli-Vergani is also supported by and have a respective MW of 48 and 50 kDa. They also WellChild (London, U.K.). Y Ma is a Dorothy Hodgkin Fellow of the Royal (Ser)Sec show that anti-tRNP – but not anti-α-enolase – cor- Society (London, U.K.). relates with anti-SLA antibody reactivity suggesting that (Ser)Sec the target of anti-SLA antibody is tRNP and not α- enolase [1-9,19]. Page 5 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:4 http://www.jautoimdis.com/content/1/1/4 20. Bogdanos DP, Pares A, Baum H, Caballeria L, Rigopoulou EI, Ma Y, References Burroughs AK, Rodes J, Vergani D: Disease-specific cross-reactiv- 1. Manns M, Gerken G, Kyriatsoulis A, Staritz M, Meyer zum Buschen- ity between mimicking peptides of heat shock protein of felde KH: Characterisation of a new subgroup of autoimmune mycobacterium gordonae and dominant epitope of E2 subu- chronic active hepatitis by autoantibodies against a soluble nit of pyruvate dehydrogenase is common in Spanish but not liver antigen. Lancet 1987, 1:292-294. British patients with primary biliary cirrhosis. J Autoimmun 2. Wies I, Brunner S, Henninger J, Herkel J, Kanzler S, Meyer zum 2004, 22:353-362. Buschenfelde KH, Lohse AW: Identification of target antigen for SLA/LP autoantibodies in autoimmune hepatitis. Lancet 2000, 355:1510-1515. 3. Costa M, Rodriguez-Sanchez JL, Czaja AJ, Gelpi C: Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec complex recognized by autoanti- bodies from patients withtype-1 autoimmune hepatitis. Clin Exp Immunol 2000, 121:364-374. 4. Wen L, Ma Y, Bogdanos DP, Wong FS, Demaine A, Mieli-Vergani G, Vergani D: Pediatric autoimmune liver diseases: the molecu- lar basis of humoral and cellular immunity. Curr Mol Med 2001, 1:379-389. 5. Volkmann M, Martin L, Baurle A, Heid H, Strassburg CP, Trautwein C, Fiehn W, Manns MP: Soluble liver antigen: isolation of a 35- kd recombinant protein (SLA-p35) specifically recognizing sera from patients with autoimmune hepatitis. Hepatology 2001, 33:591-596. 6. Ma Y, Okamoto M, Thomas MG, Bogdanos DP, Lopes AR, Portmann B, Underhill J, Durr R, Mieli-Vergani G, Vergani D: Antibodies to conformational epitopes of soluble liver antigen define a severe form of autoimmune liver disease. Hepatology 2002, 35:658-664. 7. Vitozzi S, Djilali-Saiah I, Lapierre P, Alvarez F: Anti-soluble liver antigen/liver-pancreas (SLA/LP) antibodies in pediatric patients with autoimmune hepatitis. Autoimmunity 2002, 35:485-492. 8. Czaja AJ, Shums Z, Norman GL: Frequency and significance of antibodies to soluble liver antigen/liver pancreas in variant autoimmune hepatitis. Autoimmunity 2002, 35:475-483. 9. Baeres M, Herkel J, Czaja AJ, Wies I, Kanzler S, Cancado EL, Porta G, Nishioka M, Simon T, Daehnrich C, Schlumberger W, Galle PR, Lohse AW: Establishment of standardised SLA/LP immunoassays: specificity for autoimmune hepatitis, worldwide occurrence, and clinical characteristics. Gut 2002, 51:259-264. 10. Vergani D, Choudhuri K, Bogdanos DP, Mieli-Vergani G: Pathogen- esis of autoimmune hepatitis. Clin Liver Dis 2002, 6:439-449. 11. Ballot E, Bruneel A, Labas V, Johanet C: Identification of rat tar- gets of anti-soluble liver antigen autoantibodies by serologic proteome analysis. Clin Chem 2003, 49:634-643. 12. Adamus G, Amundson D, Seigel GM, Machnicki M: Anti-enolase- alpha autoantibodies in cancer-associated retinopathy: epitope mapping and cytotoxicity on retinal cells. J Autoimmun 1998, 11:671-677. 13. Lee KH, Chung HS, Kim HS, Oh SH, Ha MK, Baik JH, Lee S, Bang D: Human alpha-enolase from endothelial cells as a target anti- gen of anti-endothelial cell antibody in Behcet's disease. Arthritis Rheum 2003, 48:2025-2035. 14. Moodie FD, Leaker B, Cambridge G, Totty NF, Segal AW: Alpha- enolase: a novel cytosolic autoantigen in ANCA positive vasculitis. Kidney Int 1993, 43:675-681. 15. Pratesi F, Moscato S, Sabbatini A, Chimenti D, Bombardieri S, Migl- iorini P: Autoantibodies specific for alpha-enolase in systemic autoimmune disorders. J Rheumatol 2000, 27:109-115. 