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Vol. 52, Issue 6, 1034-1040, 1997
Department of Medical Biochemistry and Biophysics and Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden
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Summary
Introduction
Procedures
Results
Discussion
References
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In many black African populations, the capacity for CYP2D6-dependent drug metabolism is generally reduced. A specific variant of the CYP2D6 gene (CYP2D6*17) that carries three functional mutations (T107I, R296C, and S486T) has been found to be present in Zimbabwean subjects with impaired CYP2D6-dependent hydroxylase activity. To evaluate whether the CYP2D6*17 allele was the major cause behind the decreased rate of drug metabolism and to examine the role of the different mutations, CYP2D6 cDNAs containing all eight combinations of the mutations were created. Expression of the cDNAs in COS-1 cells revealed that the CYP2D6 17 enzyme displayed only 20% of the wild-type (CYP2D6 1) activity, whereas the T107I substitution on its own had no significant effect on enzyme function. Expression in yeast showed that the three possible single amino-acid mutant CYP2D6 variants all had properties similar to CYP2D6 1 when the kinetics of bufuralol hydroxylation was examined. However, enzymes containing both the T107I and R296C mutations exhibited a more than 5-fold higher Km for bufuralol than the wild-type enzyme, whereas the S486T mutation was of little importance. In contrast, when codeine was used as a substrate, the T107I substitution alone was sufficient to cause a significant increase in the apparent Km, indicating a differential effect for this substitution depending on the CYP2D6 substrate. In conclusion, the CYP2D6*17 allele represents the first human cytochrome P450 polymorphic variant in which a combination of substitutions is required to alter the enzyme's catalytic properties and is the first case in which a decreased CYP2D6 activity, as monitored in vivo, has been documented to be caused by an enzyme with altered affinity for CYP2D6 substrates.
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Introduction |
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Summary
Introduction
Procedures
Results
Discussion
References
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The
human P450 2D6 (CYP2D6; debrisoquine hydroxylase) catalyzes the
oxidative metabolism of several important classes of drugs, including
tricyclic antidepressants and some of the novel selective serotonin
reuptake inhibitors, most antipsychotic agents, antiarrhythmic drugs,
-adrenergic antagonists, and codeine (1-3). The CYP2D6-dependent conversion of codeine to morphine has been shown to be required for the
analgesic effect of codeine (4). Moreover, this enzyme has been
implicated to be involved in the pathogenesis of several cancer forms
and other diseases (5, 6).
A tremendous variation in CYP2D6 enzyme activity has been shown among individuals, mainly because of genetic polymorphism of the CYP2D6 gene. Historically speaking, the population has been divided into two phenotypes (7): extensive metabolizers (EMs), who exhibit CYP2D6 activity, and PMs, who lack CYP2D6 activity. In addition, ultrarapid metabolizers have been identified who carry multiple copies of active CYP2D6 genes (8). In Caucasian populations, the prevalence of PMs is 5-10% (9), the majority of whom (90-95%) are homozygous for any of the known defective CYP2D6 alleles or combinations thereof (10). Alleles that cause reduced but not abolished activity, CYP2D6*91 [C; (11)] and CYP2D6*10 [J, Ch1, Ch2; (12)], have also been found. Here, the reduced rate of drug metabolism seen in vivo is explained by mutations in the CYP2D6 gene that cause a less stable enzyme.
Besides interindividual differences in CYP2D6-dependent drug metabolism, there are also remarkable interethnic differences. In this respect, essentially no PMs are seen in the Asian (13) and black African populations (14 and references therein), but the capacity for CYP2D6-dependent drug metabolism is generally diminished. Among Asians, this is caused by the frequent distribution of the CYP2D6*10B allele encoding the unstable CYP2D6 10 enzyme (12).
