Three mutants (deletion of E196, G291S, and
R483P) of steroid 21-hydroxylase (P450c21) from patients with inherited
congenital adrenal hyperplasia had reduced activity toward progesterone
and 17-hydroxyprogesterone after transient expression in cultured mammalian cells. In addition, both the E196 deletion and the R483P mutant had shorter half-lives than the wild-type enzyme, whereas the
half-life of the G291S mutant was comparable with that of the normal
protein. These results directly link the clinical situation with the
three mutations and suggest that G291 is important for the catalytic
activity of P450c21.
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INTRODUCTION |
Cytochrome P450s are a superfamily of enzymes essential for the
oxidative metabolism of different endogenous compounds and xenobiotics
(1). For many members of the family, enzyme activity varies between
individuals due to polymorphisms in the coding parts of the genes. Four
cytochrome P450 enzymes are involved in the synthesis of cortisol in
the adrenal cortex (2). Impaired production of cortisol due to reduced
function of any of these enzymes causes increased secretion of
adrenocorticotropic hormone (ACTH) from the pituitary and congenital
adrenal hyperplasia (CAH)1
(3). Of particular interest is the cytochrome P450 steroid 21-hydroxylase (P450c21) since more than 90% of all cases of CAH are
due to mutations in the gene encoding the enzyme. P450c21 participates
in the synthesis of both glucocorticoids and mineralocorticoids. In
addition to lack of cortisol and aldosterone, P450c21 deficiency leads
to overproduction of adrenal androgens. The disorder has a wide
spectrum of manifestations (4), ranging from a life-threatening neonatal condition in both sexes with ambiguous genitalia in girls, to
inconspicuous symptoms of hyperandrogenism in adult women.
P450c21 is a protein of 494 amino acids (5, 6). The gene locus has a
complicated structure, with an active gene (CYP21) and a
highly homologous inactive pseudogene (CYP21P) (5-8).
Misalignment followed by recombination events between the two
homologous genes during meiosis with transfer of sequences from
CYP21P to CYP21 account for around 95% of the
mutations of P450c21 that lead to CAH (9). Rarely, mutations also arise
independently of the pseudogene. We have charcterized three such rare
missense mutations, which were found in patients with CAH. To
investigate the molecular mechanisms behind the impaired enzyme
function displayed by the patients, the mutations were reconstructed by
in vitro site-directed mutagenesis, and normal and mutant
enzyme was transiently expressed in COS-1 cells. Enzyme activities
toward the two natural substrates, 17-hydroxyprogesterone (17-OHP) and
progesterone, were determined. In addition, to assess whether the
impaired function resulted from reduced stability of the mutant
proteins, their half-lives were determined by pulse-chase experiments
followed by immunoprecipitation.
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EXPERIMENTAL PROCEDURES |
Patients--
The CYP21 mutations were found in five
patients with CAH. The siblings numbers B39 and B40, as well as patient
number B103, have been reported previously (10, 11), whereas the
younger brother of B103, patient E. I., as well as patient number
1293 have not been described. Genotyping was performed by
allele-specific polymerase chain reaction (12) and direct sequencing of
CYP21 (10). Numbering of nucleotides and amino acids follows
the reference sequence of White et al. (5). Subjects numbers
B39 and B40 were compound heterozygotes, with the unique R483P mutation
on one allele and the well-known I172N mutation (13) on the other. They
had a moderate form of the disease, with early clitoral enlargement in
the female sibling but without salt-wasting. Subject number B103 and
his younger brother E. I. had inherited the unique G291S mutation
on their paternal allele, whereas the maternal allele was deleted. They
had the most severe, salt-wasting form of CAH. Patient number 1293 was
a compound heterozygote for the I172N mutation and a novel sequence
aberration, where nucleotides 1158-1160 (AGG) in exon 5 of
CYP21 were deleted, causing loss of a glutamic acid residue
at position 196 in P450c21, delE196 (Fig.
1). This female patient was born in Norway in 1968 and was affected
with a moderate form of CAH with some signs of prenatal virilization but without salt-wasting.

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Fig. 1.
