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

Casciola-Rosen L, Wigley F, Rosen A - J. Exp. Med. (1997)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

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.

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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.
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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.

Mentions: The site-specific nature of metal- catalyzed oxidation reactions has previously indicated that these reactions are “caged” processes in which amino acid residues at metal-binding sites are specific targets of highly reactive free radical species generated at that site during a Fenton reaction (25, 35, 36). Sequestration of metals by chelators in the bulk solution, although only minimally influencing the capacity of the metal to catalyze a Fenton reaction, prevents the metal from doing so at the metal-binding site, thus inhibiting highly localized protein oxidation (25, 35). We therefore evaluated the effects of metal chelators on the fragmentation reactions. Fe-induced cleavages were markedly inhibited by 1 mM desferroxamine (Fig. 3 A, lanes 2 and 4), 1 mM D-penicillamine (Fig. 3 A, lanes 2 and 8), or 1 mM EDTA (data not shown). Cu-induced fragmentation was entirely abolished by 200 μM bathocuproine disulfonate (a Cu[I]-specific chelator; Fig. 3 A, lanes 3 and 7), and 1 mM EDTA (data not shown), and was diminished by 1 mM desferroxamine or 1 mM D-penicillamine (Fig. 3 A, lanes 3, 5, and 9). These data strongly indicate that metal binding to the autoantigen itself is required for the fragmentation reactions to occur. Since fragmentation requires exogenous metals capable of supporting a Fenton reaction, we addressed whether zinc, a metal that potentially binds to the same site, but is unable to support Fenton chemistry, might influence the ability of Fe or Cu to induce oxidative fragmentation. Increasing concentrations of zinc were added to cell lysates before the addition of Fe/ ascorbate, and the effects on autoantigen fragmentation were assessed (Fig. 3 B). Cleavage of topoisomerase I and RNA polymerase II large subunit was inhibited by zinc in a dose-dependent manner, with fragmentation entirely abolished by 300 μM zinc (IC50 of 30–50 μM) (Fig. 3 B). A similar IC50 for inhibition by zinc was observed for Cu/ H2O2-induced fragmentation of these autoantigens; in contrast, the specific aggregation of RNA polymerase II large subunit induced by Cu/H2O2 was not prevented (data not shown).


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

Casciola-Rosen L, Wigley F, Rosen A - J. Exp. Med. (1997)

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.
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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.
Mentions: The site-specific nature of metal- catalyzed oxidation reactions has previously indicated that these reactions are “caged” processes in which amino acid residues at metal-binding sites are specific targets of highly reactive free radical species generated at that site during a Fenton reaction (25, 35, 36). Sequestration of metals by chelators in the bulk solution, although only minimally influencing the capacity of the metal to catalyze a Fenton reaction, prevents the metal from doing so at the metal-binding site, thus inhibiting highly localized protein oxidation (25, 35). We therefore evaluated the effects of metal chelators on the fragmentation reactions. Fe-induced cleavages were markedly inhibited by 1 mM desferroxamine (Fig. 3 A, lanes 2 and 4), 1 mM D-penicillamine (Fig. 3 A, lanes 2 and 8), or 1 mM EDTA (data not shown). Cu-induced fragmentation was entirely abolished by 200 μM bathocuproine disulfonate (a Cu[I]-specific chelator; Fig. 3 A, lanes 3 and 7), and 1 mM EDTA (data not shown), and was diminished by 1 mM desferroxamine or 1 mM D-penicillamine (Fig. 3 A, lanes 3, 5, and 9). These data strongly indicate that metal binding to the autoantigen itself is required for the fragmentation reactions to occur. Since fragmentation requires exogenous metals capable of supporting a Fenton reaction, we addressed whether zinc, a metal that potentially binds to the same site, but is unable to support Fenton chemistry, might influence the ability of Fe or Cu to induce oxidative fragmentation. Increasing concentrations of zinc were added to cell lysates before the addition of Fe/ ascorbate, and the effects on autoantigen fragmentation were assessed (Fig. 3 B). Cleavage of topoisomerase I and RNA polymerase II large subunit was inhibited by zinc in a dose-dependent manner, with fragmentation entirely abolished by 300 μM zinc (IC50 of 30–50 μM) (Fig. 3 B). A similar IC50 for inhibition by zinc was observed for Cu/ H2O2-induced fragmentation of these autoantigens; in contrast, the specific aggregation of RNA polymerase II large subunit induced by Cu/H2O2 was not prevented (data not shown).

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

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.

Show MeSH
Related in: MedlinePlus