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The Journal of Immunology, 1999, 162: 2422-2426.
Copyright © 1999 by The American Association of Immunologists

A Conformation-Dependent Epitope in Addison’s Disease and Other Endocrinological Autoimmune Diseases Maps to a Carboxyl-Terminal Functional Domain of Human Steroid 21-Hydroxylase1

Andrej Nikoshkov2,*, Alberto Falorni{dagger}, Svetlana Lajic*,{ddagger}, Stefano Laureti{dagger}, Anna Wedell*, Åke Lernmark§ and Holger Luthman*

* Department of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden; {dagger} Department of Internal Medicine and Endocrine and Metabolic Sciences, University of Perugia, Perugia, Italy; {ddagger} Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden; and § Department of Medicine, University of Washington, Seattle, WA 98195-7790


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Idiopathic Addison’s disease develops as a consequence of autoimmune destruction of steroid-producing cells in the adrenal gland. A major autoantigen is 21-hydroxylase (21OH; P450c21), which is involved in the biosynthesis of cortisol and aldosterone in the adrenal cortex. We selected a number of functionally important 21OH amino acid substitutions, found in patients with congenital adrenal hyperplasia, to study their effects on the binding of 21OH autoantibodies (21OHAb) to 21OH. The ability of 21OHAb to bind in vitro transcribed and translated wild-type 21OH and five different 21OH mutant proteins was quantified by liquid-phase assays. Sera from 21OHAb-positive patients with idiopathic Addison’s disease (n = 24), Graves’ disease (n = 3), and insulin-dependent diabetes mellitus (n = 1) were used. While the P105L, delE196, and G291S mutations had no effect on autoantibody binding, the P453S mutation had a considerable effect, and the R483P mutation almost completely abolished binding. Synthetic peptides corresponding to linear epitopes defined by amino acids 447–461 and 477–491 were unable to compete with wild-type 21OH for binding to autoantibodies. Direct 21OH DNA sequencing could not reveal any specific genetic variation in alleles found in 21OHAb-positive patients. We conclude that the region involving R483 plays a key role in the formation of a three-dimensional epitope in a functionally important C-terminal domain of the enzyme.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Understanding the selection of epitopes in an Ag in relation to its three-dimensional structure is important for understanding the development of autoimmunological reactions. Recently, it was suggested that the nonrandom location of autoantibody epitopes reflects their colocalization with functional domains in autoantigens 1 . Therefore, we studied the influence of different functionally important mutations in the steroid 21-hydroxylase (21OH)3 (P450c21) on the binding of 21OH autoantibodies (21OHAb). Sera from patients with different autoimmune endocrine diseases were used. All of the investigated mutations have been found in patients with different forms of congenital adrenal hyperplasia (CAH), and their influences on enzyme activity have been investigated previously 2, 3 . These data allowed us to relate the level of 21OHAb binding with the phenotypic and biochemical impairments caused by amino acid substitutions in different domains in the enzyme.

Adrenocortical deficiency in idiopathic Addison’s disease is a consequence of autoimmune destruction of steroid-producing cells and is sometimes associated with Graves’ disease and type 1 diabetes mellitus 4 . It has been shown that 21OH, which is involved in the biosynthesis of cortisol and aldosterone in the adrenal cortex, is a major autoantigen associated with adrenal autoimmunity 5 .

The gene for 21OH, CYP21, is located together with its pseudogene, CYP21P, in the HLA class III gene region on chromosome 6p21.3. The two genes are 98% homologous, and their location in tandem is associated with a high frequency of mutations due to increased misalignment and unequal crossing-over during meiosis. The type of mutation in the CYP21 gene determines the enzyme activity and thereby the clinical phenotype of CAH. CAH has a wide spectrum of severity. In the salt-wasting form of the disease the synthesis of both mineralocorticoids and glucocorticoids is affected, leading to renal salt loss and prenatal virilization of affected female fetuses. The simple virilizing form results in prenatal virilization, but residual mineralocorticoids protect the child from hypotonic shock due to salt-loss. In the mildest manifestations of CAH, the affected individuals are diagnosed due to precocious puberty or hyperandrogenism in women 6 . In general, 21OH with mutations identified in patients with CAH displays biochemical activities that correspond to the clinical severity of the disease 2, 3, 7 . In this study we have tested five of these disease-causing mutations to determine their effects on the ability of 21OHAb to precipitate 21OH. Studies using deletion mutants of human 21OH suggested that 21OHAb epitopes in Addison’s disease are localized in the central and carboxyl-terminal regions of the enzyme 8, 9 . However, the use of deletion mutants complicates the interpretation of the results due to severe distortion of the three-dimensional structures of the epitopes. It has also been shown in Western blot experiments that the R339H and P453S mutations, associated with mild CAH, impair binding of 21OHAb 10 .

