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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1937
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Right arrow Adrenal and Hypertension
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 2148-2153
Copyright © 2005 by The Endocrine Society

Not All Amino Acid Substitutions of the Common Cluster E6 Mutation in CYP21 Cause Congenital Adrenal Hyperplasia

Tiina Robins, Michela Barbaro, Svetlana Lajic and Anna Wedell

Department of Molecular Medicine, Center of Molecular Medicine, Karolinska Institutet/Karolinska University Hospital, 171 76 Stockholm, Sweden

Address all correspondence and requests for reprints to: Tiina Robins, Department of Molecular Medicine, Center of Molecular Medicine (CMM) L8:02, Karolinska Institutet/Karolinska University Hospital, 171 76 Stockholm, Sweden. E-mail: tiina.robins{at}cmm.ki.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
More than 90% of all cases of congenital adrenal hyperplasia result from steroid 21-hydroxylase (CYP21) gene mutations. Around 95% of these are either gene deletions or any of nine sequence aberrations that have been transferred from the nearby pseudogene through apparent gene conversions. One such recurrent pseudogene-derived mutation is Cluster E6, a combination of three amino acid substitutions in exon 6: I236N, V237E, and M239K. Cluster E6 is associated with the most severe, salt-wasting form of congenital adrenal hyperplasia. We studied the functional consequences of each missense mutation individually as well as the combined effect of the three mutations comprising Cluster E6. V237E abolished enzyme function and is thus a null mutation, whereas very low but measurable activity remained for I236N. M239K, on the other hand, had no effect on enzyme activity and consequently does not contribute to the disease. Although no allele has been reported yet to contain only one or two missense mutations of Cluster E6, it is a well-known feature of CYP21 that it can harbor many different combinations of pseudogene-derived mutations. The exclusion of M239K as a disease-causing mutation is thus relevant when designing protocols for genetic diagnostics.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CONGENITAL ADRENAL HYPERPLASIA (CAH) is a group of autosomal recessive disorders characterized by disturbed adrenal cortisol synthesis in which the majority of all cases (around 90–95%) are due to defects in the steroid 21-hydroxylase (CYP21) gene causing steroid 21-hydroxylase deficiency (for review see Ref. 1). Steroid 21-hydroxylation is an essential step in the biosynthesis of both cortisol and aldosterone; thus, a complete absence of CYP21 function leads to the most severe, salt-wasting (SW) form of CAH, with life-threatening salt loss in the neonatal period. A lack of feedback inhibition results in high levels of ACTH and adrenal hyperplasia and stimulation of steroidogenesis up to the enzyme block. Accumulated steroid metabolites proximal to the 21-hydroxylation step are further shunted into the androgen pathway, leading to various degrees of hyperandrogenic symptoms.

SW CAH together with a slightly less severe form of the disease with apparently normal aldosterone biosynthesis, simple virilizing (SV) CAH, are both referred to as classical CAH. In classical CAH, there is prenatal virilization of external genitalia in females including clitoromegaly and fusion of the labiae majorae. These genital malformations are graded according to severity into Prader stages I-V (2). There is also progressive postnatal virilization in both sexes including accelerated growth and advancement of bone age during the first years of life. Without proper treatment, patients with classical CAH develop as short adults due to early epiphyseal closure. Milder forms of CAH are referred to as nonclassic (NC) or late-onset CAH. Symptoms of NC CAH include precocious pubarche in children and acne, hirsutism, and menstrual irregularities in women. The severity of hyperandrogenic symptoms is variable in these patients. Males especially are usually asymptomatic.

The incidence of classical 21-hydroxylase deficiency is around 1/10,000 in most populations (3), whereas the NC form has been estimated to occur in 0.1–3.7% of Caucasians (4), making CAH one of the most frequent inborn errors of metabolism in humans.