16. Roozendaal C, Zhao MH, Horst G, Lockwood CM, Kleibeuker JH, Publish with Bio Med Central and every Limburg PC, Nelis GF, Kallenberg CG: Catalase and alpha-eno- lase: two novel granulocyte autoantigens in inflammatory scientist can read your work free of charge bowel disease (IBD). Clin Exp Immunol 1998, 112:10-16. "BioMed Central will be the most significant development for 17. Saulot V, Vittecoq O, Charlionet R, Fardellone P, Lange C, Marvin L, Machour N, Le Loet X, Gilbert D, Tron F: Presence of autoanti- disseminating the results of biomedical researc h in our lifetime." bodies to the glycolytic enzyme alpha-enolase in sera from Sir Paul Nurse, Cancer Research UK patients with early rheumatoid arthritis. Arthritis Rheum 2002, 46:1196-1201. Your research papers will be: 18. Tanaka S, Tatsumi KI, Takano T, Murakami Y, Takao T, Yamakita N, available free of charge to the entire biomedical community Tahara S, Teramoto A, Hashimoto K, Kato Y, Amino N: Anti-alpha- peer reviewed and published immediately upon acceptance enolase antibodies in pituitary disease. Endocr J 2003, 50:697-702. cited in PubMed and archived on PubMed Central 19. Bogdanos DP, Bianchi I, Ma Y, Mitry RR, Mieli-Vergani G, Vergani D: yours — you keep the copyright Targets of antibodies to soluble liver antigen in patients with autoimmune hepatitis. Clin Chem 2004, 50:682-3; author reply BioMedcentral Submit your manuscript here: 683-4. http://www.biomedcentral.com/info/publishing_adv.asp Page 6 of 6 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Autoimmune Diseases Springer Journals

Antibodies to soluble liver antigen and α-enolase in patients with autoimmune hepatitis

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Springer Journals
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Copyright © 2004 by Bogdanos et al; licensee BioMed Central Ltd.
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Biomedicine; Immunology; Internal Medicine
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1740-2557
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10.1186/1740-2557-1-4
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Abstract

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 (Ser)Sec protein complex (tRNP ), through the screening of cDNA libraries. A recent report questioned the identity (Ser)Sec of tRNP 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 (Ser)Sec against α-enolase and tRNP 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 (Ser)Sec polyclonal anti-α-enolase IgG antibody was used as reference serum sample. Anti-tRNP antibody reactivity (Ser)Sec was detected by ELISA or dot blot using recombinant tRNP antigen. Results and Discussion: The affinity purified IgG antibody directed to human α-enolase gave a band of (Ser)Sec approximately 48 kDa in both human and rat liver homogenates. A high titre anti-tRNP 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 (Ser)Sec (Ser)Sec recombinant full length tRNP protein. None of the anti-SLA negative sera reacted with tRNP . Anti- SLA positive, and anti-SLA negative sera reacted equally against recombinant α-enolase by immunoblot. Pre- (Ser)Sec incubation of anti-SLA positive sera with tRNP completely abolished the 50 kDa band. The findings of the (Ser)Sec present study indicate that α-enolase and tRNP are both expressed in primate and rat liver and have a (Ser)Sec respective MW of 48 and 50 kDa. They also show that anti-tRNP – but not anti-α-enolase – correlates with anti-SLA antibody reactivity. (Ser)Sec Conclusion: Our findings indicate that tRNP is the most likely target of anti-SLA. Page 1 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:4 http://www.jautoimdis.com/content/1/1/4 3. What is the reactivity of SLA positive and negative sera Background Antibodies to a cytosolic soluble liver antigen (SLA), against recombinant α-enolase? detected originally by an inhibition ELISA using cytosolic liver fractions in a sub-group of patients with autoim- 4. How do we explain the apparent paradox of SLA being mune hepatitis (AIH) negative for other autoantibodies, identified as α-enolase by proteomic analysis and as (Ser)Sec have recently been also reported in adult patients with tRNP by the screening of cDNA libraries? Do α-eno- (Ser)Sec anti-nuclear and/or smooth muscle antibody (ANA/SMA) lase and tRNP cross-react? positive type 1 AIH and in seronegative patients with a form of cryptogenic hepatitis resembling type 1 AIH [1-6]. In the present study, we have investigated reactivity of SLA (Ser)Sec In pediatric patients, anti-SLA has been described not only positive sera against α-enolase and tRNP using rat in type 1 AIH but also in anti-liver kidney microsomal-1 and primate liver homogenate and the recombinant antibody positive type 2 AIH and autoimmune sclerosing antigens. cholangitis [7-10]. Anti-SLA is specific for these autoim- mune liver diseases, where it is associated with a more Methods Patients severe course and is virtually absent in non-hepatic autoimmune disorders [1-9]. The target of anti-SLA has Thirty-three serum samples, 11 from SLA-positive patients been identified by several groups as a ~50 kDa UGA serine and 22 from SLA negative controls were investigated. SLA- (Ser)Sec tRNA-associated protein complex (tRNP ), through positive patients included 8 paediatric patients with AIH1 (Ser)Sec the screening of cDNA libraries [2-4,7]. Anti-tRNP [7 female, median age 12, range 5–17 years, all ANA pos- antibodies have been detected in up to 90% of serum itive, median immunofluorescence (IFL) titre: 1/320, samples positive for SLA by the original inhibition ELISA range 1/80–1/1280] and 3 adults with AIH/PBC overlap [1-8]. syndrome, (2 female, median age 56, range 47–65), all but one AMA positive (1/5120), and ANA positive Using anti-SLA positive sera against rat liver cytosolic frac- (median tire 1/320, range 1/80–1/640). Eleven case- tion in one and two-dimensional immunoblotting analy- matched SLA negative patients were tested as pathological ses and through peptide mass fingerprint analysis, controls including 8 with AIH1 and 3 with AIH/PBC over- following MALDI-TOF mass spectrometry, Ballot et al. lap syndrome. Eleven demographically matched healthy [11] identified four isoforms of α-enolase, – a cytosolic subjects including 8 children (7 female, median age 11, antigen of 48–50 kDa –, as the major target of anti-SLA range 6–16) and 3 adults (2 female, median age 53, range positive sera. These findings challenge the notion that 42–63) negative for SLA were also tested as controls. (Ser)Sec tRNP is the sole target of anti-SLA antibodies [2-8]. Critically, no absorption studies were performed with Antibody Detection purified α-enolase to confirm this proposal [11]. Moreo- All sera have been tested for conventional antibodies by ver, α-enolase has been described as an antigen in several indirect IFL using rodent liver, kidney, stomach tissues. autoimmune disorders totally unrelated to autoimmune hepatitis [12-18]. SLA antibodies were detected by a modified inhibition ELISA [1] and confirmed by immunoblot using human (Ser)Sec Using recombinant tRNP antigen as competitor in liver homogenate [20]. Autoantibody reactivity was fur- inhibition experiments it has been found removal of the ther evaluated using preparations of primate (Euroim- 50 kDa band immunofixed by SLA positive sera from mun, Lubeck, Germany) and rat (AID Autoimmun immunoblots of primate liver homogenate [19]. Though Diagnostika GmbH, Strassberg, Germany) liver homoge- (Ser)Sec this finding indicates tRNP as a major component of nates, according to manufacturers' instructions. Anti-α- SLA, a view apparently shared by Ballot et al, several ques- enolase antibody reactivity has been tested by immunob- tions still remain unanswered: lot using recombinant α-enolase, as described previously [17]. Briefly, the complete complementary DNA (cDNA) 1. Are there any differences in α-enolase expression encoding human α-enolase was isolated from a cDNA between rat – used by Ballot et al [11] – and primate liver expression library derived from synoviocytes obtained homogenate – used by our study [19] – that could explain from a patient with rheumatoid arthritis (Stratagene, La the discrepancy between these studies? Jolla, CA) and immunoscreened with goat anti-enolase antibodies. This cDNA was subcloned in frame in the 2. Is it true that failure of proteomic analysis to detect pSPUTK in vitro translation vector (Stratagene) using the (Ser)Sec tRNP is due to its presence in trace amounts in the Apa I and Bam HI restriction sites. The translation product supernatant of liver homogenate [11]? was synthesized as a separated biotinylated