Nucleotide sequence in the vicinity of the
delE196 mutation. Part of the sequence of exon 5 of
CYP21 is shown, including the novel mutation found in
patient number 1293. Nucleotides 1158-1160 (AGG) are deleted,
resulting in a protein lacking residue glutamic acid 196. The
identification of the other two mutations described in this paper
(G291S and R483P) has been reported previously (10, 11).
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Construction of Plasmids, Site-directed Mutagenesis, in Vitro
Expression, and Assay of Enzyme Activity--
pGEM constructs as well
as pCMV4 expression vectors were constructed to contain the cDNAs
encoding the wild-type and the three mutant enzymes, essentially as
described previously (14). The plasmids thus generated were named
pGEM-CYP21 (encoding the wild-type enzyme), pGEM-CYP21(delE196),
pGEM-CYP21(G291S), pGEM-CYP21(R483P), and pCMV4-CYP21,
pCMV4-CYP21(delE196), pCMV4-CYP21(G291S), and pCMV4-CYP21(R483P). All constructs were sequenced to verify the correct incorporation of the mutations and to exclude additional aberrations. Enzymatic activities of the mutants and the wild-type enzyme were determined in intact COS-1 cells after transient
expression, using 17-OHP and progesterone as substrates (14).
In Vitro Transcription/Translation--
Estimation of
translation efficiency was performed using the TNT Coupled Reticulocyte
Lysate System (Promega, Madison, WI) according to the instructions of
the manufacturer. An ATG sequence was present in the multiple cloning
site of pGEM-CYP21, upstream of CYP21, but in a different
reading frame. This initiation codon was destroyed by SphI
cleavage, T4 polymerase treatment, and blunt-end ligation, which
resulted in the removal of four nucleotides in this region. Two µg of
plasmids pGEM-CYP21, pGEM-CYP21(delE196), pGEM-CYP21(G291S), and
pGEM-CYP21(R483P) were used for transcription/translation, incubation
times were 5, 10, 15, and 20 min, and [35S]methionine
incorporation was measured by scintillation counting after
trichloroacetic acid precipitation of translation products. The
translation products obtained after a 60-min incubation were subjected
to SDS-PAGE to verify the integrity of the 55-kDa 21-hydroxylase protein.
Immunoprecipitation and Assay of Enzyme Stability--
COS-1
cells were transfected (14) with the expression constructs pCMV4-CYP21,
pCMV4-CYP21(delE196), pCMV4-CYP21(G291S), or pCMV4-CYP21(R483P) and
incubated for 30 h. Cells were then labeled (100 µCi of
[35S]methionine) and immunoprecipitated as described
(15), and the immunoprecipitates were analyzed on 10% SDS-PAGE gels.
The rabbit P450c21 antiserum used was kindly provided by Dr. Peter F. H. Hall, Prince of Wales Hospital, Randwick, Australia. Labeled proteins were visualized by autoradiography, and their intensities were
quantified using a phosphoimager (Fujix Bas 1000 Bio-Imaging Analyzer,
Fuji, Japan). For each time point examined (within each mutant),
immunoprecipitated material corresponded to equal total cell protein as
determined by Bradford (16). The degradation profiles and half-lives of
the different P450c21s were calculated using a curve-fitting program
(FigP, Elsevier Biosoft Ver. 5.0).
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RESULTS |
Enzyme Activity of Mutants--
Table
I shows that all mutants had a fraction
of the activity of the wild-type enzyme. The delE196 mutant retained
6% activity toward 17-OHP and 23% activity toward progesterone. The
G291S mutant displayed 0.8% of normal activity in both cases, whereas the R483P mutant displayed 1% of normal activity toward 17-OHP and
2.2% activity toward progesterone. The activity of the wild-type enzyme was defined as 100%.
In Vitro Translation--
To investigate whether the three
sequence alterations of CYP21 had any effect on the
translation of the gene, an in vitro-coupled transcription/translation assay was employed. None of the mutations affected the rate of translation in this in vitro system
(data not shown).
Stability of Mutant Versus Wild-type
Protein--
Fig. 2 shows the
degradation profiles of immunoprecipitated wild-type and mutant
P450c21s for chase periods of up to 4 h. Fig. 3 shows the same results after
quantification of the immunoreactive bands with a phosphoimager. The
degradation of each enzyme species is expressed as percentage of
remaining counts at each time point, with the level at point 0 defined
as 100%. The level of expression at 0 h in terms of actual counts
was comparable between the normal and mutant enzymes (data not shown).