The gene encoding 21-hydroxylase, CYP21 (also called CYP21A2), is located in a locus with a complicated structure. There are two CYP21 genes located in the highly polymorphic HLA histocompatibility complex region on chromosome 6p21.3 in very near proximity (30 kb) to each other (5, 6). Both genes are located immediately adjacent to and alternating with the two genes encoding the fourth component of complement, factors C4A and C4B. Both genes contain 10 exons, and their nucleotide sequences are 98% identical in exons and approximately 96% identical in introns (7, 8). However, only one of these genes is functional, whereas the other, CYP21P (also called CYP21A1P), is an inactive pseudogene that has accumulated a number of deleterious sequence changes during evolution.

The presence of CYP21P in such close proximity to CYP21 predisposes to exchange of material between these highly homologous genes. Misalignment during meiosis followed by recombination (unequal crossing over) can lead to a complete deletion of C4B and a net deletion of CYP21 (9, 10). This mechanism can also generate chromosomes with more than two C4/CYP21 repeat units, complicating genetic diagnostics (11). In addition, sequences can be transferred between the genes by apparent gene conversion events (12, 13). Pseudogene-derived sequences that in this way can compromise the function of CYP21 are P30L, an A/C to G substitution 13 bases upstream of exon 3 (I2 splice), an 8-bp deletion in exon 3 (E3del8bp), I172N, a cluster of three amino acid alterations in exon 6 (Cluster E6), V281L, a T-insertion in exon 7 causing a shift of the reading frame (L307insT), Q318X, and R356W.

Of all disease-causing mutations found in patients with CAH, around 95% are represented by either a deletion of the entire CYP21 gene or conversion of CYP21 into pseudogene sequences in one or more of the nine positions described above. This general mutational spectrum is similar in most populations (14, 15, 16, 17, 18, 19, 20, 21, 22), although the relative distribution of individual mutations can vary somewhat in different ethnic groups. The remaining 5% of disease-causing CYP21 alleles harbor rare mutations that do not originate from the pseudogene.

Generally, there is a good relationship between clinical disease presentation and a patient’s underlying combination of mutations (17, 18, 21, 23), although rare exceptions to these genotype-phenotype relationships are sometimes seen. In vitro assays of recombinant mutant enzyme activity after expression in cultured cells have also been helpful to classify mutations according to severity, especially for rare mutations where large numbers of patients are not available (15, 24, 25, 26, 27, 28, 29, 30).

The Cluster E6 mutation belongs to the group of common, pseudogene-derived mutations that are found in patients with classical CAH from all major populations. Cluster E6 consists of a combination of three amino acid substitutions in exon 6: I236N, V237E, and M239K. These are assumed to be transferred together from CYP21P to CYP21 in a continuous stretch of DNA. To establish whether all three mutations contribute to the disease, we have constructed each individual mutation, as well as the whole cluster, by in vitro site-directed mutagenesis. Activities toward the two natural substrates, 17-hydroxyprogesterone (17OHP) and progesterone, were determined for each mutant and compared with the normal enzyme after transient transfection of CYP21 proteins in COS-1 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Introduction of CYP21 mutations in the pCMV4-CYP21 expression vector

Construction of the pCMV4 expression vector containing the CYP21 cDNA, pCVM4-CYP21, has previously been described (31). The V237E and Cluster E6 mutation as a unit (I236N + V237E + M239K) were introduced using the pALTER-CYP21 mutagenesis vector and the Altered Sites II in vitro mutagenesis system (Promega, SDS Scandinavian Diagnostic Services, Falkenberg, Sweden) as previously described (25). Mutagenesis primers covering the specific point mutations were: 5'-CTG CAT CTC CTC GAT GTG ATC CC for V237E and 5'-CTG CCT CAG CTG CTT CTC CTC GTT GTG ATC CCT C for Cluster E6. BglII-KpnI fragments of pALTER-CYP21(V237E) and pALTER-CYP21(Cluster E6) were transferred into pCMV4, thereby generating pCMV4-CYP21(V237E) and pCMV4-CYP21(Cluster E6). The entire CYP21 cDNA was sequenced to verify the correct incorporation of mutations and exclude additional sequence aberrations.

