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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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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.
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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.

Mentions: Substantial evidence exists for the in vitro production of reactive oxygen species (including hydroxyl radical [OH•]) via the metal-catalyzed Fenton (H2O2 + Fe2+(Cu+) → Fe3+(Cu2+) + OH− + OH•) and HaberWeiss reactions (O2−• + H2O2Fe/Cu → O2 + OH− + OH•) (see reviews in references 24 and 25). In initial studies, we added ferrous sulfate (Fe) to NP-40 lysates of HeLa cells to generate free radical species by Fenton chemistry (26–29), and addressed whether several of the major autoantigens in diffuse scleroderma were fragmented (30–33). After incubating in vitro for 30 min in the absence of added metal, topoisomerase I remained stable, and migrated as a single 100-kD species (Fig. 1, lane 1). In these metal-free conditions, addition of either ascorbate or H2O2 failed to generate any fragments (Fig. 1, lanes 2 and 3). Addition of 100 μM Fe induced the specific fragmentation of topoisomerase I, generating a major fragment of 95 kD, and several minor, discrete species migrating between 65 and 90 kD (Fig. 1, lane 4). In the presence of 1.7 mM ascorbate, generation of these fragments was enhanced (Fig. 1, lane 5); the reaction went to completion (that is, total loss of the intact protein) when 17 mM ascorbate was added (data not shown). In contrast, 1 mM H2O2 resulted in a slight, but reproducible, decrease in fragmentation when compared to Fe alone (Fig. 1, lane 6 versus lane 4). Fe also induced specific fragmentation of three additional scleroderma autoantigens: the large subunit of RNA polymerase II, upstream binding factor (UBF/ NOR90), and the 70 kD protein component of the U1 small nuclear ribonucleoprotein (U1-70kDa). Although the sensitivity of the different proteins to metal-induced fragmentation varied (e.g., the large subunit of RNA polymerase II was efficiently fragmented using <10 μM Fe), cleavage was enhanced by the addition of ascorbate in all cases: (a) the large subunit of RNA polymerase II (a protein doublet of 220 and 240 kD) was specifically fragmented, generating major species of 190, 160, and 140 kD, as well as several minor species of 200, 180, 170, and 130 kD (Fig. 1, lane 5); (b) Fragmentation of UBF/NOR90 (a doublet of 90 and 100 kD in control lysates) yielded a poorly resolved smear of fragments between 70 and 90 kD (Fig. 1, lane 5), (c) U1-70kDa fragmentation in response to Fe generated a few discrete fragments that migrated between 33 and 38 kD (Fig. 1, lane 5); this Fe-induced fragmentation was inefficient compared to that of the other autoantigens. Identical U1-70kDa fragments were also observed when [35S]methionine-labeled U1-70kDa was generated by coupled in vitro transciption/translation, and then subjected to Fe/ ascorbate treatment (data not shown). In contrast to the autoantigens described above, fibrillarin, the major scleroderma ribonucleoprotein autoantigen, was not fragmented under identical conditions (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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196102&req=5

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.
Mentions: Substantial evidence exists for the in vitro production of reactive oxygen species (including hydroxyl radical [OH•]) via the metal-catalyzed Fenton (H2O2 + Fe2+(Cu+) → Fe3+(Cu2+) + OH− + OH•) and HaberWeiss reactions (O2−• + H2O2Fe/Cu → O2 + OH− + OH•) (see reviews in references 24 and 25). In initial studies, we added ferrous sulfate (Fe) to NP-40 lysates of HeLa cells to generate free radical species by Fenton chemistry (26–29), and addressed whether several of the major autoantigens in diffuse scleroderma were fragmented (30–33). After incubating in vitro for 30 min in the absence of added metal, topoisomerase I remained stable, and migrated as a single 100-kD species (Fig. 1, lane 1). In these metal-free conditions, addition of either ascorbate or H2O2 failed to generate any fragments (Fig. 1, lanes 2 and 3). Addition of 100 μM Fe induced the specific fragmentation of topoisomerase I, generating a major fragment of 95 kD, and several minor, discrete species migrating between 65 and 90 kD (Fig. 1, lane 4). In the presence of 1.7 mM ascorbate, generation of these fragments was enhanced (Fig. 1, lane 5); the reaction went to completion (that is, total loss of the intact protein) when 17 mM ascorbate was added (data not shown). In contrast, 1 mM H2O2 resulted in a slight, but reproducible, decrease in fragmentation when compared to Fe alone (Fig. 1, lane 6 versus lane 4). Fe also induced specific fragmentation of three additional scleroderma autoantigens: the large subunit of RNA polymerase II, upstream binding factor (UBF/ NOR90), and the 70 kD protein component of the U1 small nuclear ribonucleoprotein (U1-70kDa). Although the sensitivity of the different proteins to metal-induced fragmentation varied (e.g., the large subunit of RNA polymerase II was efficiently fragmented using <10 μM Fe), cleavage was enhanced by the addition of ascorbate in all cases: (a) the large subunit of RNA polymerase II (a protein doublet of 220 and 240 kD) was specifically fragmented, generating major species of 190, 160, and 140 kD, as well as several minor species of 200, 180, 170, and 130 kD (Fig. 1, lane 5); (b) Fragmentation of UBF/NOR90 (a doublet of 90 and 100 kD in control lysates) yielded a poorly resolved smear of fragments between 70 and 90 kD (Fig. 1, lane 5), (c) U1-70kDa fragmentation in response to Fe generated a few discrete fragments that migrated between 33 and 38 kD (Fig. 1, lane 5); this Fe-induced fragmentation was inefficient compared to that of the other autoantigens. Identical U1-70kDa fragments were also observed when [35S]methionine-labeled U1-70kDa was generated by coupled in vitro transciption/translation, and then subjected to Fe/ ascorbate treatment (data not shown). In contrast to the autoantigens described above, fibrillarin, the major scleroderma ribonucleoprotein autoantigen, was not fragmented under identical conditions (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