(Received for publication, July 7, 1995; and in revised form, December 5, 1995)
From the
We have investigated the structure and function of P450c21 with
regard to a conserved site around Ile-172 by site-directed mutagenesis
making single amino acid substitutions of residues 169-173.
Substitutions of Ile-171 and -172 resulted in production of mutant
proteins with dramatic reductions in enzymatic activities, indicating
the importance of these two residues in maintaining the structure and
function of P450c21. The I171N protein was present at a slightly lower
level, due to a decreased rate of protein synthesis. The I172N
apoprotein was synthesized at the normal rate, but its heme-bound P450
form was present at a much lower level. This I172N protein was tightly
integrated into the membrane of endoplasmic reticulum, similar to the
wild type P450c21, as shown by immunofluorescence detection, alkaline
extraction, and cellular fractionation. Kinetic studies indicated that
I172N had a lower V value. In addition, the
I172N protein was more sensitive to proteinase K digestion, indicating
a possible alteration of conformation. This conformational change may
result in the lower yield of the I172N hemoprotein and the reduced
catalytic activity.
Cytochrome P450 is a protein superfamily of more than 200 members (1) . Every member in the superfamily binds heme molecules at their active site to carry out mixed-function monooxygenation reactions at the end of the electron transport chain(2, 3) . These proteins are termed cytochromes P450, because the reduced heme in the protein shows characteristic absorption at 450 nm upon binding to carbon monoxide (4) .
In contrast to the bacterial P450s, eukaryotic P450s are associated
with the membrane of the endoplasmic reticulum (ER) ()or
mitochondria. The topology of P450s relative to the membrane has not
been elucidated due to a lack of knowledge about the three-dimensional
structure. It is generally believed that the bulk of the P450s have a
structure similar to that of the bacterial P450s, with an N-terminal
hydrophobic domain integrated into the membrane. This N-terminal
hydrophobic domain not only serves as the signal for ER membrane
targeting and integration(5, 6) , it also participates
in the determination of the overall structure and stability of
P450c21(7) . The rest of the polypeptide probably forms a
globular structure located at the cytoplasmic phase of the
membrane(8, 9, 10, 11) , although a
part of the polypeptide might interact with the membrane(8) .
Cytochrome P450c21 as a microsomal protein is integrated into the membrane of the smooth ER. It catalyzes the conversion of progesterone and 17-hydroxyprogesterone (17-OHP) into deoxycorticosterone and 11-deoxycortisol, two essential steps of steroid hormone synthesis(3) . Its deficiency is the main cause of congenital adrenal hyperplasia, due to decreased cortisol synthesis, leading to virilization and sometimes salt loss. 21-Hydroxylase deficiency is mainly caused by mutations of the CYP21A2 (c21B) gene that encodes P450c21(12, 13) . c21B and its neighboring CYP21A1P (c21A) genes are more than 98% identical in sequence, but the c21A gene does not encode an active protein due to multiple mutations throughout the gene(14, 15) . Because of their proximity, these two genes exchange their sequences frequently through gene conversion events. This gene conversion is the main cause for the mutations found in the c21B gene (16) and can be used as the basis for the detection of known mutations in patients(17) .
The loss of enzymatic activity as a result of mutations of the c21B gene is shown by the expression of mutant proteins corresponding to each mutation in various cell types in mammalian(18) , vaccinia(19) , or yeast vectors(20) . Some mutations cause the complete loss of enzymatic activity, resulting in the severe salt-wasting type of the disease. Other mutations have less deleterious effects, resulting in mutant proteins with some residual activities, causing the milder form of the disease(21, 22) . The availability of the expression systems enables the examination of parameters affecting the structure and function of wild type and mutated cytochromes P450. One common mutation is the Ile-172 to Asn substitution(23, 24) . This mutation causes 100-fold loss of enzymatic activity resulting in the simple virilizing form of the disease(24) . In this report, we investigated the structural perturbation caused by the substitution. Despite the 100-fold loss of enzymatic activity causing the simple virilizing form of the disease, the Ile-172 mutation did not grossly affect the integration of the protein into the endoplasmic reticulum. Instead, the mutant protein became more sensitive to proteinase K digestion. This result indicated that the mutant protein had an altered conformation.