Another site-directed mutagenesis system, the Stratagene QuickChange site-directed mutagenesis kit (AH Diagnostics, Skärholmen, Sweden) was used for the I236N and M239K mutations. Mutagenesis primers were: 5'-GAG AAG AGG GAT CAC AAC GTG GAG ATG CAG CTG AGG for I236N and 5'-GG GAT CAC ATC GTG GAG AAG CAG CTG AGG CAG CAC AAG G for M239K. The oligonucleotide primers were annealed to the pCMV4-CYP21 vector and extended during temperature cycling (an initial 30 sec at 95 C was followed by 20 cycles of 95 C for 30 sec, 55 C for 1 min, and 68 C for 14 min) using high-fidelity Pfu Turbo DNA polymerase, generating a mutated plasmid containing staggered nicks. After temperature cycling, products were treated with DpnI, specific for methylated DNA, digesting parental DNA template and leaving the mutation-containing newly synthesized DNA intact. The nicked pCMV4-CYP21 vector containing CYP21 mutations was then used to transform XL1-Blue supercompetent cells by a heat pulse. Bacteria were grown in ampicillin-containing medium, repairing nicks of the mutated plasmids generating pCMV4-CYP21(I236N) and pCMV4-CYP21(M239K). BglII-KpnI fragments of mutagenized plasmid were thereafter subcloned into native pCMV4 expression vector to circumvent the risk of having any additional mutations in the vector generated during mutagenesis. The complete CYP21 cDNA was finally sequenced in both cases to verify the correct incorporation of mutations and exclude additional sequence aberrations.

Cell culturing conditions

COS-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 0.2 mg/ml gentamicin, and 0.2% 100 x L-glutamine (all reagents supplied by Gibco, Invitrogen Life Technologies, Lidingö, Sweden). Cells were cultured at 37 C, 5% CO2 in 225-cm2 cell culture flasks (Costar, Lab Design AB, Täby, Sweden) and replated every third to fourth day.

Transient expression of CYP21 in COS-1 cells

Transfection of COS-1 cells with mutant pCMV4-CYP21 constructs, wild-type pCMV4-CYP21 (run as a reference), and native pCMV4 without CYP21 cDNA (mock transfection run as a negative control) was performed using liposomes (FuGENE 6 transfection reagent; Roche Diagnostics Scandinavia AB, Bromma, Sweden).

Approximately 2 x 105 cells were plated in six-well plates (Costar, Lab Design) on a surface area of 9.5 cm2 per well, with 2 ml medium to have a cell confluency of 50–70% on the following day when transfection experiments were performed. Cells were cotransfected with 1 µg of each pCMV4 construct and 0.25 µg ß-galactosidase vector pCH110 (Pharmacia, Uppsala, Sweden) using 3 µl FuGENE according to the manufacturer’s instructions and incubated at 37 C for 48 h for optimal CYP21 expression.

Assay of CYP21 enzyme activity

To determine in vitro 21-hydroxylase activity, 1 nCi/µl 3H-labeled substrate, 17OHP, or progesterone (Amersham Biosciences, Uppsala, Sweden) was added to each well of cells together with 2.0 µmol/liter unlabeled steroid and 4 mmol/liter of the naturally occurring cofactor nicotinamide adenine dinucleotide phosphate reduced (Sigma-Aldrich, Stockholm, Sweden). Conversion of substrates to the corresponding products, 11-deoxycortisol (11-DOL) and deoxycorticosterone, respectively, was allowed to proceed for 15 min at 37 C, whereupon 240 µl of medium was collected in duplicate samples. Steroids were extracted with 300 µl dichloromethane (Merck Eurolab, Stockholm, Sweden), evaporated to dryness, and dissolved in 15 µl ethanol containing size markers (17OHP + 11-DOL/progesterone + deoxycorticosterone). Separation of substrates and products was achieved by thin-layer chromatography using chloroform and ethyl acetate (Merck Eurolab) (80:20) as the mobile phase. Steroid spots were visualized by UV light, cut, and mixed with 4 ml Ultima Gold scintillation liquid (Packard Bioscience Co., Chemical Instruments AB, Lidingö, Sweden), and subsequently radioactivity was measured by liquid scintillation spectrophotometry. Background activity, i.e. activity of mock transfection, was deducted in each experiment and substrate conversion rates for mutant CYP21 were compared with those of wild-type (WT) enzyme after correction for total protein content. Enzyme activities were expressed as percentage of WT CYP21-activity that was defined as 100%.

