Characterization of Mutations Causing CYP21A2 Deficiency in Brazilian and Portuguese Populations.

Deficiency of 21-hydroxylase enzyme (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction and functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V, and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of CYP21A2 deficiency.

Identification and circumvention of bottlenecks in CYP21A2-mediated premedrol production using recombinant Escherichia coli.

Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo- and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone-precursor premedrol by optimizing the CYP21A2-based whole-cell system on a laboratory scale. We successfully improved the whole-cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N-terminally truncated version of the bovine NADPH-dependent cytochrome P450 reductase (bCPR-27 ) and coexpression of microsomal cytochrome b5 ; (b) enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c) circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed-batch protocol. By overcoming the presented limitations in whole-cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L-1 ·d-1 premedrol.

How do mutations affect the structural characteristics and substrate binding of CYP21A2? An investigation by molecular dynamics simulations.

Congenital adrenal hyperplasia (CAH) is one of the most frequent inborn errors of metabolism, inherited as an autosomal recessive trait. Above 95% of CAH cases are caused by mutations in cytochrome P450 21A2 (CYP21A2). It is a pity that how these mutations affect the structural characteristics and substrate binding of CYP21A2 is still unclear. To this end, molecular dynamics (MD) simulations and binding free energy calculations are performed to investigate the effects of single point mutations (L108R, G292C, G292S, G293D, and T296N) in CYP21A2. The results indicate that mutations could cause the local conformational changes of CYP21A2, affecting the substrate binding by changing the interaction between the protein and heme, changing the charge environment of residues, or introducing steric hindrance. In addition, our work gives a wonderful explanation of the phenomenon that though the substrate binding ability increases, the reaction activity decreases in T296N. The present study provides detailed atomistic insights into the structure-function relationships of CYP21A2, which could contribute to further understanding about 21-hydroxylase deficiency and also provide a theoretical basis for CAH prediction and treatment.

In Silico Structural and Biochemical Functional Analysis of a Novel CYP21A2 Pathogenic Variant.

Classical congenital adrenal hyperplasia (CAH) caused by pathogenic variants in the steroid 21-hydroxylase gene (CYP21A2) is a severe life-threatening condition. We present a detailed investigation of the molecular and functional characteristics of a novel pathogenic variant in this gene. The patient, 46 XX newborn, was diagnosed with classical salt wasting CAH in the neonatal period after initially presenting with ambiguous genitalia. Multiplex ligation-dependent probe analysis demonstrated a full deletion of the paternal CYP21A2 gene, and Sanger sequencing revealed a novel de novo CYP21A2 variant c.694-696del (E232del) in the other allele. This variant resulted in the deletion of a non-conserved single amino acid, and its functional relevance was initially undetermined. We used both in silico and in vitro methods to determine the mechanistic significance of this mutation. Computational analysis relied on the solved structure of the protein (Protein-data-bank ID 4Y8W), structure prediction of the mutated protein, evolutionary analysis, and manual inspection. We predicted impaired stability and functionality of the protein due to a rotatory disposition of amino acids in positions downstream of the deletion. In vitro biochemical evaluation of enzymatic activity supported these predictions, demonstrating reduced protein levels to 22% compared to the wild-type form and decreased hydroxylase activity to 1-4%. This case demonstrates the potential of combining in-silico analysis based on evolutionary information and structure prediction with biochemical studies. This approach can be used to investigate other genetic variants to understand their potential effects.

Endogenous insertion of non-native metalloporphyrins into human membrane cytochrome P450 enzymes.

Human cytochrome P450 enzymes are membrane-bound heme-containing monooxygenases. As is the case for many heme-containing enzymes, substitution of the metal in the center of the heme can be useful for mechanistic and structural studies of P450 enzymes. For many heme proteins, the iron protoporphyrin prosthetic group can be extracted and replaced with protoporphyrin containing another metal, but human membrane P450 enzymes are not stable enough for this approach. The method reported herein was developed to endogenously produce human membrane P450 proteins with a nonnative metal in the heme. This approach involved coexpression of the P450 of interest, a heme uptake system, and a chaperone in Escherichia coli growing in iron-depleted minimal medium supplemented with the desired trans-metallated protoporphyrin. Using the steroidogenic P450 enzymes CYP17A1 and CYP21A2 and the drug-metabolizing CYP3A4, we demonstrate that this approach can be used with several human P450 enzymes and several different metals, resulting in fully folded proteins appropriate for mechanistic, functional, and structural studies including solution NMR.

Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia.

Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone (17α-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH-progesterone). We found that the 17α-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (Ks) for four variants were 37-13,000-fold higher than for WT P450 21A2. Cytochrome b5, which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (≥ 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d3-progesterone, indicating that C-H bond breaking is a rate-limiting step over a 104-fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme.

Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase: STRUCTURE OF THE ENZYME·PROGESTERONE SUBSTRATE COMPLEX AND RATE-LIMITING C-H BOND CLEAVAGE.

Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613-10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C-H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 10(7) M(-1) s(-1)) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 10(7) M(-1) s(-1)) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants.

A rare CYP 21 mutation (p.E431K) induced deactivation of CYP 21A2 and resulted in congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is caused by mutations in the CYP21A2 gene. The residual enzyme activity is strongly associated with the phenotype. We describe a rare case of CAH with a rare CYP21A2 mutation. The patient was a one-year-old Japanese boy. At 16 days old, he was referred to our hospital because of elevated serum 17-OH-progesterone (17-OHP) levels in neonatal screening. The compound heterozygous mutations (IVS2-13 A/C>G, and p.E431K) in CYP21A2 were identified at 2 months old, and we diagnosed non-classical CAH, since he did not have significant physical signs (pigmentation and salt-wasting). However, his body weight decreased, and his serum 17-OHP level (99.5 ng/mL) was elevated at 3 months old. Steroid replacement therapy was started at 3 months old. Our patient's clinical course resembled simple virilizing (SV) CAH, but classification was difficult because the patient showed increased renin activity indicating an aldosterone deficiency, and late onset of symptoms. While the IVS 2-13 A/C>G mutation is common in the classical form of CAH, p.E431K is a rare point mutation. Functional analysis revealed that the residual enzyme activity of p.E431L was 5.08±2.55% for 17-OHP and 4.12±2.37% for progesterone, which is consistent with SV CAH. p.E431 is localized in the L-helix near the heme-binding site. The mutation might interfere with heme binding, leading to deactivation of CYP21A2. This report showed that CYP21A2 p.E431 has an important effect on enzyme activity.

Epoxidation activities of human cytochromes P450c17 and P450c21.

Some cytochrome P450 enzymes epoxidize unsaturated substrates, but this activity has not been described for the steroid hydroxylases. Physiologic steroid substrates, however, lack carbon-carbon double bonds in the parts of the pregnane molecules where steroidogenic hydroxylations occur. Limited data on the reactivity of steroidogenic P450s toward olefinic substrates exist, and the study of occult activities toward alternative substrates is a fundamental aspect of the growing field of combinatorial biosynthesis. We reasoned that human P450c17 (steroid 17-hydroxylase/17,20-lyase, CYP17A1), which 17- and 16α-hydroxylates progesterone, might catalyze the formation of the 16α,17-epoxide from 16,17-dehydroprogesterone (pregna-4,16-diene-3,20-dione). CYP17A1 catalyzed the novel 16α,17-epoxidation and the ordinarily minor 21-hydroxylation of 16,17-dehydroprogesterone in a 1:1 ratio. CYP17A1 mutation A105L, which has reduced progesterone 16α-hydroxylase activity, gave a 1:5 ratio of epoxide:21-hydroxylated products. In contrast, human P450c21 (steroid 21-hydroxylase, CYP21A2) converted 16,17-dehydroprogesterone to the 21-hydroxylated product and only a trace of epoxide. CYP21A2 mutation V359A, which has significant 16α-hydroxylase activity, likewise afforded the 21-hydroxylated product and slightly more epoxide. CYP17A1 wild-type and mutation A105L do not 21- or 16α-hydroxylate pregnenolone, but the enzymes 21-hydroxylated and 16α,17-epoxidized 16,17-dehydropregnenolone (pregna-5,16-diene-3ß-ol-20-one) in 4:1 or 12:1 ratios, respectively. Catalase and superoxide dismutase did not prevent epoxide formation. The progesterone epoxide was not a time-dependent, irreversible CYP17A1 inhibitor. Our substrate modification studies have revealed occult epoxidase and 21-hydroxylase activities of CYP17A1, and the fraction of epoxide formed correlated with the 16α-hydroxylase activity of the enzymes.

