Transfection and Overexpression of the Human 60-kDa Ro/SS-A Autoantigen in HEp-2 Cells

Keech, Catherine L.; McCluskey, James; Gordon, Thomas P.

doi:10.1006/clin.1994.1181

Cited by 53 publications

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“…Native Ro 60 inhibited the binding of these IgG by a median of 43% (range 31-68%) compared with a BSA control. Furthermore, the expression of the apotope was increased on HEp-2 cells that overexpress the Ro 60 protein (13), with anti-Ro 60 apotope-positive IgG showing an ϳ2-fold enhancement of binding compared with nontransfected HEp-2 cells, suggesting that the mechanism for translocating Ro 60 to the apoptotic cell surface is not limiting. Therefore, in a subset of anti-Ro 60-positive SLE patients, a surface-exposed Ro 60 epitope (apotope) on early apoptotic cells that is present within the aa 82-244 region of Ro 60 is recognized.…”

Section: Resultsmentioning

confidence: 99%

A B cell apotope of Ro 60 in systemic lupus erythematosus

Reed¹,

Jackson²,

Gordon³

2008

Arthritis & Rheumatism

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Objective. Previous studies have attempted to segregate anti-60-kd Ro/SSA (anti-Ro 60) responses in systemic lupus erythematosus (SLE) and primary Sjö-gren's syndrome (SS) but have shown limited disease preference. The aim of the present study was to determine whether the presence of autoantibodies against an Ro 60 apotope (an epitope expressed on apoptotic cells) distinguishes anti-Ro 60 responses in SLE and primary SS.Methods. Multiparameter flow cytometry was used to select early apoptotic cells and measure the simultaneous binding of annexin V, propidium iodide, and anti-Ro 60-positive IgG from SLE patients (n ‫؍‬ 21) and patients with primary SS (n ‫؍‬ 19). The specificity of the Ro 60 apotope was determined by inhibition experiments with recombinant and native Ro 60.Results. Autoantibodies against the Ro 60 apotope were prevalent in SLE patients (13 of 21, 62%) and were rarely observed in patients with primary SS (1 of 19, 5%) (P ‫؍‬ 0.0002). Further, within SLE patients, autoantibodies to the Ro 60 apotope strongly distinguished patients with anti-Ro 60 alone (12 of 13, 92%) from those with both anti-Ro 60 and anti-La (1 of 8, 13%) (P ‫؍‬ 0.0005). When we considered all patients with anti-Ro 60 alone, the presence of autoantibodies to the Ro 60 apotope had both high sensitivity (92.3%) and high specificity (85.7%) for SLE compared with primary SS (P ‫؍‬ 0.0012). The presence of autoantibodies to the Ro 60 apotope may therefore be of diagnostic value in patients with isolated anti-Ro 60 responses.Conclusion. The preferential targeting of an Ro 60 apotope exposed on early apoptotic cells in a subset of SLE patients implies disease-specific pathways for the induction of anti-Ro 60 autoimmunity.

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Section: Resultsmentioning

confidence: 99%

A B cell apotope of Ro 60 in systemic lupus erythematosus

Reed¹,

Jackson²,

Gordon³

2008

Arthritis & Rheumatism

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“…Jurkat and HEp-2 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), glutamate, penicillin, and streptomycin. A full-length cDNA encoding human Ro 60 protein was transfected into and overexpressed in HEp-2 cells as described previously (23). HEp-2 Ro 60 transfectants were maintained in RPMI 1640 medium, 10% FCS, glutamate, penicillin, streptomycin, and 0.4 mg/ml G418 (Sigma).…”

