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Naturally occurring mutations in human steroid 21-hydroxylase influence adrenal autoantibody binding
T Asawa1, N Wedlock, A Baumann-Antczak
1Endocrine Immunology Unit, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom.
Abstract:
Human 21-hydroxylase (21-OH) genes containing various mutations, truncations, and deletions were expressed in yeast, and autoantibody binding was studied by Western blotting using patient sera and rabbit antibodies to 21-OH. 21-OH autoantibodies in 13 Addisonian sera showed a marked reduction in their ability to recognize 21-OH mutated at Pro453-->Ser (mean +/- SD, 31 +/- 9% of binding to wild type), whereas the effect on rabbit antibody binding was small (88 +/- 11% of binding to wild type; n = 7). Mutation at Arg339-->His had a less pronounced effect on autoantibody binding (85 +/- 11% of binding to wild type; n = 13) and caused a small enhancement of rabbit antibody binding (124 +/- 16% of binding to wild type; n = 7). These studies indicate that Pro453 has a key role in forming an autoantigenic epitope on 21-OH. It is important to note, however, that the Pro453 mutation caused only partial loss of autoantibody binding, i.e. all Addisonian sera studied still reacted with the mutated protein. This may indicate that each serum sample contains at least two different populations of 21-OH autoantibodies, only one of which recognizes a site dependent on Pro453. A series of more extensive modifications of the 21-OH sequence, including truncations (amino acids 460-494, 448-494, and 418-494) and deletions (amino acids 165-379, 142-240, and 142-280) indicated that most of the sequence of amino acids from 241-494 is important for autoantibody binding. The involvement of such an extensive region of the molecule suggests that the binding sites are generated by three-dimensional folding, with Pro453 having a critical role in forming at least one major autoantigenic epitope.
Insights
Addisonian sera autoantibodies targeting 21-hydroxylase (21-OH) show reduced binding to mutated Pro453, indicating its critical role in autoantigenic epitope formation.
Area of Science:
- Endocrinology
- Immunology
- Molecular Biology
Background:
- Autoimmune Addison's disease is often associated with autoantibodies against steroidogenic enzymes.
- Human 21-hydroxylase (21-OH) is a key enzyme in cortisol synthesis and a target of autoantibodies in Addison's disease.
- Understanding the specific epitopes recognized by these autoantibodies is crucial for diagnosing and potentially treating the condition.
Purpose of the Study:
- To investigate the role of specific amino acid residues, particularly Proline 453, in the autoantigenic epitopes of human 21-hydroxylase (21-OH).
- To characterize the binding patterns of autoantibodies from Addisonian patients and rabbit antibodies to wild-type and mutated 21-OH.
- To identify regions of 21-OH critical for autoantibody recognition.
Main Methods:
- Expression of various mutated, truncated, and deleted human 21-OH genes in yeast.
- Western blotting analysis of autoantibody binding using patient sera and rabbit anti-21-OH antibodies.
- Quantitative assessment of antibody binding to wild-type versus mutated 21-OH proteins.
Main Results:
- Mutation of Proline 453 to Serine in 21-OH significantly reduced binding by autoantibodies from Addisonian sera (31% of wild-type binding).
- Rabbit antibodies to 21-OH showed minimal reduction in binding to the Pro453 mutant (88% of wild-type binding).
- Extensive modifications, including truncations and deletions, indicated that amino acids 241-494 are important for autoantibody binding, suggesting a role for three-dimensional structure.
Conclusions:
- Proline 453 plays a critical role in forming at least one major autoantigenic epitope on 21-hydroxylase.
- The continued reactivity of Addisonian sera with the Pro453 mutant suggests the presence of multiple autoantibody populations recognizing different epitopes.
- The extensive region involved in autoantibody binding implies that conformational epitopes, dependent on protein folding, are significant targets in autoimmune responses against 21-OH.