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Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 1994 Mar;95(3):397–407. doi: 10.1111/j.1365-2249.1994.tb07010.x

Epitope mapping with synthetic peptides of 52-kD SSA/Ro protein reveals heterogeneous antibody profiles in human autoimmune sera.

V Ricchiuti 1, J P Briand 1, O Meyer 1, D A Isenberg 1, G Pruijn 1, S Muller 1
PMCID: PMC1535087  PMID: 7511075

Abstract

The reactivity of autoantibodies present in the sera of 489 patients with Sjögren's syndrome (SS), systemic lupus erythematosus (SLE) and other autoimmune diseases was investigated by ELISA using recombinant 52-kD SSA/Ro protein (rRo52) and 39 overlapping synthetic peptides representing the entire sequence of Ro52. We report that IgG antibodies reacting with rRo52 were present in the sera of a large number of patients with SS (67% of patients with primary SS and 46% of patients with SS associated with SLE), whereas they were less frequent (10-25%) in SLE, rheumatoid arthritis (RA), juvenile chronic arthritis (JCA) and mixed connective tissue disease (MCTD), and absent in scleroderma. Among the 39 peptides tested, five were recognized by sera from 30-65% of patients with SS, namely peptides representing residues 2-11, 107-122, 107-126, 277-292 and 365-382. Patients with JCA had raised levels of IgG antibodies reacting with peptides 2-11 and 365-382, and 51% of patients with MCTD had raised levels of IgG antibodies reacting with peptide 365-382. None of the five peptides was recognized by more than 20% of sera from patients with SLE and RA. Interestingly, and of importance in the field of diagnostic tests based on peptides, the reactivity of antibodies to the Ro52 synthetic peptides varied greatly according to the origin of sera. Inhibition experiments using either patients' sera or antibodies induced in rabbits against Ro52 peptides showed that the four domains 2-11, 107-122, 277-292 and 365-382 are accessible on the surface of the Ro52 protein. These regions may thus be involved in the induction of specific antibodies in autoimmune patients.