Figure 1: Alignment of P450c21 protein sequence from residues 166 to 177 with other P450s. The sequence of the human P450c21 is on the first line with its residue number on top. The conserved residues are boxed. Amino acids in positions 171 and 172 are shown in bold type. All P450 sequences were taken from published data(15, 47, 48, 49, 50, 51, 52, 53, 54) .
Figure 2: Production of wild type and mutant P450c21 proteins in yeast as detected by immunoblotting. Yeast cells (1 ml) harboring vector pYE8, wild type (WT), or mutant P450c21 cDNA plasmid as indicated on top of each lane were lysed in gel electrophoresis buffer, and proteins were separated by gel electrophoresis. The gel was transferred to a membrane and reacted with anti-P450c21 and anti-hsp60 antibodies simultaneously. The bands corresponding to P450c21 and hsp60 are marked.
To determine synthetic rates of proteins, mutant
proteins were pulse-labeled with [S]Met for 5
min followed by immunoprecipitation. Fig. 3showed that most of
the mutant proteins were synthesized at normal rate except the I171N
mutant. Scanning of protein intensities from four separate experiments
showed that the I171N protein was produced at 70 ± 16% of the
normal rate.
Figure 3:
Amounts of wild type and mutant P450c21
proteins in yeast detected by immunoprecipitation. Yeast cells
harboring vector pYE8, wild type (WT), or mutant P450c21 cDNA plasmid
as indicated on top of each lane were labeled with
[S]Met for 5 min, lysed, and immunoprecipitated
with anti-P450c21 and anti-hsp60 antisera. The precipitated proteins
were analyzed by gel electrophoresis. The bands corresponding to
P450c21 and hsp60 are marked.
Figure 4:
Activities of P450c21 and mutant proteins
expressed in yeast cells. A, serial mutations. B,
mutations at 171 and 172. Yeast cells containing P450c21 and mutant
proteins were incubated with C-labeled 17-OHP for 30 min
or progesterone (Prog) for 2 h. The amount of enzymatic
activity of each mutant relative to that of the wild type P450c21 is
shown.
The kinetic properties of the mutated and wild type enzymes in the yeast microsomes were determined further (Table 1). Some P420 forms, which usually represent denatured proteins with altered interaction with heme(30) , were associated especially with the mutant protein during purification after cells were broken. Similar to other P450s like P450cam (33) and P450c11(34) , adding the substrate 17-OHP during purification stabilized P450c21 and increased yield of the P450 form for both wild type and mutated P450c21 (data not shown).
We obtained similar amounts of P450s (about 2 nmol/liter) for most of the mutant forms, except the I171N and I172N mutant proteins. The yield for the I171N mutant (1.26 nmol/liter) was about 50% of the wild type protein (Table 1), consistent with the decreased abundance of the apoprotein in the cell. The yield for the I172N P450 (0.29 nmol/liter) was about 10% of that of the wild type. Since P450 content is a measure of the amount of hemoprotein(4) , it indicated that there is less hemoprotein although the I172N apoprotein was produced at the normal amount (Fig. 2).
The K values of
all mutants were similar to that of the wild type protein with a slight
elevation of the I171N protein (Table 1). This result indicates
similar affinity of the mutant proteins toward the substrate. As the
I172N mutation does not drastically affect the ability of the protein
to bind substrate, the Ile-172 residue of P450c21 does not seem to be
involved directly in substrate binding. The V
value of the I172N mutant protein, however, was decreased to
about 1/6 to 1/10 that of the normal protein when equal amounts of the
P450 were compared. This together with about 10% yield of the I172N
P450 results in overall low enzymatic activity.
