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Anti-Nuclear Antibody Screening Using HEp-2 Cells
Published on: June 24, 2014
Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by autoantibodies
C Szostecki1, H H Guldner, H J Netter
1Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Insights
Researchers identified autoantibodies targeting the Sp100 nuclear antigen, predominantly found in primary biliary cirrhosis patients. They isolated cDNA coding for Sp100, revealing its potential role in autoimmune diseases and its structural similarities to MHC class I antigens.
Area of Science:
- Immunology
- Molecular Biology
- Hepatology
Background:
- Autoantibodies to nuclear antigens are markers for autoimmune diseases.
- Sp100 is a novel nuclear antigen recognized by autoantibodies, associated with a distinct nuclear dot-like distribution.
- Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease.
Purpose of the Study:
- To isolate and characterize the cDNA encoding the Sp100 autoantigen.
- To identify the clinical significance of anti-Sp100 autoantibodies.
- To investigate the structural and functional properties of the Sp100 autoantigen.
Main Methods:
- Immunoscreening of a HeLa cell cDNA expression library using anti-Sp100 serum.
- ELISA and Western blot analysis to detect autoepitopes and confirm protein identity.
- Isolation of full-length cDNA from human liver and placenta libraries.
- In vitro expression and characterization of the Sp100 autoantigen.
- Protein sequence analysis using computational methods.
Main Results:
- A 0.7-kb cDNA fragment (Sp26) encoding a major autoepitope of Sp100 was isolated.
- Anti-Sp100 autoantibodies were predominantly found in patients with primary biliary cirrhosis (50/184).
- Full-length Sp100 cDNA was obtained, coding for a 53 kDa protein with aberrant electrophoretic mobility (95-100 kDa).
- Sequence analysis revealed similarities to MHC class I antigens and transcriptional regulatory proteins.
Conclusions:
- The Sp100 autoantigen is a significant target in primary biliary cirrhosis.
- The isolated Sp100 cDNA provides a tool for further research into its function and role in disease.
- Sp100's structural homology to MHC class I antigens suggests potential roles in immune regulation or cellular stress responses.
Abstract:
Autoantibodies to a novel nuclear Ag, Sp100, have recently been described that recognize a nuclear protein with an apparent molecular mass of 95 to 100 kDa and a dot-like distribution within cell nuclei. By immunoscreening of a lambda gt11 cDNA expression library derived from HeLa cells with an anti-Sp100 autoimmune serum a 0.7-kb cDNA (Sp26) coding for a fragment of Sp100 was isolated. Expression of this cDNA and use of the recombinant protein in ELISA revealed that the fragment carries major Sp100 autoepitopes and that anti-Sp100 autoantibodies predominantly occur in patients suffering from primary biliary cirrhosis (50/184). The Sp26 cDNA was used as hybridization probe for isolation of longer cDNA from human liver- and placenta-derived lambda gt10 cDNA libraries. Overlapping fragments were assembled to generate a full length cDNA coding for a protein with a molecular mass of 53 kDa and an isoelectric point of 4.7. The Sp100 autoantigen expressed in vitro from this cDNA and authenticated by a capture immunoblot assay, comigrated in SDS-PAGE with the authentic HeLa autoantigen of 95 to 100 kDa and thus showed an aberrant electrophoretic mobility. Computer based protein sequence analysis of the Sp100 autoantigen revealed regions of striking sequence similarities to the alpha 1 and alpha 2 domains of various human and non-human MHC class I Ag and to several transacting transcriptional regulatory proteins.
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