Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I

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.

Involvement of lysosomal cathepsins in the cleavage of DNA topoisomerase I during necrotic cell death

“…The concentration ranges of cathepsins and the incubation conditions used in these experiments were based on reported studies (26,27). It should be noted that untreated topo I, either in purified form or in the isolated nuclei, underwent limited degradation into a 70-kd product during protein or nuclear preparation (Figure 3), consistent with previous reports (3)(4)(5). Nuclear preparations were done under limited protease inhibitory conditions to prevent inhibition of cathepsins.…”

Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription

“…It should be noted that the antibodies against RNA polymerase I react predominantly with the M, 190000 polypeptide of the polymerase and that monoclonal antibodies against the M, 190000 subunit display an analogous pattern to those of the polyclonal antibodies. Other experiments using the anti-topoisomerase I antibodies indicate that the enzyme can be located in the " caps " of segregated nucleoli of actinomycin D treated cells, in the fibrillar centers of nucleoli of tissue prepared for electron microscopy and at the nucleolar organizer regions of metaphase chromosomes (not shown; see also Guldner et al 1986). In no case can we distinguish the nucleolar pattern from that using anti-RNA polymerase I antibodies.…”

“…As shown in Figure 2, lane 3, the antibodies recognized the 110000 M, polypeptide. Two smaller polypeptides, not visible on the proteinstained gel, were also detected on the immunoblots ; presumably these were cleavage products since they are not observed in freshly prepared enzyme preparations (see also Schmitt et al 1984;Guldner et al 1986 ;Shero et al 1986). …”

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

“…10; Scheer and Rose, 1984) and topoisomerase I ( Fig. 12; Rose et al, 1988; see also Guldner et al, 1986) remain bound to the NORs during mitosis. Apparently rRNA genes together with the essential transcriptional machinery (perhaps in the form of 'frozen' transcriptional complexes) are transmitted from one cell generation to the next one, thereby ensuring a rapid reactivation of rRNA transcription following mitosis.…”