polypeptide, purified by SoftLink Soft Release Avidin Resin (Promega, Madison, WI), migrated in SDS-PAGE, according to the Page 2 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:4 http://www.jautoimdis.com/content/1/1/4 1 2 50 kDa Human liver ~ 48 kDa Rat liver (Ser)Sec tRNP ag (Ser)Sec Goat polyclonal anti-Į enolase ab 1 2 Anti-tRNP ab positive serum (Ser)Sec(S er)Sec and rat liv Figure 1 Immunoblot p er homogenates and dot b atterns produced by an lot results ti-α-enolawith recombin ses and anti-tRN ant tRNP P antibodies on electrophoretically separated primate (Ser)Sec Immunoblot patterns produced by anti-α-enolases and anti-tRNP antibodies on electrophoretically separated primate (Ser)Sec and rat liver homogenates and dot blot results with recombinant tRNP . 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 (Ser)Sec (Ser)Sec serum containing a high-titre anti-tRNP antibody. Anti-α-enolase antibody does not recognize tRNP by dot blot analysis. method of Laemmli, and electrotransferred onto a nitro- boxyl-terminus of human α-enolase, which is common to cellulose membrane [17]. The filters were then incubated α, β, and γ isoforms of mouse, rat and human enolase with goat anti-enolase antibodies (see below), a mono- (200 µg/ml; Santa Cruz Biotechnology, Santa Cruz, Cali- specific anti-α-enolase antibody positive serum from a fornia, USA) was used as reference serum sample at a dilu- patient with rheumatoid arthritis or with individual tion of 1:100, according to the manufacturer's serum samples, in Tris buffered saline, 0.05% Tween 20, instructions. 5% dry milk for 2 hours. After washing, the filters were (Ser)Sec incubated for 1 hour with 1:15,000-diluted peroxidase- Anti-tRNP antibody reactivity was detected by ELISA (Ser)Sec conjugated goat anti-human IgG (Sigma-Aldrich) in or dot blot using recombinant tRNP antigen (Ser)Sec 0.05% TBST-milk. The filters were washed and revealed by (Euroimmun). A high-titre anti-tRNP antibody pos- a chemiluminescence reaction (Supersignal; Pierce, Rock- itive serum was used as a positive control. ford, IL) [17]. An affinity purified goat polyclonal anti-α-enolase IgG antibody raised against a peptide mapping near the car- Page 3 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:4 http://www.jautoimdis.com/content/1/1/4 Rat liver homogenate 12 12 335446 (Ser)Sec an (la Immunoblot p Figure 2 ti nes 2–5) four -tRNP atterns an representative anti-solubl tiboproduced on dy rat liver hom e liver o antigen (SLA) posit genate by (lane 1) ia polyclon ve sera; (laal g ne o 6) a at Ig reference serum G anti-α-enolaseconta speciifning h ic antibo igh-titr dy; e Immunoblot patterns produced on rat liver homogenate by (lane 1) a polyclonal goat IgG anti-α-enolase specific antibody; (lanes 2–5) four representative anti-soluble liver antigen (SLA) positive sera; (lane 6) a reference serum containing high-titre (Ser)Sec anti-tRNP antibody. Page 4 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:4 http://www.jautoimdis.com/content/1/1/4 Anti-Į -enolase ab SLA positive sera SLA negative sera Immunoblot p Figure 3 atterns obtained using 9 SLA positive and 9 SLA negative serum samples against recombinant α-enolase Immunoblot patterns obtained using 9 SLA positive and 9 SLA negative serum samples against recombinant α-enolase. A poly- clonal goat IgG anti-α-enolase specific antibody has been used as a reference positive serum. Ab, antibody; ag, antigen Inhibition Studies However, Ballot et al [11] state that α-enolase is a major To investigate whether the 50 kDa band immunofixed by SLA antigen since rat α-enolases have MW of 47.4 to 47.5 (Ser)Sec anti-SLA is tRNP , inhibition experiments were per- and Pi values of 5.8 to 6.2. These characteristics do not (Ser)Sec formed using 3 anti-SLA positive serum samples, diluted match the MW (48.8) and Pi (8.6) of tRNP . Ballot et at 1/1000, and pre-incubated with solid phase recom- al [11], however, show a Coomassie stained 2D-gel over (Ser)Sec binant tRNP (Euroimmun, UK), as previously the Pi range 6 to 11 and an immunoblot of this gel with described [19]. several bands of a Pi above 8, but have not investigated these bands by MALDI-TOF analysis being therefore una- (Ser)Sec Results and Discussion