Scleroderma autoantigens are uniquely fragmented by metal-catalyzed oxidation reactions: implications for pathogenesis

Abstract

The observation that revelation of immunocryptic epitopes in self antigens may initiate the autoimmune response has prompted the search for processes which induce novel fragmentation of autoantigens as potential initiators of autoimmunity. The reversible ischemia reperfusion which characterizes scleroderma has focused attention on reactive oxygen species as molecules which might induce autoantigen fragmentation. We demonstrate that several of the autoantigens targeted in diffuse scleroderma are uniquely susceptible to cleavage by reactive oxygen species, in a metal-dependent manner. Multiple features of the fragmentation reaction and its inhibition indicate that these autoantigens possess metal-binding sites, which focus metal-catalyzed oxidation reactions (and consequent fragmentation) to specific regions of the antigens. These data suggest that the autoantibody response in scleroderma is the immune marker of unique protein fragmentation, induced by ischemia reperfusion in the presence of appropriate metals, and focus attention on abnormal metal status as a potential pathogenic principle in this disease.

Figure 1

Several scleroderma autoantigens are uniquely fragmented by Fe/ascorbate or Cu/H₂O₂ oxidation reactions. HeLa lysates were prepared as described in the Materials and Methods section, and metal-catalyzed oxidation reactions were performed by adding the following: no additions (lane 1), 1.7 mM ascorbate (lane 2), 1 mM H₂O₂ (lane 3), 100 μM Fe(II)SO₄ (lane 4), 100 μM Fe(II)SO₄ + 1.7 mM ascorbate (lane 5), 100 μM Fe(II)SO₄ + 1 mM H₂O₂ (lane 6), 100 μM Cu(II)SO₄ (lane 7), 100 μM Cu(II)SO₄ + 1.7 mM ascorbate (lane 8) and 100μM Cu(II)SO₄ + 1 mM H₂O₂ (lane 8). Samples were immunoblotted with the sera denoted on the left side of each panel. Equal amounts of protein were electrophoresed in the lanes of each panel. Migration positions of molecular weight standards are indicated on the right.

Figure 2

Fragmentation of scleroderma autoantigens by oxidation is a highly specific event. (A and B) Equal protein amounts of control HeLa lysates (lanes 1 and 3), and HeLa lysates incubated with 100 μM Fe + 1.7 mM ascorbate (lanes 2 and 4) were electrophoresed. (A) Gels were stained with Coomassie blue to visualize the total protein profile of each sample. Oxidation catalyzed by Fe/ascorbate caused no striking alterations in the Coomassie pattern, with the exception of the decreased staining of an ∼85 kD protein (arrow). Migration positions of the molecular weight standards are indicated to the right of lane 2. (B) Electrophoresed proteins were immunoblotted with patient sera recognizing NuMA, La, and 52 and 60-kD Ro, or a monoclonal antibody to fodrin (Chemicon International Inc., Temecula, CA). None of these antigens were fragmented by Fe/ascorbate oxidation reactions (lanes 3 and 4). (C) Fragmentation of RNA polymerase II and topoisomerase I is decreased in O₂-depleted lysates. O₂ depletion was performed as described in Materials and Methods, before adding 100 μM Fe and 1.7 mM ascorbate. Equal protein amounts were electrophoresed and immunoblotted with antibodies to RNA polymerase II and topoisomerase I.

Figure 2

Fragmentation of scleroderma autoantigens by oxidation is a highly specific event. (A and B) Equal protein amounts of control HeLa lysates (lanes 1 and 3), and HeLa lysates incubated with 100 μM Fe + 1.7 mM ascorbate (lanes 2 and 4) were electrophoresed. (A) Gels were stained with Coomassie blue to visualize the total protein profile of each sample. Oxidation catalyzed by Fe/ascorbate caused no striking alterations in the Coomassie pattern, with the exception of the decreased staining of an ∼85 kD protein (arrow). Migration positions of the molecular weight standards are indicated to the right of lane 2. (B) Electrophoresed proteins were immunoblotted with patient sera recognizing NuMA, La, and 52 and 60-kD Ro, or a monoclonal antibody to fodrin (Chemicon International Inc., Temecula, CA). None of these antigens were fragmented by Fe/ascorbate oxidation reactions (lanes 3 and 4). (C) Fragmentation of RNA polymerase II and topoisomerase I is decreased in O₂-depleted lysates. O₂ depletion was performed as described in Materials and Methods, before adding 100 μM Fe and 1.7 mM ascorbate. Equal protein amounts were electrophoresed and immunoblotted with antibodies to RNA polymerase II and topoisomerase I.

