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Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 1990 Apr;80(1):38–43. doi: 10.1111/j.1365-2249.1990.tb06438.x

A recombinant topoisomerase I used for autoantibody detection in sera from patients with systemic sclerosis.

R Verheijen 1, F Van den Hoogen 1, R Beijer 1, A Richter 1, E Penner 1, W J Habets 1, W J van Venrooij 1
PMCID: PMC1535233  PMID: 1691063

Abstract

We report the expression of a cDNA clone encoding 695 carboxyl-terminal amino acids of human DNA topoisomerase I (topoI) in Escherichia coli. More than 96% of the anti-HeLa topoI-positive sera from patients with a connective tissue disease displayed also an immunoreactivity with this recombinant protein (the HTopoA protein). Sera from patients with a definite diagnosis systemic sclerosis and reacting with HeLa topoI, all reacted with the HTopoA protein as well. Sera from patients with systemic sclerosis that did not contain anti-topoI antibodies (about 30% of the systemic sclerosis sera), as concluded from HeLa immunoblot, displayed also no immunoreactivity with our recombinant antigen. By expressing different fragments of HTopoA, we were able to assign at least three different autoimmune epitope regions on the HTopoA protein and we show that over a period of 5 years the amount of anti-topoI antibodies against these regions may fluctuate.