Figure 5: Cellular location of wild type P450c21 and its I172N mutant protein by immunofluorescence detection. Rat-1 cells expressing P450c21 or the I172N mutant were incubated with anti-P450c21 antibodies followed by fluorescein isothiocyanate-conjugated anti-rabbit IgG before observing the immunofluorescence under the microscope.
In addition to cytological examination, we also used a biochemical method to examine cellular localization of the I172N mutant protein. Cellular proteins were fractionated into the microsomal and soluble portions. As shown in Fig. 6A, wild type P450c21 sedimented in the pellet portion, indicative of its membrane association. Likewise, the I172N protein was also fractionated into the pellet portion. Therefore, the I172N mutant was associated with the membrane as well. There was no immunoreactivity from the yeast strain harboring vector pYE8 only.
Figure 6:
Membrane association of P450c21. A, fractionation. Yeast proteins from yeast cells harboring
wild type P450c21 (15 µg) or its I172N mutant (60 µg) were
fractionated into the particulate (P) and soluble portions (S) as described under ``Experimental Procedures,''
electrophoresed, and reacted with anti-P450c21 antisera. Lane 1
contains lysate from cells harboring vector pYE8. B, membrane
integration assay. Plasmids harboring wild type (WT-c21), I172N, or
truncated (52) forms of P450c21 cDNA were transcribed and
translated in vitro in the presence of membranes. The total
translation products (T) were extracted with alkali and
centrifuged to separate into the supernatant (S) and pellet (P) forms. The full-length translation products are indicated
by arrows.
Some peripheral membrane proteins can associate with the membrane
not by direct membrane integration, but by charge interaction. These
proteins can be extracted into the soluble fraction by salt in alkaline
pH. To separate peripheral from integral membrane proteins, we
translated proteins in vitro in the presence of microsomes,
followed by alkaline extraction and centrifugation. Fig. 6B showed that proteins translated from both wild type plasmid WT-c21
and the mutant I172N still sedimented into the pellet after alkaline
extraction, indicating tight membrane integration. Another 52
protein of P450c21, which is truncated at the N terminus by 52 amino
acids, could not integrate into the membrane and remained in the
supernatant. This experiment further established the nature of the
I172N mutant protein as an integral membrane protein. Therefore, the
biochemical procedure, together with cytological detection, showed that
the I172N mutant protein was localized in the ER through tight membrane
integration. Since the N-terminal membrane targeting signal of I172N is
intact, it is reasonable that this protein can still target into the ER
membrane with equal efficiency.
Figure 7: Proteinase K digestion of wild type P450c21 and its I172N mutant expressed in yeast. Yeast microsomes (120 µg of proteins) containing either wild type P450c21 or the I172N mutant protein was digested with various concentrations of proteinase K (from 0.02 to 1 mg/ml) as indicated on top of each lane at room temperature for 30 min. The digestion products were separated by gel electrophoresis before immunoblotting.
The I172N mutant protein isolated from yeast microsomes was present at a slightly lower amount (lane 6, Fig. 7). This protein was more sensitive to proteinase K digestion. It was digested to completion at a low concentration (0.2 mg/ml) when proteinase K just began to digest the wild type P450c21 into three fragments. The lack of visible bands in lane 8 of Fig. 7was not due to the lower level of the I172N protein, as overexposure of the gel did not show any band. The complete sensitivity of the I172N protein toward proteinase K digestion indicated that it was partially unfolded so as to allow penetration of the protease into the protein. The I172N mutant expressed in COS-1 cells was also more sensitive to trypsin digestion than the wild type P450c21 (data not shown). These results indicate that the I172N mutant protein had partially unfolded conformation which could be detected by higher sensitivity toward proteinase K digestion, irrespective of the cell types which expressed the proteins.
The Ile-172 to Asn substitution is one of the most common mutations of the CYP21A2 gene causing congenital adrenal hyperplasia. This residue seems to be in a functionally important motif as its sequence is conserved in different microsomal P450s (Fig. 1). We have in this report characterized wild type and mutant P450c21 with point mutations in the vicinity of Ile-172, with respect to the amount of protein production, the rate of protein synthesis, enzymatic activity, and kinetic properties. Our data showed that replacing Ile-171 and -172 causes enzyme deficiency.