Figure 3

Metal chelators, zinc, or EDAC strongly inhibit or abolish fragmentation of the scleroderma autoantigens induced by both Fe/ascorbate and Cu/H₂O₂. The following additions were made to equal protein amounts of HeLa cell lysate, before immunoblotting with the indicated antibodies: (A) none (lane 1), 100 μM Fe + 1.7 mM ascorbate (lanes 2, 4, 6, and 8); 100 μM Cu + 1 mM H₂O₂ (lanes 3, 5, 7, and 9); 1 mM desferroxamine (lanes 4 and 5); 200 μM bathocuproine disulfonate (lanes 6 and 7), or 1 mM D-penicillamine (lanes 8 and 9). The band migrating at 66 kD in the UBF/NOR90 immunoblot is nonspecific. (B) Increasing concentrations of ZnCl₂ (0–300 μM) were added to HeLa lysates before addition of 100 μM Fe + 1.7 mM ascorbate. The IC₅₀ value for inhibition of Fe/ascorbate-induced fragmentation of topoisomerase I or RNA polymerase II large subunit was 30–50 μM. (C) HeLa lysates were incubated in the absence (−) or presence (+) of 5 mM EDAC, before adding 100 μM Fe and 1.7 mM ascorbate. (A–C) Equal protein amounts were loaded in each lane. Results are representative of two to six separate experiments.

Figure 3

Metal chelators, zinc, or EDAC strongly inhibit or abolish fragmentation of the scleroderma autoantigens induced by both Fe/ascorbate and Cu/H₂O₂. The following additions were made to equal protein amounts of HeLa cell lysate, before immunoblotting with the indicated antibodies: (A) none (lane 1), 100 μM Fe + 1.7 mM ascorbate (lanes 2, 4, 6, and 8); 100 μM Cu + 1 mM H₂O₂ (lanes 3, 5, 7, and 9); 1 mM desferroxamine (lanes 4 and 5); 200 μM bathocuproine disulfonate (lanes 6 and 7), or 1 mM D-penicillamine (lanes 8 and 9). The band migrating at 66 kD in the UBF/NOR90 immunoblot is nonspecific. (B) Increasing concentrations of ZnCl₂ (0–300 μM) were added to HeLa lysates before addition of 100 μM Fe + 1.7 mM ascorbate. The IC₅₀ value for inhibition of Fe/ascorbate-induced fragmentation of topoisomerase I or RNA polymerase II large subunit was 30–50 μM. (C) HeLa lysates were incubated in the absence (−) or presence (+) of 5 mM EDAC, before adding 100 μM Fe and 1.7 mM ascorbate. (A–C) Equal protein amounts were loaded in each lane. Results are representative of two to six separate experiments.

Figure 3

Metal chelators, zinc, or EDAC strongly inhibit or abolish fragmentation of the scleroderma autoantigens induced by both Fe/ascorbate and Cu/H₂O₂. The following additions were made to equal protein amounts of HeLa cell lysate, before immunoblotting with the indicated antibodies: (A) none (lane 1), 100 μM Fe + 1.7 mM ascorbate (lanes 2, 4, 6, and 8); 100 μM Cu + 1 mM H₂O₂ (lanes 3, 5, 7, and 9); 1 mM desferroxamine (lanes 4 and 5); 200 μM bathocuproine disulfonate (lanes 6 and 7), or 1 mM D-penicillamine (lanes 8 and 9). The band migrating at 66 kD in the UBF/NOR90 immunoblot is nonspecific. (B) Increasing concentrations of ZnCl₂ (0–300 μM) were added to HeLa lysates before addition of 100 μM Fe + 1.7 mM ascorbate. The IC₅₀ value for inhibition of Fe/ascorbate-induced fragmentation of topoisomerase I or RNA polymerase II large subunit was 30–50 μM. (C) HeLa lysates were incubated in the absence (−) or presence (+) of 5 mM EDAC, before adding 100 μM Fe and 1.7 mM ascorbate. (A–C) Equal protein amounts were loaded in each lane. Results are representative of two to six separate experiments.

Figure 4

Topoisomerase I is fragmented in intact keratinocytes chronically exposed to 20 μM Cu. Confluent monolayers of human foreskin keratinocytes were cultured for 18 h (lanes 3 and 4) or 2 h (lanes 5 and 6) in keratinocyte growth medium supplemented with 20 μM CuSO₄. Control cultures were maintained in the absence of added Cu (lanes 1 and 2). Before harvesting the cells, 2 mM H₂O₂ was added to some of the cultures (lanes 2, 4, and 6), but not others (lanes 1 3, and 5), for 30 min. Cells were subsequently harvested and immunoblotted with anti-topoisomerase I serum as described in Fig. 1. Equal amounts of protein were electrophoresed in each lane. Results are representative of those obtained in three separate experiments.

Figure 5

Metal ions and topoisomerase I are concentrated in punctate intranucleolar structures. (A) HeLa cells were fixed in 1% lead acetate, and subsequently stained with 1% ammonium sulfide. Bright field microscopy demonstrates intense staining of punctate intranucleolar structures (nucleolini). (B) HeLa cells were stained with a monospecific human serum recognizing topoisomerase I and FITC-goat anti– human IgG, and were examined by confocal fluoresence microscopy. Diffuse nuclear staining, as well as punctate intranucleolar structures (nucleolini), are seen. Similar results were obtained with six other monospecific topoisomerase I sera. Nucleolini were never stained when similar experiments were performed using sera obtained from healthy individuals, or from Ro/La-positive lupus patients. Bar, 10 μM.