Functional and structural analysis of four novel mutations of CYP21A2 gene in Italian patients with 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the 21-hydroxylase gene (CYP21A2), coding for the enzyme 21-hydroxylase (21-OH). About 95% of the mutations arise from gene conversion between CYP21A2 and the inactive pseudogene CYP21A1P: only 5% are novel CYP21A2 mutations, in which functional analysis of mutant enzymes has been helpful to correlate genotype-phenotype. In the present study, we describe 3 novel point mutations (p.L122P, p.Q481X, and p.E161X) in 3 Italian patients with CAH: the fourth mutation (p.M150R) was found in the carrier state. Molecular modeling suggests a major impact on 21-hydroxylase activity, and functional analysis after expression in COS-7 cells confirms reduced enzymatic activity of the mutant enzymes. Only the p.M150R mutation affected the activity to a minor extent, associated with NC CAH. CYP21A2 genotyping and functional characterization of each disease-causing mutation has relevance both for treatment and genetic counseling to the patients.

Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants.

Steroid 21-hydroxylase (cytochrome P450 21A2, CYP21A2) deficiency accounts for ∼95% of individuals with congenital adrenal hyperplasia, a common autosomal recessive metabolic disorder of adrenal steroidogenesis. The effects of amino acid mutations on CYP21A2 activity lead to impairment of the synthesis of cortisol and aldosterone and the excessive production of androgens. In order to understand the structural and molecular basis of this group of diseases, the bovine CYP21A2 crystal structure complexed with the substrate 17-hydroxyprogesterone (17OHP) was determined to 3.0 Šresolution. An intriguing result from this structure is that there are two molecules of 17OHP bound to the enzyme, the distal one being located at the entrance of the substrate access channel and the proximal one bound in the active site. The substrate binding features locate the key substrate recognition residues not only around the heme but also along the substrate access channel. In addition, orientation of the skeleton of the proximal molecule is toward the interior of the enzyme away from the substrate access channel. The 17OHP complex of CYP21A2 provides a good relationship between the crystal structure, clinical data, and genetic mutants documented in the literature, thereby enhancing our understanding of congenital adrenal hyperplasia. In addition, the location of certain CYP21A2 mutations provides general understanding of structure/function relationships in P450s.

Role of microsomal steroid hydroxylases in Δ7-steroid biosynthesis.

CYP17 (steroid 17α-hydroxylase/17,20-lyase) is a key enzyme in steroid hormone biosynthesis. It catalyzes two independent reactions at the same active center and has a unique ability to differentiate Δ(4)-steroids and Δ(5)-steroids in the 17,20-lyase reaction. The present work presents a complex experimental analysis of the role of CYP17 in the metabolism of 7-dehydrosteroids. The data indicate the existence of a possible alternative pathway of steroid hormone biosynthesis using 7-dehydrosteroids. The major reaction products of CYP17 catalyzed hydroxylation of 7-dehydropregnenolone have been identified. Catalytic activity of CYP17 from different species with 7-dehydropregnenolone has been estimated. It is shown that CYP21 cannot use Δ(5)-Δ(7) steroids as a substrate.

Minor activities and transition state properties of the human steroid hydroxylases cytochromes P450c17 and P450c21, from reactions observed with deuterium-labeled substrates.