The CYP2D6*17 (CYP2D6Z) allele, common among black Zimbabweans, has been described recently (15). This gene contains four mutations, of which one is silent, thus yielding the three amino acid exchanges T107I, R296C, and S486T. The distribution of this allele correlates well with decreased CYP2D6 activity seen in vivo measured as metabolic ratio of the probe drug debrisoquine. We considered it of interest to study whether the CYP2D6 17 enzyme exhibited altered catalytic properties and to investigate the role of the different mutations in this respect using two different heterologous expression systems, mammalian COS-1 cells and yeast cells. The data indicate that CYP2D6 17 exhibits an altered substrate affinity for CYP2D6 substrates and that a combination of the T107I and R296C amino-acid substitutions are required for altered catalytic properties of the enzyme toward hydroxylation of bufuralol.
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Experimental Procedures |
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Summary
Introduction
Procedures
Results
Discussion
References
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Site-Directed Mutagenesis and Expression Vectors
Mutant CYP2D6 cDNAs that contained the C1111T, C2938T, and
G4268C mutations (numbering based on genomic DNA) were generated with
the USE mutagenesis kit (Pharmacia Biotech, Uppsala, Sweden), using a
wild-type CYP2D6 cDNA subcloned into a pBluescript KS vector
(Stratagene, LaJolla, CA), and mutagenic primers 5
-GTG CCC ATC
ATC CAG ATC CTG-3, 5-GAG AAC CTG TGC ATA GTG
GT-3, and 5-CCT GGT GAC CCC ATC CCC CT-3 respectively
(mutations underlined). The reactions were carried out according to the
manufacturer's recommendations; the incorporation of the mutations
were confirmed by DNA sequencing. Using the restriction enzymes
BssHII and Tth111 I, eight constructs that
contained all the possible combinations of these three mutations were
created. The variant cDNAs were subsequently subcloned into the V60
(pYeDP60) yeast expression vector (16) and into the mammalian cell
expression vector pCMV4 (17) using the restriction enzymes
BamHI and KpnI.
Expression of Mutated cDNAs in COS-1 Cells
COS-1 cells were transfected with the pCMV4-2D6 plasmids
described above as reported previously (12). After a 50-hr incubation, cells for Western blot analysis and bufuralol 1-hydroxylase activity measurements were harvested in 100 mM sodium phosphate
buffer, pH 7.4, sonicated for 20 × 1 sec, and then centrifuged at
10,000 × g for 10 min at 4°.
Quantification of CYP2D6 mRNA Levels, Apoprotein Levels, and Catalytic Activities in COS-1 Cells
Total RNA was prepared from transfected COS-1 cells using Trizol
Reagent (Life Technologies, Rockville, MD). Northern Blot analysis was
carried out with 7 µg of total RNA using
32P-labeled CYP2D6 cDNA and -actin cDNA as
probes. For apoprotein quantification, cell supernatant (10,000 × g) corresponding to 1 µg of protein was subjected to
sodium dodecyl sulfate gel electrophoresis using 8.7% polyacrylamide
gels. The proteins were transferred to a nitrocellulose filter (BioRad,
Hemel Hempstead, UK), incubated with the monoclonal mouse-anti-2D6
antibody 114/2 and subsequently a horseradish-peroxidase linked goat
anti-mouse antibody (DAKO AS, Glostrup, Denmark). The enhanced
chemiluminescence method (Amersham, Buckinghamshire, UK) was used to
visualize the proteins. Quantification of the Northern and Western blot
results was done using a personal densitometer (Molecular Dynamics,
Sunnyvale, CA).
Cell supernatant corresponding to 100 µg of protein was incubated at 37° in 100 mM sodium phosphate buffer, pH 7.4, with 100 µM (+)-bufuralol, 75 µg NADPH, and 200 pmol of rat P450 reductase [purified as described previously (18)] in a total volume of 75 µl for 30 min; the reaction was terminated by the addition of 7.5 µl of 70% perchloric acid. The samples were centrifuged at 15,000 × g for 5 min and the supernatants subjected to reversed-phase HPLC as described previously (19). Linearity of the reaction for at least 30 min was established.