Determination of total protein content and ß-galactosidase assay

Total protein content was determined directly after cell harvest, which was achieved by a 5-min incubation at 37 C with 0.25% trypsin in PBS lacking calcium and magnesium salts (Gibco, Invitrogen Life Technologies), and activated with 1% of 0.5 mol/liter EDTA (Merck Eurolab). Cells were washed twice with PBS, centrifuged at 172 x g for 10 min, and resuspended in 200 µl PBS. Extracts from cells were obtained by 10 sec of sonication followed by centrifugation at 10,000 x g for 5 min at 4 C for removal of cell debris. Protein concentration was determined using a protein assay (Bio-Rad Laboratories AB, Sundyberg, Sweden) based on the method of Bradford (32) using BSA as a protein standard.

ß-Galactosidase activity was measured according to standard procedures (33). The ratio of ß-galactosidase activity to total protein content was measured in each experiment to verify the reproducibility of transfection.

Analysis of protein expression by Western blot

COS-1 cells were transiently expressed with each of the pCMV4-CYP21 constructs and incubated for 48 h as described above. Because cell harvesting by trypsination can lead to degradation of proteins, cell extracts for Western blot analyses were lysed directly on the plate. Medium was aspirated, and cells were rinsed twice with PBS and lysed in sodium dodecyl sulfate (SDS) buffer containing 50 mmol/liter Tris-HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue, 10% glycerol, 100 mmol/liter dithiothreitol (Sigma-Aldrich), 50 mmol/liter NaCl, 5 mmol/liter Tris-HCl (pH 7.5), and 0.5 mmol/liter EDTA. Lysates were denatured at 70 C for 5 min and run on a 10% SDS-PAGE (Nupage Novex, Invitrogen Life Technologies) according to the manufacturer’s instructions. The size-separated proteins were blotted to a nitrocellulose membrane (Hybond ECL) (Amersham Biosciences) according to recommendations from the supplier and washed in 5 mol/liter Tris-HCl and 20 mmol/liter NaCl [Tris-buffered saline (TBS) buffer] supplemented with 1% Tween 20 (Sigma-Aldrich) for 10 min. Membranes were stained with Ponceau S solution (Sigma-Aldrich) for 5 min to ensure equal amounts of protein loading and washed with nonionized water. Blocking with TBS buffer supplemented with 1% Tween 20 and 10% milk powder was carried out for 2 h thereafter. Membranes were immunostained overnight at 4 C with primary antibody diluted in TBS supplemented with Tween 20 and milk as above. Two different primary antibodies were used in different immunoblot experiments: polyclonal antibodies raised in rabbit against a synthetic peptide derived from human CYP21 (28) in a 1:2000 dilution and serum from a patient with Addison’s disease having natural antibodies against CYP21 (kindly provided by Dr. A. Falorni, University of Perugia, Perugia, Italy) in a 1:100 dilution. After washing and blocking with above-mentioned buffers, a 2-h incubation with secondary goat antihuman IgG-horseradish peroxidase or antirabbit IgG-horseradish peroxidase antibody (Santa Cruz Biotechnology Inc., Scandinavian Diagnostic Services) was performed in a 1:1500 dilution. Finally, membranes were washed and incubated with enhanced chemiluminescent substrate (ECL Western blotting detection reagent) (Amersham Biosciences). A charge-coupled device camera and an image analyzer (Image Reader LAS-1000; Fuji Film, Tokyo, Japan) were subsequently used to visualize the proteins.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enzyme activity of mutants