ble to rule out their possible relation to tRNP . The affinity purified IgG antibody directed to human α- enolase gave a band of approximately 48 kDa in both Conclusion (Ser)Sec human and rat liver homogenates as shown in Figure 1. A All the above observations indicate that tRNP is the (Ser)Sec antibody serum gave a single most likely target of anti-SLA. high titre anti-tRNP band of ~50 kDa in both liver preparations (Figure 1). All but one anti-SLA antibody positive sera reacted with a ~50 List of Abbreviations kDa band similar to that obtained by the high titre anti- AIH, autoimmune hepatitis; SLA, soluble liver antigen; (Ser)Sec (Ser)Sec tRNP antibody serum in rat liver preparations (Fig- tRNP , tRNA-associated antigenic protein ure 2) but none immunofixed a 48 kDa band. All anti-SLA antibody positive sera reacted strongly with the recom- Competing Interests (Ser)Sec binant full length tRNP protein both in ELISA The author(s) declare that they have no competing (mean titre 87 ± 23 RU/ml, cut off: 20 RU/ml) and dot interests. blot. None of the anti-SLA negative sera reacted with (Ser)Sec tRNP . Anti-SLA positive, and anti-SLA negative sera Authors' Contributions reacted equally against recombinant α-enolase by immu- DPB designed the study, performed the ELISA and inhibi- tion experiments and contributed in writing the report. noblot with 5/9 cases in each group giving a strong band (Figure 3). DG performed the immunoblot testing of anti-enolase antibodies. IB, SL & YM performed the immunoblot (Ser)Sec Pre-incubation of anti-SLA positive sera with tRNP experiments involving liver preparations. RRM helped completely abolished the 50 kDa band immunofixed in with the artwork. GMV provided clinical material and either rat or human liver preparation. In contrast, a paral- contributed in writing the report. DV supervised the study lel experiment where the anti-α-enolase antiserum was and wrote the report. (Ser)Sec pre-incubated with recombinant tRNP left unaltered the reactivity to the 48 kDa band. Acknowledgements We thank Professor Huub J.M. Op den Camp (Department of Microbiol- ogy, University of Nijmegen, The Netherlands) for helpful comments. DP The findings of the present study indicate that α-enolase Bogdanos and G Mieli-Vergani are supported by the Children's Liver Dis- (Ser)Sec and tRNP are both expressed in primate and rat liver ease Foundation (Birmingham, U.K.). G Mieli-Vergani is also supported by and have a respective MW of 48 and 50 kDa. They also WellChild (London, U.K.). Y Ma is a Dorothy Hodgkin Fellow of the Royal (Ser)Sec show that anti-tRNP – but not anti-α-enolase – cor- Society (London, U.K.). relates with anti-SLA antibody reactivity suggesting that (Ser)Sec the target of anti-SLA antibody is tRNP and not α- enolase [1-9,19]. Page 5 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2004, 1:4 http://www.jautoimdis.com/content/1/1/4 20. Bogdanos DP, Pares A, Baum H, Caballeria L, Rigopoulou EI, Ma Y, References Burroughs AK, Rodes J, Vergani D: Disease-specific cross-reactiv- 1. 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Saulot V, Vittecoq O, Charlionet R, Fardellone P, Lange C, Marvin L, Machour N, Le Loet X, Gilbert D, Tron F: Presence of autoanti- disseminating the results of biomedical researc h in our lifetime." bodies to the glycolytic enzyme alpha-enolase in sera from Sir Paul Nurse, Cancer Research UK patients with early rheumatoid arthritis. Arthritis Rheum 2002, 46:1196-1201. Your research papers will be: 18. Tanaka S, Tatsumi KI, Takano T, Murakami Y, Takao T, Yamakita N, available free of charge to the entire biomedical community Tahara S, Teramoto A, Hashimoto K, Kato Y, Amino N: Anti-alpha- peer reviewed and published immediately upon acceptance enolase antibodies in pituitary disease. Endocr J 2003, 50:697-702. cited in PubMed and archived on PubMed Central 19. Bogdanos DP, Bianchi I, Ma Y, Mitry RR, Mieli-Vergani G, Vergani D: yours — you keep the copyright Targets of antibodies to soluble liver antigen in patients with autoimmune hepatitis. 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Journal of Autoimmune Diseases – Springer Journals

Published: Nov 19, 2004

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