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  1. Adam S. A., Nakagawa T., Swanson M. S., Woodruff T. K., Dreyfuss G. mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol Cell Biol. 1986 Aug;6(8):2932–2943. doi: 10.1128/mcb.6.8.2932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barakat S., Briand J. P., Abuaf N., van Regenmortel M. H., Muller S. Mapping of epitopes on U1 snRNP polypeptide A with synthetic peptides and autoimmune sera. Clin Exp Immunol. 1991 Oct;86(1):71–78. doi: 10.1111/j.1365-2249.1991.tb05776.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barakat S., Briand J. P., Weber J. C., van Regenmortel M. H., Muller S. Recognition of synthetic peptides of Sm-D autoantigen by lupus sera. Clin Exp Immunol. 1990 Aug;81(2):256–262. doi: 10.1111/j.1365-2249.1990.tb03327.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barakat S., Meyer O., Torterotot F., Youinou P., Briand J. P., Kahn M. F., Muller S. IgG antibodies from patients with primary Sjögren's syndrome and systemic lupus erythematosus recognize different epitopes in 60-kD SSA/Ro protein. Clin Exp Immunol. 1992 Jul;89(1):38–45. doi: 10.1111/j.1365-2249.1992.tb06874.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bellini M., Lacroix J. C., Gall J. G. A putative zinc-binding protein on lampbrush chromosome loops. EMBO J. 1993 Jan;12(1):107–114. doi: 10.1002/j.1460-2075.1993.tb05636.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ben-Chetrit E., Fox R. I., Tan E. M. Dissociation of immune responses to the SS-A (Ro) 52-kd and 60-kd polypeptides in systemic lupus erythematosus and Sjögren's syndrome. Arthritis Rheum. 1990 Mar;33(3):349–355. doi: 10.1002/art.1780330307. [DOI] [PubMed] [Google Scholar]
  7. Black C., Isenberg D. A. Mixed connective tissue disease--goodbye to all that. Br J Rheumatol. 1992 Oct;31(10):695–700. doi: 10.1093/rheumatology/31.10.695. [DOI] [PubMed] [Google Scholar]
  8. Boire G., Lopez-Longo F. J., Lapointe S., Ménard H. A. Sera from patients with autoimmune disease recognize conformational determinants on the 60-kd Ro/SS-A protein. Arthritis Rheum. 1991 Jun;34(6):722–730. doi: 10.1002/art.1780340613. [DOI] [PubMed] [Google Scholar]
  9. Bozic B., Pruijn G. J., Rozman B., van Venrooij W. J. Sera from patients with rheumatic diseases recognize different epitope regions on the 52-kD Ro/SS-A protein. Clin Exp Immunol. 1993 Nov;94(2):227–235. doi: 10.1111/j.1365-2249.1993.tb03436.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Briand J. P., Van Dorsselaer A., Raboy B., Muller S. Total chemical synthesis of ubiquitin using BOP reagent: biochemical and immunochemical properties of the purified synthetic product. Pept Res. 1989 Nov-Dec;2(6):381–388. [PubMed] [Google Scholar]
  11. Buyon J. P., Ben-Chetrit E., Karp S., Roubey R. A., Pompeo L., Reeves W. H., Tan E. M., Winchester R. Acquired congenital heart block. Pattern of maternal antibody response to biochemically defined antigens of the SSA/Ro-SSB/La system in neonatal lupus. J Clin Invest. 1989 Aug;84(2):627–634. doi: 10.1172/JCI114208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Buyon J. P., Slade S. G., Chan E. K., Tan E. M., Winchester R. Effective separation of the 52 kDa SSA/Ro polypeptide from the 48 kDa SSB/La polypeptide by altering conditions of polyacrylamide gel electrophoresis. J Immunol Methods. 1990 May 25;129(2):207–210. doi: 10.1016/0022-1759(90)90440-7. [DOI] [PubMed] [Google Scholar]
  13. Chan E. K., Hamel J. C., Buyon J. P., Tan E. M. Molecular definition and sequence motifs of the 52-kD component of human SS-A/Ro autoantigen. J Clin Invest. 1991 Jan;87(1):68–76. doi: 10.1172/JCI115003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Isenberg D. A., Rowe D., Tookman A., Hopp A., Griffiths M., Paice E., Stewart J., Beverley P. C. An immunohistological study of secondary Sjögren's syndrome. Ann Rheum Dis. 1984 Jun;43(3):470–476. doi: 10.1136/ard.43.3.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Itoh K., Itoh Y., Frank M. B. Protein heterogeneity in the human Ro/SSA ribonucleoproteins. The 52- and 60-kD Ro/SSA autoantigens are encoded by separate genes. J Clin Invest. 1991 Jan;87(1):177–186. doi: 10.1172/JCI114968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Itoh Y., Itoh K., Frank M. B., Reichlin M. Autoantibodies to the Ro/SSA autoantigen are conformation dependent. II: Antibodies to the denatured form of 52 kD Ro/SSA are a cross reacting subset of antibodies to the native 60 kD Ro/SSA molecule. Autoimmunity. 1992;14(2):89–95. doi: 10.3109/08916939209083125. [DOI] [PubMed] [Google Scholar]
  17. Lieu T. S., Newkirk M. M., Capra J. D., Sontheimer R. D. Molecular characterization of human Ro/SS-A antigen. Amino terminal sequence of the protein moiety of human Ro/SS-A antigen and immunological activity of a corresponding synthetic peptide. J Clin Invest. 1988 Jul;82(1):96–101. doi: 10.1172/JCI113607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mamula M. J., Silverman E. D., Laxer R. M., Bentur L., Isacovics B., Hardin J. A. Human monoclonal anti-La antibodies. The La protein resides on a subset of Ro particles. J Immunol. 1989 Nov 1;143(9):2923–2928. [PubMed] [Google Scholar]