To pinpoint which of the three missense mutations is responsible for the disease phenotype seen in CAH patients carrying the Cluster E6 mutation, each of the involved mutations was studied separately. The three individual mutations as well as the whole cluster were reconstructed by in vitro site-directed mutagenesis, and the resulting mutant proteins were transiently expressed in mammalian COS-1 cells. Assays of enzyme activity were performed in vitro using 17OHP and progesterone, the two natural substrates of CYP21. The effects of the different amino acid substitutions on 21-hydroxylase activity are shown in Fig. 1Go, with the WT enzyme activity defined as 100%. As shown, Cluster E6, containing all three missense mutations, as well as V237E on its own resulted in enzymes with no detectable activity. I236N caused a severely reduced but measurable function, with 1.0 ± 0.7% (mean ± SD) activity toward 17OHP and 2.4 ± 1.4% activity using progesterone as a substrate. The activities of the I236N and V237E mutants were significantly different from each other (P < 0.05 for both substrates, Student’s t test). The third amino acid substitution included in Cluster E6, M239K, did not cause a significantly reduced substrate conversion. Activity was 95.4 ± 24.7% for 17OHP and 97.7 ± 7.7% for progesterone, compared with normal CYP21 (P > 0.05 for both substrates, Student’s t test).



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  		FIG. 1. Enzymatic activities of CYP21 mutants in COS-1 cells. Enzymatic activities of CYP21 mutants (I236N, V237E, M239K, and Cluster E6) in COS-1 cells are expressed in relative values, i.e. as a percentage of WT activity that is defined as 100%. Substrate conversion rates (percent) were determined for 17OHP to 11-DOL (A) and progesterone to deoxycorticosterone (B), using substrate concentrations of 2 µmol/liter in both cases. All values are mean ± 1 SD of four to nine independent transfections/assays.

 
Expression of normal and mutant CYP21 in COS-1 cells

To investigate whether the CYP21 mutations affected the levels of protein expression, immunoblotting was performed from homogenates of cells transfected with the different constructs using rabbit antiserum raised against a peptide derived from human CYP21 (28). Comparable amounts of protein were detected for the complete Cluster E6 as well as for the V237E and M239K mutants (data not shown). The I236N mutant, on the other hand, repeatedly resulted in a barely detectable protein of the expected size. To confirm these results, we performed Western blot analyses using serum from a patient with Addison’s disease. Again, the I236N mutant was barely detectable, whereas the V237E and M239K mutant as well as the protein containing the complete Cluster E6 mutation resulted in protein amounts that were comparable with normal (Fig 2Go). Finally, we repeated the construction of the pCMV4-CYP21(I236N) expression vector, made novel transfections, and repeated the Western blotting experiments using both antisera. Again, low protein concentrations were detected for the I236N mutant. These results indicated that the cells containing the I236N mutant hardly produce any CYP21 protein. Another potential mechanism behind the low levels of protein is an accelerated degradation of the mRNA or the translated protein.



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  		FIG. 2. Immunoblot of mutant CYP21 proteins expressed in COS-1 cells. CYP21 protein containing the I236N, V237E, M239K, and Cluster E6 mutations as well as WT CYP21 was transiently expressed in COS-1 cells that were subsequently harvested after 48 h with SDS lysis buffer. Lysates were separated on a 10% SDS-PAGE, and protein expression was determined by immunoblot analyses using serum from a patient with Addison’s disease containing autoantibodies against the CYP21 protein. A reduced amount of protein was detected for the I236N mutant in comparison with all other CYP21 variants in repeated experiments. An arrow indicates the size of the CYP21 protein (55 kDa).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Cluster E6 mutation was first described in a patient with SW CAH in 1988 (34). The CYP21 gene was cloned from this patient, and the resulting protein was expressed in COS-7 cells using the pKCRH-2-expression vector. No enzymatic activity was detectable for the mutant protein. The Cluster E6 mutation has also been studied after expression of CYP21 in COS-1 cells using recombinant vaccinia virus. Also, in this system, the Cluster E6 mutant had no detectable activity (24). Cluster E6 has since been found in CAH patients of most populations in which the mutational spectrum of CYP21 has been investigated in detail. Clinical observations invariably show a correlation to the most severe, SW phenotype. Thus, there has been little doubt that the three missense mutations in exon 6 in combination cause a completely abolished CYP21 function, and our results confirm that Cluster E6 belongs to the null group of mutations. It has not been known, however, whether all three components of the cluster have the same deleterious effect on the enzyme.