The steroid hydroxylases CYP17A1 (P450c17, 17-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) catalyze progesterone hydroxylation at one or more sites within a 2 Å radius. We probed their hydrogen atom abstraction mechanisms and regiochemical plasticity with deuterium-labeled substrates: 17-[(2)H]-pregnenolone; 17-[(2)H]-, 16α-[(2)H]-, 21,21,21-[(2)H(3)]-, and 21-[(2)H]-progesterone; and 21,21,21-[(2)H(3)]-17-hydroxyprogesterone. Product distribution and formation rates with recombinant human P450-oxidoreductase and wild-type human CYP17A1 or mutation A105L (reduced progesterone 16α-hydroxylation) and wild-type human CYP21A2 or mutation V359A (substantial progesterone 16α-hydroxylation) were used to calculate intramolecular and intermolecular kinetic isotope effects (KIEs). The intramolecular KIEs for CYP17A1 and mutation A105L were 4.1 and 3.8, respectively, at H-17 and 2.9 and 5.1, respectively, at H-16α. Mutation A105L 21-hydroxylates progesterone (5% of products), and wild-type CYP17A1 also catalyzes a trace of 21-hydroxylation, which increases with 16α-[(2)H]- and 17-[(2)H]-progesterone. The intramolecular KIEs with CYP21A2 mutation V359A and progesterone were 6.2 and 3.8 at H-21 and H-16α, respectively. Wild-type CYP21A2 also forms a trace of 16α-hydroxyprogesterone, which increased with 21,21,21-[(2)H(3)]-progesterone substrate. Competitive intermolecular KIEs paralleled the intramolecular KIE values, with (D)V values of 1.4-5.1 and (D)V/K values of 1.8-5.1 for these reactions. CYP17A1 and CYP21A2 mutation V359A both 16α-hydroxylate 16α-[(2)H]-progesterone with 33-44% deuterium retention, indicating stereochemical inversion. We conclude that human CYP17A1 has progesterone 21-hydroxylase activity and human CYP21A2 has progesterone 16α-hydroxylase activity, both of which are enhanced with deuterated substrates. The transition states for C-H bond cleavage in these hydroxylation reactions are either significantly nonlinear and/or asymmetric, and C-H bond breakage is partially rate-limiting for all reactions.

Why human cytochrome P450c21 is a progesterone 21-hydroxylase.

Human cytochrome P450c21 (steroid 21-hydroxylase, CYP21A2) catalyzes the 21-hydroxylation of progesterone (P4) and its preferred substrate 17α-hydroxyprogestrone (17OHP4). CYP21A2 activities, which are required for cortisol and aldosterone biosynthesis, involve the formation of energetically disfavored primary carbon radicals. Therefore, we hypothesized that the binding of P4 and 17OHP4 to CYP21A2 restricts access of the reactive heme-oxygen complex to the C-21 hydrogen atoms, suppressing oxygenation at kinetically more favorable sites such as C-17 and C-16, which are both hydroxylated by cytochrome P450c17 (CYP17A1). We reasoned that expansion of the CYP21A2 substrate-binding pocket would increase substrate mobility and might yield additional hydroxylation activities. We built a computer model of CYP21A2 based principally on the crystal structure of CYP2C5, which also 21-hydroxylates P4. Molecular dynamics simulations indicate that binding of the steroid nucleus perpendicular to the plane of the CYP21A2 heme ring limits access of the heme oxygen to the C-21 hydrogen atoms. Residues L107, L109, V470, I471, and V359 were found to contribute to the CYP21A2 substate-binding pocket. Mutation of V470 and I471 to alanine or glycine preserved P4 21-hydroxylase activity, and mutations of L107 or L109 were inactive. Mutations V359A and V359G, in contrast, acquired 16α-hydroxylase activity, accounting for 40% and 90% of the P4 metabolites, respectively. We conclude that P4 binds to CYP21A2 in a fundamentally different orientation than to CYP17A1 and that expansion of the CYP21A2 substrate-binding pocket allows additional substrate trajectories and metabolic switching.

Interactions of galeterone and its 3-keto-Δ4 metabolite (D4G) with one of the key enzymes of corticosteroid biosynthesis - steroid 21-monooxygenase (CYP21A2).