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Selected References

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  1. Brill S. J., DiNardo S., Voelkel-Meiman K., Sternglanz R. Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA. 1987 Mar 26-Apr 1Nature. 326(6111):414–416. doi: 10.1038/326414a0. [DOI] [PubMed] [Google Scholar]
  2. Bullock P., Champoux J. J., Botchan M. Association of crossover points with topoisomerase I cleavage sites: a model for nonhomologous recombination. Science. 1985 Nov 22;230(4728):954–958. doi: 10.1126/science.2997924. [DOI] [PubMed] [Google Scholar]
  3. Crowl R., Seamans C., Lomedico P., McAndrew S. Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli. Gene. 1985;38(1-3):31–38. doi: 10.1016/0378-1119(85)90200-8. [DOI] [PubMed] [Google Scholar]
  4. D'Arpa P., Machlin P. S., Ratrie H., 3rd, Rothfield N. F., Cleveland D. W., Earnshaw W. C. cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2543–2547. doi: 10.1073/pnas.85.8.2543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Douvas A. S., Achten M., Tan E. M. Identification of a nuclear protein (Scl-70) as a unique target of human antinuclear antibodies in scleroderma. J Biol Chem. 1979 Oct 25;254(20):10514–10522. [PubMed] [Google Scholar]
  6. Eng W. K., Pandit S. D., Sternglanz R. Mapping of the active site tyrosine of eukaryotic DNA topoisomerase I. J Biol Chem. 1989 Aug 15;264(23):13373–13376. [PubMed] [Google Scholar]
  7. Garg L. C., DiAngelo S., Jacob S. T. Role of DNA topoisomerase I in the transcription of supercoiled rRNA gene. Proc Natl Acad Sci U S A. 1987 May;84(10):3185–3188. doi: 10.1073/pnas.84.10.3185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Guldner H. H., Szostecki C., Vosberg H. P., Lakomek H. J., Penner E., Bautz F. A. Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I. Chromosoma. 1986;94(2):132–138. doi: 10.1007/BF00286991. [DOI] [PubMed] [Google Scholar]
  9. Habets W. J., Sillekens P. T., Hoet M. H., Schalken J. A., Roebroek A. J., Leunissen J. A., van de Ven W. J., van Venrooij W. J. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B" antigen. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2421–2425. doi: 10.1073/pnas.84.8.2421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Habets W. J., de Rooij D. J., Salden M. H., Verhagen A. P., van Eekelen C. A., van de Putte L. B., van Venrooij W. J. Antibodies against distinct nuclear matrix proteins are characteristic for mixed connective tissue disease. Clin Exp Immunol. 1983 Oct;54(1):265–276. [PMC free article] [PubMed] [Google Scholar]
  11. Jarzabek-Chorzelska M., Blaszczyk M., Jablonska S., Chorzelski T., Kumar V., Beutner E. H. Scl 70 antibody--a specific marker of systemic sclerosis. Br J Dermatol. 1986 Oct;115(4):393–401. doi: 10.1111/j.1365-2133.1986.tb06233.x. [DOI] [PubMed] [Google Scholar]
  12. Kumar V., Kowalewski C., Koelle M., Qutaishat S., Chorzelski T. P., Beutner E. H., Jarzabek-Chorzelska M., Kolacinska Z., Jablonska S. Scl-70 antigen stability and its effect on antibody detection in scleroderma. J Rheumatol. 1988 Oct;15(10):1499–1502. [PubMed] [Google Scholar]
  13. Kurata N., Tan E. M. Identification of antibodies to nuclear acidic antigens by counterimmunoelectrophoresis. Arthritis Rheum. 1976 May-Jun;19(3):574–580. doi: 10.1002/art.1780190309. [DOI] [PubMed] [Google Scholar]
  14. Lynn R. M., Bjornsti M. A., Caron P. R., Wang J. C. Peptide sequencing and site-directed mutagenesis identify tyrosine-727 as the active site tyrosine of Saccharomyces cerevisiae DNA topoisomerase I. Proc Natl Acad Sci U S A. 1989 May;86(10):3559–3563. doi: 10.1073/pnas.86.10.3559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maul G. G., French B. T., van Venrooij W. J., Jimenez S. A. Topoisomerase I identified by scleroderma 70 antisera: enrichment of topoisomerase I at the centromere in mouse mitotic cells before anaphase. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5145–5149. doi: 10.1073/pnas.83.14.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Maul G. G., Jimenez S. A., Riggs E., Ziemnicka-Kotula D. Determination of an epitope of the diffuse systemic sclerosis marker antigen DNA topoisomerase I: sequence similarity with retroviral p30gag protein suggests a possible cause for autoimmunity in systemic sclerosis. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8492–8496. doi: 10.1073/pnas.86.21.8492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McCoubrey W. K., Jr, Champoux J. J. The role of single-strand breaks in the catenation reaction catalyzed by the rat type I topoisomerase. J Biol Chem. 1986 Apr 15;261(11):5130–5137. [PubMed] [Google Scholar]
  18. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  19. Oddou P., Schmidt U., Knippers R., Richter A. Monoclonal antibodies neutralizing mammalian DNA topoisomerase I activity. Eur J Biochem. 1988 Nov 15;177(3):523–529. doi: 10.1111/j.1432-1033.1988.tb14404.x. [DOI] [PubMed] [Google Scholar]
  20. Osheroff N. Biochemical basis for the interactions of type I and type II topoisomerases with DNA. Pharmacol Ther. 1989;41(1-2):223–241. doi: 10.1016/0163-7258(89)90108-3. [DOI] [PubMed] [Google Scholar]
  21. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Arthritis Rheum. 1980 May;23(5):581–590. doi: 10.1002/art.1780230510. [DOI] [PubMed] [Google Scholar]
  22. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shero J. H., Bordwell B., Rothfield N. F., Earnshaw W. C. Antibodies to topoisomerase I in sera from patients with scleroderma. J Rheumatol Suppl. 1987 Jun;14 (Suppl 13):138–140. [PubMed] [Google Scholar]
  25. Shero J. H., Bordwell B., Rothfield N. F., Earnshaw W. C. High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients. Science. 1986 Feb 14;231(4739):737–740. doi: 10.1126/science.3003910. [DOI] [PubMed] [Google Scholar]
  26. Sillekens P. T., Habets W. J., Beijer R. P., van Venrooij W. J. cDNA cloning of the human U1 snRNA-associated A protein: extensive homology between U1 and U2 snRNP-specific proteins. EMBO J. 1987 Dec 1;6(12):3841–3848. doi: 10.1002/j.1460-2075.1987.tb02721.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Snapka R. M. Topoisomerase inhibitors can selectively interfere with different stages of simian virus 40 DNA replication. Mol Cell Biol. 1986 Dec;6(12):4221–4227. doi: 10.1128/mcb.6.12.4221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  29. Thrash C., Bankier A. T., Barrell B. G., Sternglanz R. Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4374–4378. doi: 10.1073/pnas.82.13.4374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Uemura T., Yanagida M. Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J. 1984 Aug;3(8):1737–1744. doi: 10.1002/j.1460-2075.1984.tb02040.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Verheijen R., Kuijpers H., Vooijs P., Van Venrooij W., Ramaekers F. Distribution of the 70K U1 RNA-associated protein during interphase and mitosis. Correlation with other U RNP particles and proteins of the nuclear matrix. J Cell Sci. 1986 Dec;86:173–190. doi: 10.1242/jcs.86.1.173. [DOI] [PubMed] [Google Scholar]
  32. Yang L., Wold M. S., Li J. J., Kelly T. J., Liu L. F. Roles of DNA topoisomerases in simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1987 Feb;84(4):950–954. doi: 10.1073/pnas.84.4.950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. de Rooij D. J., van de Putte L. B., Habets W. J., Verbeek A. L., van Venrooij W. J. The use of immunoblotting to detect antibodies to nuclear and cytoplasmic antigens. Clinical and serological associations in rheumatic diseases. Scand J Rheumatol. 1988;17(5):353–364. doi: 10.3109/03009748809105271. [DOI] [PubMed] [Google Scholar]
  34. van Venrooij W. J., Stapel S. O., Houben H., Habets W. J., Kallenberg C. G., Penner E., van de Putte L. B. Scl-86, a marker antigen for diffuse scleroderma. J Clin Invest. 1985 Mar;75(3):1053–1060. doi: 10.1172/JCI111767. [DOI] [PMC free article] [PubMed] [Google Scholar]

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