Recently we developed a conformation-sensitive radioligand-binding assay for detection of 21OHAb in Addison’s disease, which showed that 21OHAb are most sensitively detected in liquid-phase assays 11 , as is the case also for other autoantigens 12, 13 . We used this assay to investigate the effects of single, naturally occurring, amino acid substitutions in 21OH on the binding of 21OHAb in sera from patients with Addison’s disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients

Serum samples were obtained from 24 patients with idiopathic Addison’s disease characterized by the presence of 21OHAb (6 individuals with isolated Addison’s disease and 18 individuals with type 2 autoimmune polyendocrine syndrome (APS)). DNA was obtained from 17 (6 Addison’s disease and 11 type 2 APS) of these subjects. Serum samples were also used from three patients with Graves’ disease and one patient with insulin-dependent diabetes mellitus (IDDM) positive for 21OHAb 14 . The presence of 21OHAb was associated with clinical and biochemical signs of adrenal insufficiency in two of the three individuals with Graves’ disease, while the individual with IDDM did not show any clinical signs of adrenal insufficiency. The individuals with 21OHAb, but without signs of adrenal insufficiency, have not developed Addison’s disease during 2–5 years of follow-up.

Construction of plasmids

Human full-length CYP21 cDNA, starting from the ATG codon and containing the 3'-untranslated sequence, was kindly provided by Dr. Bon-chu Chung (Institute of Molecular Biology, Academia Sinica Taipei, Taiwan) 15 . The cDNA was modified by addition of 10 bp of the 5'-untranslated region and 19 bp of the 3'-untranslated end, to allow cloning into the SalI/KpnI sites of the pGEM3Z vector (Promega). This placed the cDNA under the control of the prokaryotic SP6 promoter. The four nucleotides of the SphI sticky ends, containing an extra ATG codon in front of the CYP21 sequence in the plasmid pGEM-CYP21, were removed by T4 DNA polymerase after SphI cleavage to produce a plasmid suitable for in vitro translation of radiolabeled 21OH protein 11 .

For in vivo expression of the 21OH protein, the CYP21 cDNA was transferred from the pGEM-CYP21 plasmid to the BglII/KpnI sites of the eukaryotic expression vector pCMV4 (Invitrogen, San Diego, CA). The expression vector contained the cytomegalovirus promoter; the 3'-untranslated region, including the polyadenylation signal from the gene encoding human growth hormone; and an SV40 origin of replication. The 21OH expression vector was referred to as pCMV4-CYP21.

Site-directed mutagenesis

The Clontech transformer site-directed mutagenesis kit (Clontech Laboratories, Palo Alto, CA) was used to introduce mutations in pGEM-CYP21. Two phosphorylated primers were used, one primer corresponding to the structural part of the CYP21 gene and containing the mutation of interest, and the other (5'-CAGATCTGTGGACCTGCAG) overlapping the SalI and BglII restriction sites in front of the CYP21 sequence and introducing an impaired SalI restriction site. To confirm the introduction of mutations and to exclude other sequence aberrations, the entire cDNA in all pGEM-CYP21 constructs was sequenced.

Expression of the 21OH protein in COS-1 cells

Approximately 7 x 106 COS-1 cells were transfected by electroporation (Bio-Rad Gen Pulsor, Richmond, CA; 1200 V, 25 µF) with 10 µg of each pCMV4-CYP21 construct together with 2 µg of the ß-galactosidase vector pHC110 (Pharmacia, Sweden) and seeded in 6-cm petri dishes. After a 30–36 h incubation in DMEM supplemented with 10% FCS (DMEM-FCS), the cells were trypsinized and washed twice with PBS (Life Technologies, Grand Island, NY). Approximately 2 x 106 cells per plate were recovered for homogenization by sonication for 20 s in 500 µl of hypotonic buffer (10 mM HEPES, pH 6.2; 10 mM NaCl; and 1.5 mM MgCl2). The homogenate was centrifuged at 350 x g for 10 min to remove nuclei and whole cells, and the supernatant was collected for Western blotting, measurement of total protein content, and analysis of ß-galactosidase activity.

Western blotting

The wild-type and mutated 21OH proteins expressed in COS-1 cells were, after correction for total protein content and ß-galactosidase activity, detected by immunoblotting according to standard procedures, using sera from Addison’s disease patients with high-titer 21OHAb.