We have investigated interactions of galeterone and its pharmacologically active metabolite - 3-keto-Δ4-galeterone (D4G) - with one of the key enzymes of corticosteroid biosynthesis - steroid 21-monooxygenase (CYP21A2). It was shown by absorption spectroscopy that both compounds induce type I spectral changes of CYP21A2. Spectral dissociation constants (KS ) of complexes of CYP21A2 with galeterone or D4G were calculated as 3.1 ± 0.7 µm and 4.6 ± 0.4 µm, respectively. It was predicted by molecular docking that both ligands similarly bind to the active site of CYP21A2. We have revealed using reconstituted monooxygenase system that galeterone is a competitive inhibitor of CYP21A2 with the inhibition constant (Ki ) value of 12 ± 3 µm, while D4G at the concentrations of 10 and 25 µm does not inhibit the enzyme. Summarizing, based on the in vitro analyses we detected inhibition of CYP21A2 by galeterone and lack of the influence of D4G on this enzyme.

Is the unique benzodiazepine structure interacting with CYP enzymes to affect steroid synthesis in vitro?

The aim of this project was to investigate the endocrine disrupting effects of three γ-aminobutyric acid type A receptor (GABAAR) agonists, diazepam (DZ), oxazepam (OX) and alprazolam (AL) using the steroidogenic in vitro H295R cell line assay, a recombinant CYP17A1 assay, qPCR analysis and computational modelling. Similar effects for DZ and OX on the steroidogenesis were observed in the H295R experiment at therapeutically relevant concentrations. Progestagens and corticosteroids were increased up to 10 fold and androgens were decreased indicating CYP17A1 lyase inhibition. For DZ the inhibition on both the hydroxylase and lyase was confirmed by the recombinant CYP17A1 assay, whereas OX did not appear to directly affect the recombinant CYP17A1 enzyme. Androgens were decreased when exposing the H295R cells to AL, indicating a CYP17A1 lyase inhibition. However, this was not confirmed by the recombinant CYP17A1 assay but a down-regulation in gene expression was observed for StAR and CYP17A1. The present study showed that the three investigated benzodiazepines (BZDs) are rather potent endocrine disruptors in vitro, exerting endocrine effects close the therapeutic Cmax. Both direct and indirect effects on steroidogenesis were observed, but molecular modelling indicated no direct interactions between the heme group in the steroidogenic CYP enzymes and the unique diazepin structure. In contrast, physicochemical properties such as high log P, structure and molecular weight similar to that of steroids appeared to influence the endocrine disrupting abilities of the investigated pharmaceuticals in vitro. Docking of the three BZDs in CYP17A1 and CYP21A2 confirmed that shape complementarity and hydrophobic effects seem to determine the binding modes.

21-Hydroxylase epitopes are targeted by CD8 T cells in autoimmune Addison's disease.

In autoimmune adrenal deficiency, autoantibodies target the 21-hydroxylase (21OH) protein. However, it is presumed that autoreactive T cells, rather than antibodies, are the main effectors of adrenal gland destruction, but their identification is still lacking. We performed a T-cell epitope mapping study using 49 overlapping 20mer peptides covering the 21OH sequence in patients with isolated Addison's disease, Autoimmune Polyendocrine Syndrome 1 and 2. IFNγ ELISPOT responses against these peptides were stronger, broader and more prevalent among patients than in controls, whatever the disease presentation. Five peptides elicited T-cell responses in patients only (68% sensitivity, 100% specificity). Blocking experiments identified IFNγ-producing cells as CD8 T lymphocytes, with two peptides frequently recognized in HLA-B8+ patients and a third one targeted in HLA-B35+ subjects. In particular, the 21OH(431-450) peptide was highly immunodominant, as it was recognized in more than 30% of patients, all carrying the HLA-B8 restriction element. This 21OH(431-450) region contained an EPLARLEL octamer (21OH(431-438)) predicted to bind to HLA-B8 with high affinity. Indeed, circulating EPLARLEL-specific CD8 T cells were detected at significant frequencies in HLA-B8+ patients but not in controls by HLA tetramer staining. This report enlightens disease-specific T-cell biomarkers and epitopes targeted in autoimmune adrenal deficiency.