Our assays indicate that V237E is as deleterious as the complete cluster, in itself capable of completely destroying enzyme function. V237E is thus also a null mutation and would be expected to result in the most severe, SW form of CAH if present on its own in CYP21.

I236N also resulted in a drastic impairment of 21-hydroxylase function, but a low, albeit significant, degree of enzyme activity remained. Investigations of the I172N mutation, the most common mutation associated with a SV phenotype (35), have indicated that around 1–2% of normal activity is sufficient to prevent SW (24), and in vitro assays of additional rare mutants detected in patients with SV phenotypes have indicated that less than 5–10% of normal activity is typical for this group of mutations (28). I236N had a residual activity of around 2% toward progesterone, the substrate for aldosterone production, leading us to believe that I236N is not a null mutation and that it is conceivable that a patient carrying I236N as his or her mildest CYP21 mutation would escape SW. Firm evidence for this, however, requires the identification of CAH patients representing such genotypes.

The mutant protein containing I236N was hardly detectable after transfection of COS-1 cells. This is surprising because the protein containing the complete Cluster E6 mutation was readily produced. It is possible that the mRNA corresponding to the I236N mutant adopts an unfavorable secondary structure preventing translation or that it is highly unstable. Another possibility is that the mutant protein adopts an abnormal structure causing it to be rapidly degraded in the cells, even though we omitted trypsination in an attempt to reduce protein degradation. We have previously shown that some specific mutant CYP21 forms seem to be less stable than the normal protein, as estimated by pulse-labeling followed by immunoprecipitation after a chase period (26) or by blocking of protein synthesis by cycloheximide followed by immunoblotting up to 24 h thereafter (28, 29). In no case, however, have we seen this drastic reduction in protein levels without a chase period of several hours. We used two different antisera (rabbit and human) and repeated the experiment several times, using two independently constructed expression vectors. We therefore do not think our results are due to a defective expression vector or inability of the antiserum to detect this particular mutant. Whatever the reason for the low amounts of the I236N enzyme variant in transfected COS-1 cells, it is reasonable to expect that it resembles what happens in the adrenocortical cell in vivo because the general machineries involved in transcription and translation are conserved among mammalian cells. It is not surprising that the low amount of protein produced results in a severely reduced enzyme activity. We cannot exclude, however, a possible additional contribution of a specific functional effect of the I236N mutant.

When studying the function of the CYP21 protein containing M239K, we found no difference in activity, compared with the normal enzyme for either substrate. This individual amino acid substitution does thus not contribute to CAH. We have sequenced exon 6 of CYP21 from well over 100 individuals and have not encountered M239K without the simultaneous presence of I236N and V237E; it thus does not represent a common polymorphism in the population.

Cluster E6 belongs to the group of pseudogene-derived mutations that represent the predominating causes of CAH in all populations that have been studied in sufficient detail. Its frequency among mutated CYP21 alleles ranges from approximately 1% in the Nordic countries [0.9% in Sweden, 2.7% in Norway, 1% in Finland (19), and 1.5% in Denmark (36)] to a reported maximum of 5–6% [4.8% in France (37), 5.6% in Japan (38), and 6% in south Italy (39)].

Pseudogene-derived mutations such as Cluster E6 are generally assumed to have been transferred from CYP21P to CYP21 by gene-conversion, i.e. nonreciprocal recombination, although the molecular mechanisms involved are not well known. It is a recognized feature of CYP21 that different combinations of pseudogene-derived mutations can be present in the same disease-causing allele, complicating genetic diagnostics in CAH (40). The extension of the converted sequences can thus vary substantially. Therefore, we cannot rule out the possibility that only parts of the Cluster E6 mutation, i.e. only one or two of the point mutations, can be transferred from CYP21P to CYP21, although no such allele has been described to date.