21OH-radiobinding assay

A radiobinding assay was performed with in vitro transcribed and translated human 35S-labeled 21OH in a multiwell-adapted procedure (Millipore, Bedford, MA), as described previously 16 . The wild-type pGEM-CYP21 and the five different mutated constructs (Table IGo) were used for coupled in vitro transcription-translation. In this assay, autoantibody titers are expressed as a relative index (rabbit relative index, RRI), using a 21OH-specific rabbit antiserum as positive standard. RRI is defined as follows: (cpm in the unknown sample - mean cpm in the negative (healthy) standard)/(cpm in the positive standard - mean cpm in the negative standard) 11 . This procedure was justified by the fact that none of the amino acid substitutions affected the binding of the rabbit antiserum and that this serum offers a convenient way to correct for interassay variation.


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  		Table I. Effects of different amino acid substitutions on 21OH activity in relation to the phenotypes of CAH patients with 21OH deficiency

 
Synthetic peptides

Synthetic peptides corresponding to amino acids 447–461 and 477–491 of human 21OH were obtained from Innovagen (Lund, Sweden). The peptides were resuspended at a concentration of 1 mg/ml in 150 mM NaCl, 20 mM Tris-HCl, and 5% DMSO, pH 7.4. The immunoprecipitation buffer was supplemented with 0.1 mg/ml of either peptide to investigate the effect on 21OHAb binding to wild-type 21OH.

Analyses of the chromosomal CYP21 sequence in Addison’s disease patients

The CYP21 genes from patients with Addison’s disease were analyzed by direct sequencing. The primers used for amplification of genomic DNA are described elsewhere 17 .


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunoprecipitation of 21OH mutants

Analysis of in vitro translated wild-type 35S-labeled 21OH and the five different 35S-labeled 21OH mutants resulted in 320,000 ± 35,000 to 360,000 ± 37,000 cpm/µl in the TCA precipitates, corresponding to a 33 ± 3% to 37 ± 4% incorporation of [35S]methionine during translation. The degree of immunoprecipitation of 35S-labeled 21OH and 35S-labeled 21OH mutants with specific rabbit antiserum ranged from 29 ± 2% to 33 ± 2% in two assays with triplicate measurements.

The 21OHAb-positive sera from 24 individuals with idiopathic Addison’s disease demonstrated widely different 21OHAb levels (RRI) in the conformation-sensitive radioligand binding assay (Fig. 1Go). The data also demonstrated that the 21OH immunoprecipitation was unaffected by the mutations changing amino acids at position 105, 196, or 291. In marked contrast, the P453S 21OH mutant protein was only precipitated by high-titer sera from 6 of 24 (25%) patients. This observation supports and extends a previous report, suggesting that the epitopes are located in the C-terminal part of 21OH 10 . Using these six sera, we estimated that the ability of 21OHAb from patients with idiopathic Addison’s disease to immunoprecipitate the P453S-substituted 21OH was reduced by 62–84%, according to the RRI.



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  		FIGURE 1. Effects of single-amino acid substitutions in 21OH on binding of autoantibodies from 21OHAb-positive idiopathic Addison’s disease patients. The broken line indicates the upper level of the negative control (the level of immunoprecipitation when sera from healthy controls were used). SDs in these experiments range from 4% to 11% (not shown for graphical reasons).

 
The R483P substitution had, however, more dramatic effects on 21OHAb binding, since only the two sera from Addison’s disease patients with the highest 21OHAb levels remained positive (Fig. 1Go). The R483P substitution reduced the 21OHAb binding by 88 and 95% when these sera were used, showing a profoundly impaired 21OHAb binding to this particular mutant protein. We next sought to determine whether 21OH binding would be equally affected if we used sera from 21OHAb-positive Graves’ disease and IDDM patients, with or without overt clinical adrenal insufficiency. The immunoprecipitation of human 21OH by the four 21OHAb-positive sera from three Graves’ disease patients and one IDDM patient was not affected by amino acid substitutions at position 105, 196, or 291. Again, the P453S and the R483P substitutions drastically reduced immunoprecipitation (Fig. 2Go), irrespective of clinical or biochemical signs of adrenal insufficiency in the patients. Only one serum, from the Graves’ disease patient with the highest 21OHAb level, retained a 10% ability to bind the R483P variant of 21OH. Finally, to test whether the reduction of immunoprecipitation was due to linear epitopes, we performed the precipitation in the presence of synthetic peptides (Table IIGo). The synthetic peptides, corresponding to amino acids 447–461 and amino acids 477–491 of human 21OH, failed to compete with the binding of 21OHAb to wild-type 21OH when four different sera were used from patients with Addison’s disease.



