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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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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.
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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.

Mentions: Thus, several of the antigens frequently targeted in patients with diffuse scleroderma are specifically fragmented in both of the metal-catalyzed oxidation systems we have examined. The specificity of these fragmentation reactions was confirmed in several different ways. (a) Except for the decreased staining of a protein of ∼85 kD (Fig. 2 A, arrow), the coomassie blue staining of cell lysates incubated with Fe/ascorbate was not significantly different from control lysates (Fig. 2 A, lanes 1 and 2). The coomassie blue staining profile of control lysates, and those treated with Cu/H2O2 were identical (data not shown). These data are consistent with previous observations showing that although modification of proteins by metal-catalyzed reactions is frequent (22, 23, 34), fragmentation of these proteins is an extremely uncommon event (23). (b) Fragmentation reactions were absolutely dependent on metals capable of supporting Fenton chemistry (Fe, Cu); no autoantigen cleavages were observed upon addition of 100 μM zinc, cobalt, mercury, magnesium, manganese, nickel, cadmium, or silver, either alone, in the presence of H2O2, or ascorbate (data not shown). (c) None of the SLE autoantigens tested (including 52- and 60-kD Ro, La, and the Sm proteins), nor several other proteins proteolytically cleaved during apoptosis (including poly(ADP-ribose) polymerase, actin, and the nuclear mitotic apparatus protein, NuMA) were fragmented in oxidation reactions containing Fe/ascorbate or Cu/ H2O2 (Fig. 2 B). (d) Identical fragments were produced in >20 separate experiments, immunoblotted with 7 different topoisomerase I sera, and 3 different RNA polymerase II large subunits, U1-70kDa, and UBF/NOR90 sera (data not shown). Identical fragments were also generated in lysates of several different cell types, including human keratinocytes and human umbilical vein endothelial cells (data not shown). (e) Fragmentation was inhibited under O2-depleted conditions (Fig. 2 C), confirming that the fragmentation was free-radical mediated.


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

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

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.
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Related In: Results  -  Collection

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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.
Mentions: Thus, several of the antigens frequently targeted in patients with diffuse scleroderma are specifically fragmented in both of the metal-catalyzed oxidation systems we have examined. The specificity of these fragmentation reactions was confirmed in several different ways. (a) Except for the decreased staining of a protein of ∼85 kD (Fig. 2 A, arrow), the coomassie blue staining of cell lysates incubated with Fe/ascorbate was not significantly different from control lysates (Fig. 2 A, lanes 1 and 2). The coomassie blue staining profile of control lysates, and those treated with Cu/H2O2 were identical (data not shown). These data are consistent with previous observations showing that although modification of proteins by metal-catalyzed reactions is frequent (22, 23, 34), fragmentation of these proteins is an extremely uncommon event (23). (b) Fragmentation reactions were absolutely dependent on metals capable of supporting Fenton chemistry (Fe, Cu); no autoantigen cleavages were observed upon addition of 100 μM zinc, cobalt, mercury, magnesium, manganese, nickel, cadmium, or silver, either alone, in the presence of H2O2, or ascorbate (data not shown). (c) None of the SLE autoantigens tested (including 52- and 60-kD Ro, La, and the Sm proteins), nor several other proteins proteolytically cleaved during apoptosis (including poly(ADP-ribose) polymerase, actin, and the nuclear mitotic apparatus protein, NuMA) were fragmented in oxidation reactions containing Fe/ascorbate or Cu/ H2O2 (Fig. 2 B). (d) Identical fragments were produced in >20 separate experiments, immunoblotted with 7 different topoisomerase I sera, and 3 different RNA polymerase II large subunits, U1-70kDa, and UBF/NOR90 sera (data not shown). Identical fragments were also generated in lysates of several different cell types, including human keratinocytes and human umbilical vein endothelial cells (data not shown). (e) Fragmentation was inhibited under O2-depleted conditions (Fig. 2 C), confirming that the fragmentation was free-radical mediated.

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