Expression of Mutated cDNAs in Yeast Cells
Saccharomyces cerevisiae strain W(R), a strain that has been genetically engineered to overexpress the yeast reductase (Yred) (20), was transfected with the above described V60-2D6-plasmids. Expression of CYP2D6 was carried out essentially as described previously (21). Briefly, precultures of transfected yeast cells were grown in SGI medium (l g/liter Casamino acids, 7 g/liter yeast nitrogen base, 20 g/liter glucose, 20 mg/liter tryptophan) to a density of approximately 30 × 106 cells/ml, and diluted to 2.5 × 106 cells/ml in YPGE medium (10 g/liter yeast extract, 10 g/liter BactoPeptone, 5 g/liter glucose, 2% ethanol). The cells were then grown for another 22-24 hr. Galactose was subsequently added to a final concentration of 2% (w/v) to induce the galactose-driven promoter. After a 16-hr incubation, the cells were harvested by centrifugation. After mechanical disruption of the cell walls, yeast cell microsomes were prepared by polyethylene glycol precipitation of the 20,000 × g supernatants, essentially as described previously (22).
Assay of CYP2D6 Holoprotein and Reductase
Microsomal protein concentration (23), reductase levels (24), and total P450 content estimated from the CO-induced spectrum of dithionite-reduced yeast microsomes (25) were determined using previously described methods.
Kinetic analysis of CYP2D6 Activity in Yeast Microsomes
Bufuralol 1-hydroxylation.
Yeast microsomes corresponding
to 150 µg of protein were incubated at 37° in 100 mM
sodium phosphate buffer, pH 7.4, with 0-40 µM
(+)-bufuralol and 120 µg of NADPH in a total volume of 300 µl. The
reactions were terminated by the addition of 30 µl of 70% perchloric
acid, and linearity with time was ensured in all cases. The samples
were subsequently centrifuged at 15,000 × g for 5 min
and the supernatants subjected to a reversed-phase HPLC system
essentially as described by Kronbach et al. (19). 1-Hydroxybufuralol was used as an external standard. Analysis of the
pH dependency of the reactions was carried out under half-saturated enzyme conditions [2 µM and 10 µM of
(+)-bufuralol for the CYP2D6 1 and CYP2D6 17 variants, respectively]
in 100 mM sodium phosphate or 100 mM Tris·HCl
buffers with a range of different pH values.
Codeine O-demethylation. Yeast microsomes corresponding to 200 µg of protein were incubated at 37° in 100 mM sodium phosphate buffer, pH 7.4, with 0-6 mM codeine and 120 µg of NADPH in a total volume of 100 µl. The reactions were terminated by the addition of 25 µl of acetonitrile, and the samples were subsequently centrifuged at 15,000 × g for 10 min. The supernatants were transferred to fresh tubes and extractions were done according to Ladona et al. (26). After evaporation under a steam of N2, the remaining residues were dissolved in 50 µl of mobile phase and subjected to a reversed-phase HPLC system with an electrochemical detection method. The mobile phase consisted of 5% acetonitrile, 8% methanol, 0.5 mM EDTA, and 70 mM KH2PO4. Morphine was used as an external standard.
Data Analysis
Apparent Km and Vmax parameters from the kinetic studies were determined from Lineweaver-Burke plots. Student's t test was used for intergroup comparisons.
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Results |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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The three functional mutations present in the CYP2D6*17
allele were introduced using site-directed mutagenesis in all eight combinations into a CYP2D6 cDNA. Four of the different CYP2D6 cDNAs
[wild-type (CYP2D6 1), T107I, R296C + S486T, and T107I + R296C + S486T
(CYP2D6 17)] were subcloned into the pCMV4 expression vector and
expressed in COS-1 cells. Cells transfected with an empty pCMV4 vector
were used as a negative control. The CYP2D6 mRNA expression was
measured by Northern blot analysis and CYP2D6 apoprotein levels were
quantified by Western blot analysis. The CYP2D6-dependent catalytic
activities were determined by measuring NADPH-dependent
1-hydroxylation of bufuralol in 10,000 × g
supernatants of transfected cells to which purified reductase had been
added.