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  		FIGURE 2. Effects of single-amino acid substitutions in 21OH on binding of autoantibodies from 21OHAb-positive Graves’ disease ({square}) and IDDM ({blacksquare}) patients with (——) or without (- - - - -) clinical signs of adrenal insufficiency. The broken line indicates the upper level of the negative control (the level of immunoprecipitation when sera from healthy controls were used). SDs in these experiments range from 4% to 11% (not shown for graphical reasons).

 

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  		Table II. Immunoprecipitation of 35S-21OH by sera from individuals with Addison’s disease in the absence or presence of synthetic peptides (P) that correspond to amino acids 447–461 or 477–491 of human 21OH1

 
Western blot experiments

The Western blot experiments, using two sera from Addison’s disease patients with high levels of 21OHAb, gave results similar to those observed in the immunoprecipitation assays. Strong bands, corresponding in size to the 21OH protein, were obtained for the wild-type, P105L, delE196, and G291S variants, while much weaker bands were detected for the P453S and R483P mutated proteins (data not shown). Preincubation of these sera with synthetic peptides before immunoblotting did not influence the binding of the 21OHAb to the wild-type protein. These results underline the importance of the three-dimensional conformation in formation of the 21OHAb autoantigenic epitopes.

Sequence analyses of CYP21 genes from patients with Addison’s disease

We wanted to analyze whether any amino acid variants of the protein were associated with the presence of autoantibodies in patients. DNA from a total of 17 idiopathic Addison’s disease patients (6 patients with isolated Addison’s disease and 11 with APS type 2) was purified. The complete coding sequence was determined of both CYP21 alleles in two of these patients, one with isolated Addison’s disease and the other with APS type 2. Since our data showed that a conformation-dependent epitope for 21OHAb is located in the C-terminal part of the protein, encoded by exon 10 of the gene, the entire exon 10 was sequenced from 15 additional Addison’s disease patients. It was also suggested that potential epitope regions occur in exons 7 and 8 8, 9, 10 . Therefore, we sequenced these exons in four patients with isolated Addison’s disease and four patients with APS type 2. No deviations from the normal sequence of the CYP21 were found.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Autoantibodies to P450c21 (21OH) and other steroidogenic enzymes, e.g., P450c17 and P450scc, are found in patients with Addison’s disease 18 . The 21OH is suggested as the main autoantigen in Addison’s disease 5, 19 , and it is therefore of considerable interest to identify which parts (epitopes) of the 21OH protein are recognized by 21OHAb. Patients with Addison’s disease tend to be HLA DR3/4-DQ2/8 positive 20 , and a better understanding of the mechanisms for generating epitope-specific autoantibodies may uncover the etiology of this disease.

A number of studies have demonstrated that autoantibodies can be directed against specific domains of autoantigens. In myasthenia gravis, a fraction of the Abs are specifically directed to the acetylcholine binding site of the acetylcholine receptor, directly inhibiting the function of the receptor 21 . Natural anti-estrogen receptor autoantibodies decrease the hormone binding capacity and show estrogen effects in cell culture 22 . In systemic lupus erythematosus, human autoantibodies react with highly conserved regions of proteins and have the capability to inhibit the functions of both structural proteins and enzymes 1 . These, and a number of other similar findings, suggest that the epitopes recognized by autoantibodies are often functional domains of proteins.

Two autoantigenic epitopes, located in the central and C-terminal parts of 21OH, were detected using deleted variants of the protein synthesized by in vitro transcription-translation or in yeast cells 9, 10 . These studies suggested that at least one conformational epitope was defined by the 21OHAb binding sites. However, the use of deletion mutants complicates the detection of conformational epitopes, since the three-dimensional structure of the Ag is partially lost.

We therefore studied the influence of a number of naturally occurring and functionally important CYP21 gene mutations on the binding of 21OHAbs to the enzyme. The mutants were analyzed in our recently developed sensitive radiobinding assay for 21OHAb 11 . With this assay, we demonstrated that 100% of idiopathic Addison’s disease sera were positive for 21OHAb when the analysis was performed on serum samples collected within 20 years after the diagnosis 11 . In comparison with previous studies, in which immunoblotting analyses were used to quantify 21OHAb, our liquid method is associated with a higher diagnostic sensitivity and specificity for Addison’s disease. Among the five mutations investigated in this study, two mutations (delE196 and G291S) are located in the internal region of the enzyme and two (P453S and R483P) in the C-terminal region. These five mutations are also evenly spaced along the enzyme.

