Abstract
Sera of patients suffering from the autoimmune disease progressive systemic sclerosis (PSS) are known to contain autoantibodies which have been reported to recognize a 70 kDa antigenic protein, designated the Scl 70 antigen. By immunoblotting of nuclear extracts from HeLa cells with sera from scleroderma patients we observed that the size of the antigen present in such cells depends on the conditions of antigen isolation. When protease inhibitors were included in the extraction buffer, a 95 kDa protein was identified instead of a 70 kDa protein. When protease inhibitors were omitted, a number of polypeptides in the size range 66 to 95 kDa was found. Furthermore, antibodies which had been affinity purified on the 95 kDa antigen, crossreacted with the 66 to 95 kDa polypeptides. These results suggest that the smaller proteins were degradation products of the 95 kDa antigen. Immunofluorescence studies on PtK-2 cells with the antibody specific for the 95 kDa protein gave staining of nuclei, nucleoli and of chromosomes and the nucleolar organizer region in mitotic cells. Since this distribution of antigens within the nucleus was reminiscent of the intranuclear distribution of DNA topoisomerase I found by others we probed purified DNA topoisomerase I from calf thymus directly with the autoantibodies from PSS patients, and also the 95 kDa antigens of HeLa cell nuclei with antibodies raised against the bovine DNA topoisomerase I. From the crossreaction pattern observed with the different antigens and antibodies we conclude that DNA topoisomerase I is one of the antigenic components against which autoantibodies are formed in scleroderma patients.
Similar content being viewed by others
References
Akizuki M, Powers R Jr, Holman HR (1977) A soluble acidic protein of the cell nucleus which reacts with serum from patients with systemic lupus erythematosus and Sjögren's syndrome. J Clin Invest 59:264–271
Catoggio LJ, Skinner RP, Maddison PJ (1983) Frequency and clinical significance of anticentromere and anti-Scl-70 antibodies in an English connective tissue disease population. Rheumatol Int 3:19–21
Champoux JJ, McConaughy BL (1976) Purification and characterization of the DNA untwisting enzyme from rat liver. Biochemistry 15:4638–4642
Douvas AS, Achten M, Tan EM (1979) Identification of a nuclear protein (Scl 70) as a unique target of human antinuclear antibodies in scleroderma. J Biol Chem 254:10514–10522
Guldner HH, Lakomek HJ, Bautz FA (1983) Identification of human Sm and (U1)RNP antigens by immunoblotting. J Immunol Methods 64:45–59
Guldner HH, Lakomek HJ, Bautz FA (1984) Human anti-centromere sera recognize a 19.5 kD non-histone chromosomal protein from HeLa cells. Clin Exp Immunol 58:13–20
Guldner HH, Lakomek HJ, Szostecki C, Bautz FA (1985) Autoantibody-profiles in patients with SLE, scleroderma and overlapsyndromes. In: Peeters H (ed) Protides of the biological fluids Vol 33. Pergamon Press, pp 287–291
Keller W (1975) Characterization of purified DNA-relaxing enzyme from human tissue culture cells. Proc Natl Acad Sci USA 72:2550–2554
Krohne GR, Stick R, Kleinschmidt J, Moll R, Franke WW, Hausen P (1982) Immunological localization of a major karyoskeletal protein in nucleoli of oocytes and somatic cells of Xenopus laevis. J Cell Biol 94:749–754
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lerner MR, Steitz JA (1979) Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus. Proc Natl Acad Sci USA 76:5495–5499
Linn TG, Greenleaf AL, Shorenstein RG, Losick R (1973) Loss of the sigma activity of RNA polymerase of Bacillus subtilis during sporulation. Proc Natl Acad Sci USA 70:1865–1869
Liu LF (1983) DNA topoisomerases — enzymes that catalyze the breaking and rejoining of DNA. Crit Rev Biochem 15:1–24
Liu LF, Miller KG (1981) Eucaryotic DNA topoisomerases: Two forms of type I DNA topoisomerases from HeLa cell nuclei. Proc Natl Acad Sci USA 78:3487–3491
Moroi Y, Peebles C, Fritzler MJ, Steigerwald J, Tan EM (1980) Autoantibody to centromere (kinetochore) in scleroderma sera. Proc Natl Acad Sci USA 77:1627–1631
Muller MT, Pfund WP, Mehta VB, Trask DK (1985) Eucaryotic type I topoisomerase is enriched in the nucleolus and catalytically active on ribosomal DNA. EMBO J 4:1237–1243
Nakamura RM, Peebles CL, Tan EM (1978) Microhemagglutination test for detection of antibodies to nuclear Sm and ribonucleoprotein antigens in systemic lupus erythematosus and related diseases. J Clin Pathol 70:800–807
Olmsted JB (1981) Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem 256:11955–11957
Ouchterlony Ö, Nilsson LA (1978) Immunodiffusion and immunoelectrophoresis. In: Weir DM (ed) Handbook of experimental immunology. Blackwell Scientific Publications, London, pp 19.1–19.44
Pettersson I, Hinterberger M, Mimori T, Gottlieb E, Steitz JA (1984) The structure of mammalian small nuclear ribonucleoproteins. J Biol Chem 259:5907–5914
Scheer U, Rose KM (1984) Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. Proc Natl Acad Sci USA 81:1431–1435
Schmitt B, Buhre U, Vosberg HP (1984) Characterization of size variants of type I DNA topoisomerase isolated from calf thymus. Eur J Biochem 144:127–134
Sharp GC, Irvin WS, May CM, Holman HR, McDuffie FC, Hess EV, Schmid FC (1976) Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective tissue disease, systemic lupus erythematosus and other rheumatic diseases. N Engl J Med 295:1149–1154
Steen VD, Medsger TA Jr, Ziegler G, Rodnan GD (1980) Clinical comparison of two variants of progressive systemic sclerosis: diffuse scleroderma and CREST syndrome. Arthritis Rheum 23:752 (a)
Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee (1980) Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 23:581–590
Tan EM (1982) Autoantibodies to nuclear antigens (ANA): Their immunobiology and medicine. Adv Immunol 33:167–240
Tan EM, Rodnan GP, Garcia I, Moroi Y, Fritzler MJ, Peebles C (1980) Diversity of antinuclear antibodies in progressive systemic sclerosis. Arthritis Rheum 23:617–625
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Van Venrooij WJ, Stapel SO, Houben H, Habets WJ, Kallenberg CGM, Penner E, Van de Putte LB (1985) Scl-86, a marker antigen for diffuse scleroderma. J Clin Invest 75:1053–1060
Vosberg HP (1985) DNA topisomerases: Enzymes that control DNA conformation. Curr Top Microbiol Immunol 114:19–102
Wang JC (1985) DNA topoisomerases. Annu Rev Biochem 54:665–697
Weber K, Bibring T, Osborn M (1975) Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence. Exp Cell Res 95:111–120
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Guldner, HH., Szostecki, C., Vosberg, HP. et al. Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I. Chromosoma 94, 132–138 (1986). https://doi.org/10.1007/BF00286991
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00286991