Section: Monoclonal Antibodies (Mab)mentioning

confidence: 99%

Ro 60 functions as a receptor for β₂‐glycoprotein I on apoptotic cells

Reed¹,

Giannakopoulos²,

Jackson³

et al. 2009

Arthritis & Rheumatism

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Objective. The autoantigens 60-kd Ro/SSA (Ro 60) and ␤ 2 -glycoprotein I (␤ 2 GPI) are both displayed on the surface membrane of apoptotic cells. Epitopespreading experiments have suggested that these autoantigens may be present as a complex on the apoptotic cell surface. This study was undertaken to investigate whether ␤ 2 GPI interacts with Ro 60 on apoptotic cells and alters the binding of anti-Ro 60 IgG.Methods. The interaction between soluble recombinant Ro 60 fragments and ␤ 2 GPI was investigated in vitro by direct and saturation binding assays using native human ␤ 2 GPI and recombinant domain deletion mutants. Binding of ␤ 2 GPI to early and late apoptotic cells was assessed by multiparameter flow cytometry, and specificity of binding was determined by competitive inhibition with soluble recombinant Ro 60 and anti-Ro 60 IgG.Results. The Ro 60 fragment expressing a surfaceexposed epitope (apotope) bound with high affinity (K d ‫؍‬ ϳ15 nM) to domain V of ␤ 2 GPI in vitro. Beta 2 -glycoprotein I bound to the surface of apoptotic cells in a dose-dependent manner and was blocked by the Ro 60 apotope fragment. In reciprocal competitive inhibition studies, ␤ 2 GPI blocked the binding of anti-Ro 60 autoantibodies to apoptotic cells in a dose-dependent manner, and anti-Ro 60 IgG inhibited the binding of ␤ 2 GPI.Moreover, ␤ 2 GPI showed a 2-fold increase in binding to apoptotic cells that overexpress Ro 60 on the surface.Conclusion. These results demonstrate that Ro 60 functions as a novel receptor for ␤ 2 GPI on the surface of apoptotic cells. The formation of Ro 60-␤ 2 GPI complexes may protect against anti-Ro 60 autoantibodymediated tissue injury.

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“…In agreement with such a function, part of Ro60 was shown to reside in the cell nucleus, in contrast to the Ro60 contained in 1ZoP,.NP complexes, which is localized exclusively in the cytoplasm [19][20][21]. The recent identification of a Ro60 homolog in Caenorhabditis elegans [22"] will further aid to unravel the function of Ro60 via genetic approaches.…”

Section: Ro/la Rnpsmentioning

confidence: 99%

Ribonucleoprotein complexes as autoantigens

Venrooij

Pruijn

1995

Current Opinion in Immunology

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Many intracellular proteins and nucleic acids, that are involved in important biosynthetic pathways, are targeted by autoantibodies occurring spontaneously in the sera of patients with systemic autoimmune diseases. Frequently, the autoantigens are assembled into multicomponent complexes containing both nucleic acid(s) and proteins. Recently, progress has been made in the study of autoantigenic ribonucleoprotein complexes, the most important of which are spliceosomal ribonucleoproteins, nucleolar ribonucleoproteins, Ro/La ribonucleoproteins and complexes of aminoacyl-tRNA synthetase and tRNA. In addition to new structural and functional information, important results have been obtained on epitope spreading, as well as on a potential role for apoptosis during the development of an autoimmune response against these complexes.

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Transfection and Overexpression of the Human 60-kDa Ro/SS-A Autoantigen in HEp-2 Cells

Cited by 53 publications

References 0 publications

A B cell apotope of Ro 60 in systemic lupus erythematosus

A B cell apotope of Ro 60 in systemic lupus erythematosus

Ro 60 functions as a receptor for β₂‐glycoprotein I on apoptotic cells

Ribonucleoprotein complexes as autoantigens

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Transfection and Overexpression of the Human 60-kDa Ro/SS-A Autoantigen in HEp-2 Cells

Cited by 53 publications

References 0 publications

A B cell apotope of Ro 60 in systemic lupus erythematosus

A B cell apotope of Ro 60 in systemic lupus erythematosus

Ro 60 functions as a receptor for β2‐glycoprotein I on apoptotic cells

Ribonucleoprotein complexes as autoantigens

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Ro 60 functions as a receptor for β₂‐glycoprotein I on apoptotic cells