Fig. 3 indicates that the wild-type enzyme was the most stable, whereas
all mutants had decreased stability. The R483P was most rapidly
degraded and was barely detectable at the end of the 4-h chase.
Half-lives were calculated as 1 h, 10 min (wild-type P450c21), 30 min (delE196), 55 min (G291S), and 20 min (R483P).

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Fig. 2.
Degradation profiles of wild-type and mutant
P450c21s transiently expressed in COS-1 cells. Cells were
transfected with pCMV4-CYP21 (wild-type protein) and
pCMV4-CYP21(delE196) (A) and with pCMV4-CYP21(G291S) and
pCMV4-CYP21(R483P) (B), pulse-labeled, and chased for, from
right to left, 0, 1, 2, 3, and 4 h. Cells were then lysed, and their supernatants were treated with rabbit P450c21 antiserum. Immunoprecipitated material was analyzed by SDS-PAGE
(10% gels). The arrow indicates the band corresponding to
P450c21.
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Fig. 3.
Quantification of degradation profiles of
wild-type and mutant P450c21s transiently expressed in COS-1
cells. Results are expressed as percentage of remaining protein,
with the amount at time 0 (just before chase) defined as 100%. ,
wild-type P450c21; , delE196 mutant; , G291S mutant; , R483P
mutant. Values for 0, 1, 2, 3, and 4 h represent the average of
two independent pulse-chase experiments.
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DISCUSSION |
Defective function of P450c21 is the major cause of CAH. To
examine whether this was the case for three novel mutant P450c21s from
patients with CAH, we studied the enzymatic activities and half-lives
of the mutants. All three mutations impaired enzyme activity toward the
two natural substrates of the enzyme when assayed after transient
transfection in cultured cells. The delE196 mutant was partially active
toward both substrates. P450c21 G291S resulted in a protein with less
than 1% of normal activity, whereas the R483P mutant retained 1-2%
of the activity of the native enzyme. This is in agreement with the
clinical phenotypes of the patients. Those carrying delE196 and R483P
had moderate forms of CAH and were compound heterozygotes with the
I172N mutation on the other chromosome. The latter mutation is
generally associated with the moderate, simple virilizing phenotype (9,
17, 18). The brothers carrying G291S had deleted their 21-hydroxylase
gene of the other chromosome and had the most severe, salt-wasting form
of the disease.
To examine whether the reduced enzymatic activity of the mutants was a
consequence of reduced stability rather than decreased turnover of
substrate, the half-lives of the wild-type and the three mutant enzymes
were compared by pulse-chase experiments. The half-life of the
wild-type P450c21 (1 h, 10 min) was longer than those of all mutants.
The delE196 and R483P mutants were most rapidly degraded (half-lives 30 and 20 min, respectively). These data suggest that the phenotypes
resulting from the delE196 and R483P mutants can be explained in view
of little if any available P450c21. In contrast, since the half-life of
G291S (55 min) and wild-type P450c21 (1 h, 10 min) were comparable, it
seems that G291 plays an essential role in the maintenance of enzyme
activity. Generally little is known about structure-function
relationships of mammalian cytochrome P450 enzymes. One reason is that
mammalian P450s contain hydrophobic segments and are therefore
difficult to crystallize. However, three-dimensional structures of four nonmembrane-associated bacterial cytochrome P450s (P450cam (19), P450BM-3 (20), P450terp (21), and P450eryF (22)) have been determined.
A correlation of important residues among different classes of
cytochrome P450s has been made by Nelson and Strobel (23) after
alignment of 34 different P450s, including P450c21 and P450cam (24).
According to their model, G291 would be hydrogen-bonded with T295 to
form together an oxygen-binding pocket. Another model obtained from the
structural information of P450BM-3 (20) implies that residues 291-297
of human P450c21 are involved in proton transfer from water to heme.
Thus, G291 is intricately involved in catalysis according to two
different models, and our experimental data suggest that this
particular residue is essential to maintain enzyme activity.
In conclusion, enzymatic activity and stability analyses of three
naturally occurring mutants of P450c21 from patients with CAH provided
a potential explanation for the clinical symptoms and identified
residues important for the in vitro stability and catalytic
activity of the enzyme.