  19. McCauliffe D. P., Lux F. A., Lieu T. S., Sanz I., Hanke J., Newkirk M. M., Bachinski L. L., Itoh Y., Siciliano M. J., Reichlin M. Molecular cloning, expression, and chromosome 19 localization of a human Ro/SS-A autoantigen. J Clin Invest. 1990 May;85(5):1379–1391. doi: 10.1172/JCI114582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McNeilage L. J., Macmillan E. M., Whittingham S. F. Mapping of epitopes on the La(SS-B) autoantigen of primary Sjögren's syndrome: identification of a cross-reactive epitope. J Immunol. 1990 Dec 1;145(11):3829–3835. [PubMed] [Google Scholar]
  21. Miyagawa S., Dohi K., Shima H., Shirai T. Absence of HLA-B8 and HLA-DR3 in Japanese patients with Sjögren's syndrome positive for anti-SSA(Ro). J Rheumatol. 1992 Dec;19(12):1922–1924. [PubMed] [Google Scholar]
  22. Neimark J., Briand J. P. Development of a fully automated multichannel peptide synthesizer with integrated TFA cleavage capability. Pept Res. 1993 Jul-Aug;6(4):219–228. [PubMed] [Google Scholar]
  23. Papasteriades C. A., Skopouli F. N., Drosos A. A., Andonopoulos A. P., Moutsopoulos H. M. HLA-alloantigen associations in Greek patients with Sjögren's syndrome. J Autoimmun. 1988 Feb;1(1):85–90. doi: 10.1016/0896-8411(88)90079-0. [DOI] [PubMed] [Google Scholar]
  24. Pruijn G. J., Bozic B., Schoute F., Rokeach L. A., van Venrooij W. J. Refined definition of the 56K and other autoantigens in the 50-60 kDa region. Mol Biol Rep. 1992 Sep;16(4):267–276. doi: 10.1007/BF00419667. [DOI] [PubMed] [Google Scholar]
  25. Reichlin M., Rader M., Harley J. B. Autoimmune response to the Ro/SSA particle is directed to the human antigen. Clin Exp Immunol. 1989 Jun;76(3):373–377. [PMC free article] [PubMed] [Google Scholar]
  26. Rokeach L. A., Haselby J. A., Meilof J. F., Smeenk R. J., Unnasch T. R., Greene B. M., Hoch S. O. Characterization of the autoantigen calreticulin. J Immunol. 1991 Nov 1;147(9):3031–3039. [PubMed] [Google Scholar]
  27. Routsias J. G., Tzioufas A. G., Sakarellos-Daitsiotis M., Sakarellos C., Moutsopoulos H. M. Calreticulin synthetic peptide analogues: anti-peptide antibodies in autoimmune rheumatic diseases. Clin Exp Immunol. 1993 Mar;91(3):437–441. doi: 10.1111/j.1365-2249.1993.tb05921.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Scofield R. H., Harley J. B. Autoantigenicity of Ro/SSA antigen is related to a nucleocapsid protein of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3343–3347. doi: 10.1073/pnas.88.8.3343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Slobbe R. L., Pruijn G. J., Damen W. G., van der Kemp J. W., van Venrooij W. J. Detection and occurrence of the 60- and 52-kD Ro (SS-A) antigens and of autoantibodies against these proteins. Clin Exp Immunol. 1991 Oct;86(1):99–105. doi: 10.1111/j.1365-2249.1991.tb05780.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Slobbe R. L., Pruijn G. J., Van Venrooij W. J. Ro (SS-A) and La (SS-B) ribonucleoprotein complexes: structure, function and antigenicity. Ann Med Interne (Paris) 1991;142(8):592–600. [PubMed] [Google Scholar]
  31. Tan E. M. Autoantibodies in pathology and cell biology. Cell. 1991 Nov 29;67(5):841–842. doi: 10.1016/0092-8674(91)90356-4. [DOI] [PubMed] [Google Scholar]
  32. Tan E. M., Cohen A. S., Fries J. F., Masi A. T., McShane D. J., Rothfield N. F., Schaller J. G., Talal N., Winchester R. J. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982 Nov;25(11):1271–1277. doi: 10.1002/art.1780251101. [DOI] [PubMed] [Google Scholar]
  33. Tuaillon N., Muller S., Pasquali J. L., Bordigoni P., Youinou P., Van Regenmortel M. H. Antibodies from patients with rheumatoid arthritis and juvenile chronic arthritis analyzed with core histone synthetic peptides. Int Arch Allergy Appl Immunol. 1990;91(3):297–305. doi: 10.1159/000235131. [DOI] [PubMed] [Google Scholar]
  34. Vaitukaitis J. L. Production of antisera with small doses of immunogen: multiple intradermal injections. Methods Enzymol. 1981;73(Pt B):46–52. doi: 10.1016/0076-6879(81)73055-6. [DOI] [PubMed] [Google Scholar]
  35. Wahren M., Rudén U., Andersson B., Ringertz N. R., Pettersson I. Identification of antigenic regions of the human Ro 60 kDa protein using recombinant antigen and synthetic peptides. J Autoimmun. 1992 Jun;5(3):319–332. doi: 10.1016/0896-8411(92)90146-h. [DOI] [PubMed] [Google Scholar]
  36. Whittingham S. B-cell epitopes of La and Ro autoantigens. Mol Biol Rep. 1992 Jun;16(3):175–181. doi: 10.1007/BF00464705. [DOI] [PubMed] [Google Scholar]
  37. van Venrooij W. J., Slobbe R. L., Pruijn G. J. Structure and function of La and Ro RNPs. Mol Biol Rep. 1993 Aug;18(2):113–119. doi: 10.1007/BF00986765. [DOI] [PubMed] [Google Scholar]

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