A multitude of different PCR-based methods are available for detection of specific mutations in genetic diagnostics, including, for example, allele-specific oligonucleotide hybridization, analysis of amplification-created restriction sites and single-stranded conformation polymorphisms, allele-specific PCR, ligation detection reaction, and oligonucleotide arrays. Several of these approaches have been applied to steroid 21-hydroxylase deficiency (1). When employing any of these strategies for mutation detection, we suggest that M239K should not be included in the sequence target because it does not contribute to disease and putative disease-causing alleles containing I236N and/or V237E only could be missed.

Data on structure-function relationships of CYP21 are limited because crystallization and determination of the three-dimensional structure is difficult because of the membrane bound properties of eukaryotic cytochrome P450 proteins. Crystallized prokaryotic P450 enzymes (41, 42, 43, 44, 45, 46, 47) have served as models for predictions of three-dimensional structures of various eukaryotic P450 enzymes involved in steroidogenesis including CYP21 (48, 49, 50). More recently five mammalian P450 enzymes, CYP2C5, CYP2C9, CYP2B4, CYP2C8, and CYP3A4, have been successfully crystallized, and their three-dimensional structures have been determined (51, 52, 53, 54, 55, 56, 57). Three of these (2C9, 2C8, and 3A4) are human. Based on the current model of human CYP21 (50) and the structure of CYP2C5 (a 21-hydroxylating enzyme in rabbit hepatocytes that uses progesterone as one of its substrates), we suggest that the residues mutated in Cluster E6 are located in the entrance of the substrate access channel of CYP21. All residues included in the Cluster E6 mutation are found in helix G, which is important for initial substrate recognition, although they are not near the inner substrate-binding pocket itself. Two different substrate access channels connecting the surface of the protein to the active site have been proposed for CYP2C5, both involving the G helix. All amino acid changes in the Cluster E6 mutation (I236N, V237E, M239K) represent replacements of nonpolar amino acids by polar, hydrophilic residues that could theoretically disrupt the hydrophobic environment and destabilize the {alpha}-helix, resulting in disturbed enzyme function by impairing its interaction with the steroid substrate. Our functional assays suggest that V237 is a critical residue in this respect.

Alignment of human CYP21 (Swiss-Prot primary accession no. P08686) with bovine (P00191), mouse (P03940), and pig (P15540 and Q02390) orthologs shows that amino acid 237 is identical (valine) in all species. Residue 236 is always small and hydrophobic (isoleucine in human, methionine in bovine, isoleucine in mouse, leucine in pig) and is thus highly conserved. In the position corresponding to 239 in humans, however, there is no conservation. In fact, bovine and pig orthologs all have a lysine in this position, which is the same as in the mutant human enzyme. These phylogenetic comparisons are thus in complete agreement with the results of our functional assays.

In conclusion, functional investigations of recombinant CYP21 containing the three amino acid alterations involved in the common Cluster E6 mutation showed that two of the mutations, I236N and V237E, have severe effects on enzyme activity and indicate that they would result in classical CAH if found in patients. The third mutation of the cluster (M239K), on the other hand, does not contribute to the disease.


   Footnotes
 
This work was supported by the Swedish Research Council (Grant 12198), the Novo Nordic Foundation, the Ronald McDonald Foundation, Sällskapet Barnavård, and Karolinska Institutet.

First Published Online December 28, 2004

Abbreviations: CAH, Congenital adrenal hyperplasia; Cluster E6, cluster of three amino acid alterations in exon 6; CYP21, steroid 21-hydroxylase; CYP21P, pseudogene of CYP21; 11-DOL, 11-deoxycortisol; NC, nonclassic; 17OHP, 17-hydroxyprogesterone; SDS, sodium dodecyl sulfate; SV, simple virilizing; SW, salt wasting; TBS, Tris-buffered saline; WT, wild type.

Received September 30, 2004.

Accepted December 21, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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