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Updated: Jul 17, 2026
Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins
Published on: March 3, 2016
Fine specificity mapping of autoantigens targeted by anti-centromere autoantibodies
Yasmin Akbarali1, Jennifer Matousek-Ronck, Laura Hunt
1Arthritis and Immunology Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, 825 NE 13th Street, Oklahoma City, OK 73104, USA.
Abstract:
Autoantibodies to centromeric proteins are commonly found in sera of limited scleroderma and other rheumatic disease patients. To better understand the inciting events and possible pathogenic mechanisms of these autoimmune responses, this study identified the common antigenic targets of CENP-A in scleroderma patient sera. Utilizing samples from 263 anti-centromere immunofluorescence positive patients, 93.5% were found to have anti-CENP-A reactivity and 95.4% had anti-CENP-B reactivity by ELISA. Very few patient samples exclusively targeted CENP-A (2.7%) or CENP-B (4.2%). Select patient sera were tested for reactivity with solid phase overlapping decapeptides of CENP-A. Four distinct epitopes of CENP-A were identified. Epitopes 2 and 3 were confirmed by additional testing of 263 patient sera by ELISA for reactivity with these sequences constructed as multiple antigenic peptides. Inhibition CENP-A Western blots also confirmed the specificity of these humoral peptide immune responses in a subset of patient sera. The first three arginine residues (aa 4-6) of CENP-A appear essential for antibody recognition, as replacing these arginines with glycine residues reduced antibody binding to the expressed CENP-A protein by an average of 93.2% (range 80-100%). In selected patients with serial samples spanning nearly a decade, humoral epitope binding patterns were quite stable and showed no epitope spreading over time. This epitope mapping study identifies key antigenic targets of the anti-centromere response and establishes that the majority of the responses depend on key amino-terminal residues.
Insights
Autoantibodies targeting centromeric proteins, common in scleroderma, primarily recognize specific epitopes on CENP-A. Key arginine residues in CENP-A
Area of Science:
- Immunology
- Rheumatology
- Molecular Biology
Background:
- Autoantibodies to centromeric proteins are hallmarks of limited scleroderma and other rheumatic diseases.
- Understanding the specific targets and epitopes of these autoantibodies is crucial for elucidating pathogenic mechanisms.
Purpose of the Study:
- To identify common antigenic targets of CENP-A in scleroderma patient sera.
- To map the specific epitopes recognized by autoantibodies against CENP-A.
Main Methods:
- ELISA and Western blot assays were used to detect autoantibody reactivity.
- Solid-phase overlapping decapeptides and multiple antigenic peptides of CENP-A were synthesized to map epitopes.
- Site-directed mutagenesis was employed to assess the role of specific amino acid residues.
Main Results:
- Over 93% of anti-centromere positive sera showed reactivity against CENP-A.
- Four distinct epitopes on CENP-A were identified, with epitopes 2 and 3 being major targets.
- The first three arginine residues (aa 4-6) of CENP-A were found to be essential for antibody recognition.
- Epitope binding patterns remained stable over time, with no evidence of epitope spreading.
Conclusions:
- The study identifies key antigenic targets within CENP-A for the anti-centromere autoimmune response.
- The findings highlight the critical role of specific N-terminal arginine residues in CENP-A immunogenicity.
- This epitope mapping provides insights into the molecular basis of anti-centromere autoimmunity.