Our results demonstrate that 21OHAb from all sera displayed the same pattern of binding to different mutated 21OH proteins, suggesting that, irrespective of overt disease, they are directed against the same epitope, and that this epitope is located in the C-terminal part of the protein. Only 2 of 24 Addison’s and 1 of 4 Graves’ disease and IDDM sera showed a detectable level of immunoprecipitation with the R483P mutant protein (Figs. 1Go and 2Go). As these sera had only 5, 12, and 10% binding capability to the mutant protein compared with wild type, we conclude that the R483 residue plays a critical role in forming the autoantigenic epitope. This conclusion is supported by our observation, and that by others 10 , showing that the neighboring P453S substitution also impairs (although less drastically) 21OHAb binding.

In our study, we observed that synthetic peptides, corresponding to amino acids 447–461 and 477–491 of human 21OH, did not compete with the binding of 21OHAb to the wild-type autoantigen (Table IIGo). This result suggests that single-amino acid substitutions of the carboxyl-terminal region of 21OH modify the immunoreactivity of the enzyme due to a change in the three-dimensional structure. Although the Western blot experiments have not been precisely quantified, they showed the same pattern of autoantibody binding as seen in our liquid-phase assays, supporting the conclusion concerning the existence of C-terminal conformational epitope(s). In some cases, autoantigens with conformational epitopes can bind high-titer autoantibodies in Western blots, despite the denaturing conditions in the electrophoresis 23, 24 . It was also suggested that 21OH molecules can refold after SDS gel electrophoresis 9 . The sequence encompassing the P453S and R483P substitutions is adjacent to the conserved heme binding domain and shows strong sequence conservation between human, bovine, murine, and porcine 21OH proteins 25 .

Studies of the membrane topology and hydrophilic/hydrophobic structure of 21OH and related cytochromes allow the suggestion that the C-terminal part of the protein is located on the cytoplasmic side of the microsome membrane and does not interact with other structural domains 15, 26, 27 . Recently the three-dimensional structure of the prokaryotic cytochrome P450BM-3, resembling eukaryotic microsomal P450s, was determined 28 . The topological structure of this protein demonstrated also that the C-terminal part is a separate domain without contacts to the rest of the P450BM-3 protein. Although the R483P substitution, located 11 amino acids from the C-terminal end, involves a proline residue that can drastically change peptide structure, this structural change is probably local, involving only the C-terminal part of the 21OH protein.

We wanted to examine whether any amino acid variants of 21OH exist that, by chance, are prone to convert into autoantigenic epitopes due to, e.g., molecular mimicry. Therefore, we sequenced the complete 21OH coding region of four alleles from Addison’s disease patients, 30 (15 x 2) different alleles in the gene region corresponding to the autoimmune epitope identified in exon 10, and 16 (8 x 2) alleles in the region corresponding to the potential epitopes encoded by exons 7 and 8 8, 9, 10 . No deviations from the normal CYP21 gene were found. A common neutral amino acid variant S493N has been identified in the C-terminal part of 21OH 25 . The DNA sequence analysis showed that all possible genotype combinations (homo- and heterozygotes) of this variant occur in 21OHAb-positive patients. Thus, our analysis makes it possible to conclude that the development of 21OHAb is not likely to be restricted to any particular allele of CYP21.

In this report we describe the identification of an autoimmune epitope in human 21OH by studying a number of functionally important mutations found in CAH patients. Our results demonstrate that the R483P substitution, located in a conserved C-terminal region of the protein, severely impairs 21OH enzymatic activity 3 and strongly inhibits 21OHAb binding. Taken together, these results make it possible to suggest that the region around R483 in particular, but also the region involving P453, has critical enzymatic functions and considerable autoimmune capability as a three-dimensional epitope in the C-terminal end of the enzyme. This conclusion is also supported by the observation that 21OHAb from Addison’s disease patients can inhibit human 21OH in yeast microsomes 19 . The fact that 21OHAb were directed against the same 21OH epitope both in patients with and without clinical symptoms of adrenal insufficiency, shows the general nature in the selection of this epitope.


   Footnotes
 
1 This study was supported by the Swedish Medical Research Council, the National Institutes of Health (Grant DK26190), the Swedish Diabetes Association, and the Novo Nordisk, Lars Hiertas Minne, and Tore Nilsson funds. Back

2 Address correspondence and reprint requests to Dr. Andrej Nikoshkov, Department of Molecular Medicine, Karolinska Hospital, L6:02, S-171 76 Stockholm, Sweden. E-mail address: Back

3 Abbreviations used in this paper: 21OH, 21-hydroxylase; 21OHAb, autoantibodies to 21OH; IDDM, insulin-dependent diabetes mellitus; APS, autoimmune polyendocrine syndrome; CAH, congenital adrenal hyperplasia; RRI, rabbit relative index. Back

Received for publication June 24, 1998. Accepted for publication November 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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