The I172N protein had drastically reduced enzymatic activity, which could be accounted for by its lower hemoprotein yield and altered catalytic parameters. Although the I172N protein was synthesized at the normal rate (Fig. 3) and its steady state level was similar to that of the wild type protein (Fig. 2), the amount of the heme-bound P450 form of the I172N protein was only 1/10 that of the wild type form (Table 1). In addition, the I172N hemoprotein was obtained mostly as the P450 rather than the P420 form, if care was taken and substrate was added to stabilize the protein during purification. These results suggest that the decreased yield of the I172N hemoprotein was due to the decreased efficiency of the apoprotein to take up heme.
The I172N protein was partially unfolded as shown by its increased sensitivity toward proteinase K digestion. Many denatured proteins are known to be recognized by degradation enzymes (37, 38) and tend to degrade faster than native proteins(39) . The lower stability was also observed for a polymorphic form of P4502C13 with a single Ser-180 to Cys substitution(40) . Therefore, the reduced amount of the I172N protein in the microsome appears to correlate with its sensitivity toward proteinase K digestion.
The I172N protein also
had a much lower V value (Table 1), which
is another consequence of its conformational change. The combined
effects of lower hemoprotein content and reduced catalytic function
resulted in a defective protein with about 100-fold reduction in
enzymatic activity.
There has been an earlier suggestion that the
I172N mutant protein might be impaired in ER targeting(19) . We
showed that both normal P450c21 and its I172N mutant are localized to
the ER membrane by a combination of methods including
immunofluorescence (Fig. 5) and cellular fractionation (Fig. 6). This result is expected since the N-terminal membrane
targeting and anchoring domain of the I172N mutant is intact. There is,
however, speculation that the Ile-172 motif might be associated with
the ER membrane, probably not by spanning the membrane but by forming a
loop structure(8) . Our data do not rule out the possibility
that the Ile-172 motif might be involved in other types of membrane
interaction. In addition to spanning the membrane by an -helical
domain, a protein can be associated with the lipid bilayer by direct
fatty acylation(41, 42) , through a
glycosylphosphatidylinositol anchor(43) , or by noncovalent
interaction with other membrane proteins. The exact nature of the
interaction of the microsomal P450s with the ER membrane besides the
N-terminal crossing is not known yet and should be investigated
further.
A different mutant protein, I171N, also had lower enzymatic
activity. This protein had a lower steady state level (Fig. 2),
which was probably due to its lower synthetic rate (Fig. 3).
Unlike I172N, the amount of the I171N P450 roughly correlated with the
amount of apoprotein ( Table 1and Fig. 2). The V value of the I171N P450 was only slightly
lower (about 2/3) than that of the wild type protein, when the same
amount of P450s were compared (Table 1). It suggested that the
defect of the I171N protein is the decreased amount and the slightly
impaired catalytic rate.
We used mammalian and yeast cells as two separate expression systems to obtain wild type P450c21 and mutant proteins for our study. Most of the kinetic parameters presented in this report were obtained from proteins expressed in yeast, as yeast cells are easier to grow and higher amounts of P450c21 can be obtained more easily for the study. Some of the data were also confirmed using mammalian expression systems. Since similar results were obtained regarding the structure and function of the protein in spite of the expression system, different expression systems provide a cross-check for the experimental results. We are certain we were measuring the property of the protein, not any artifact resulting from the expression system.
To probe the structure of P450c21, we investigated the differential sensitivity of the wild type and mutant proteins toward proteinase K digestion. Protease digestion has been widely used to study the conformational change of a protein(44, 45, 46) . It, however, does not provide detailed structural information of the protein, which can be obtained only through crystallization and x-ray diffraction. What are the specific structural changes caused by a single substitution? How do these structural changes affect heme-binding capacity and catalytic activity? These are important questions which await further investigation.