The purification and application of biologically active recombinant steroid cytochrome P450 21-hydroxylase: the major autoantigen in autoimmune Addison's disease.

In primary adrenocortical failure (Addison's disease) caused by autoimmunity, autoantibodies to the steroidogenic cytochrome P450 enzyme 21-hydroxylase (21OH) are detected in the majority of patients. It is currently uncertain whether the autoantibodies themselves participate in the pathogenesis, or if they merely reflect an on-going T cell mediated response. The identification of T cells reactive with 21OH, if any, has been hampered by the lack of a high-quality antigen. In the current study recombinant human 21OH has been expressed in Spodoptera frugiperda insect cells using a baculovirus expression system. Recombinant enzymatically active 21OH was purified to apparent homogeneity by immobilized metal ion affinity chromatography. The purified enzyme was highly immunogenic in immunized SJL/J mice, and immune responses to 21OH-derived peptides assayed as T cell proliferation and interferon gamma production could be invoked after priming with the recombinant protein. Furthermore, purified 21OH was recognized by sera from patients with autoimmune Addison's disease, and it could block the binding of radiolabeled in vitro translated 21OH in a sensitive fluid-phase radioimmunoassay. We conclude that the recombinant preparation of 21OH presented here is of sufficient purity and quality to be used for studies of cellular and humoral immunity in autoimmune Addison's disease.

A novel 9-bp insertion detected in steroid 21-hydroxylase gene (CYP21A2): prediction of its structural and functional implications by computational methods.

BACKGROUND: Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia (CAH). Detection of underlying mutations in CYP21A2 gene encoding steroid 21-hydroxylase enzyme is helpful both for confirmation of diagnosis and management of CAH patients. Here we report a novel 9-bp insertion in CYP21A2 gene and its structural and functional consequences on P450c21 protein by molecular modeling and molecular dynamics simulations methods. METHODS: A 30-day-old child was referred to our laboratory for molecular diagnosis of CAH. Sequencing of the entire CYP21A2 gene revealed a novel insertion (duplication) of 9-bp in exon 2 of one allele and a well-known mutation I172N in exon 4 of other allele. Molecular modeling and simulation studies were carried out to understand the plausible structural and functional implications caused by the novel mutation. RESULTS: Insertion of the nine bases in exon 2 resulted in addition of three valine residues at codon 71 of the P450c21 protein. Molecular dynamics simulations revealed that the mutant exhibits a faster unfolding kinetics and an overall destabilization of the structure due to the triple valine insertion was also observed. CONCLUSION: The novel 9-bp insertion in exon 2 of CYP21A2 genesignificantly lowers the structural stability of P450c21 thereby leading to the probable loss of its function.

Novel non-classic CYP21A2 variants, including combined alleles, identified in patients with congenital adrenal hyperplasia.

OBJECTIVE: Congenital adrenal hyperplasia (CAH) is an inborn error of metabolism and a common disorder of sex development where >90% of all cases are due to 21-hydroxylase deficiency. Novel and rare pathogenic variants account for 5% of all clinical cases. Here, we sought to investigate the functional and structural effects of four novel (p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro) and three combinations of CYP21A2 variants (i.e. one allele containing two variants p.[Ile172Asn;Val358Ile], p.[Val281Leu;Arg369Gln], or p.[Asp377Tyr;Leu461Pro]) identified in patients with CAH. METHODS: All variants were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells and enzyme activities directed toward the two natural substrates (17-hydroxyprogesterone and progesterone) were determined. In parallel, in silico prediction of the pathogenicity of the variants based on the human CYP21 X-ray structure was performed. RESULTS: The novel variants, p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro exhibited residual enzymatic activities within the range of non-classic (NC) CAH variants (40-82%). An additive effect on the reduction of enzymatic activity (1-17%) was observed when two variants were expressed together, as identified in several patients, resulting in either NC or more severe phenotypes. In silico predictions were in line with the in vitro data except for p.Leu461Pro. CONCLUSIONS: Altogether, the combination of clinical data, in silico prediction, and data from in vitro studies are important for establishing a correct genotype and phenotype correlation in patients with CAH.