All cDNA variants examined, except for the pCMV4 vector alone, yielded CYP2D6 mRNA and immunodetectable CYP2D6 apoprotein. No major difference in mRNA levels was seen between cells transfected with different cDNAs (Fig. 1A). Introduction of the T107I substitution alone yielded higher CYP2D6 levels, whereas the combination of the R296C and S486T substitutions with or without the T107I exchange resulted in decreased CYP2D6 levels. This is indicative of a slightly more unstable enzyme (Fig. 1B), although a decreased translation efficiency cannot be excluded. Determination of the catalytic activity using bufuralol as a substrate revealed that the mutant enzyme having the R296C and S486T substitutions (corresponding to CYP2D6 2) exhibited somewhat reduced activity compared with CYP2D6 1, which is in agreement with recent in vivo data presented by Sachse et al. (27), whereas CYP2D6 17 had only 20% of the hydroxylase activity seen using CYP2D6 1. In contrast, the T107I substitution alone did not significantly alter the bufuralol hydroxylase activity (Fig. 1C).
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To further study the mechanism by which the three amino acid substitutions interact, all different cDNAs were subcloned into the V60 yeast expression vector (16), where the expression is driven by a galactose-inducible promoter. These were subsequently expressed in S. cerevisiae W(R), a strain genetically engineered to overexpress the yeast reductase (Yred) (20). Because the Yred gene is also under the control of a galactose-inducible promoter, the microsomal levels of Yred were measured as an indicator of induction (Table 1).
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All plasmids except for the V60 vector alone yielded immunodetectable apoprotein (Fig. 2) and had similar expression of reductase, indicating a similar extent of galactose-dependent induction in all cases. The CYP2D6 levels were determined spectrally from the reduced CO-bound form. There was a tendency, although not significant, that enzymes that contained the R296C substitution yielded somewhat lower expression levels (Table 1), consistent with the COS-1 cell expression data.
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Kinetic studies using bufuralol were carried out in microsomes isolated
from yeast expressing all eight different cDNAs. These experiments revealed that all the corresponding enzymes had similar apparent Vmax for bufuralol
1-hydroxylation (Fig. 3B). When the three amino acid substitutions present in the CYP2D6 17 mutant (T107I,
R296C, and S486T) were introduced one by one, no significant effect on
the enzyme properties was observed. However, when the amino acid
substitutions were introduced in various combinations, it was found
that both the T107I and R296C substitutions were required for an enzyme
with altered kinetic properties of bufuralol (Fig. 3A). The CYP2D6 17 enzyme exhibited a 5-fold higher apparent Km compared with CYP2D6 1.
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Also the kinetics of codeine O-demethylation were analyzed in microsomes isolated from yeast transfected with either CYP2D6 1 (wt), CYP2D6 2 (R296C and S486T) or CYP2D6 17 (T107I, R296C, and S486T) cDNA as well as with the plasmid encoding only the T107I exchange. The apparent Km for codeine was 5- to 10-fold higher for CYP2D6 17 compared with CYP2D6 1 (Fig. 4). In contrast to the finding with bufuralol, the T107I substitution alone caused a significant increase in Km. In addition, the Vmax for codeine O-demethylation catalyzed by CYP2D6 2 was significantly higher than for the other CYP2D6 enzyme variants.
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To further analyze the molecular mechanism behind the differences
between CYP2D6 1 and CYP2D6 17, the effect of pH on 1-hydroxylation of
bufuralol was examined in the yeast microsomes. The pH optimum was
significantly higher for CYP2D6 1 compared with CYP2D6 17 (7.41 ± 0.11 versus 7.22 ± 0.05, n = 4, p = 0.01). As shown in Fig. 5, there was a
statistically significant difference at higher pH values, and CYP2D6 1 was active throughout a wider pH range. This difference indicates an
important role for charged amino acid(s) in the active site.
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Discussion |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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The results from this study show that the decreased CYP2D6
activity seen in individuals with the CYP2D6*17 allele is
caused by an enzyme with a reduced affinity for some of the CYP2D6
substrates. It was found unexpectedly that a combination of the T107I
and R296C substitutions was needed to alter the kinetic properties of
the CYP2D6 17 variant, because no significant effect was seen on
bufuralol 1-hydroxylation when they were introduced one by one. In
contrast, examination of the rate of O-demethylation of codeine revealed a significant change in the apparent
Km when only the T107I substitution
was introduced. Also, in the COS-1 expression system, there was a
combined effect of the substitutions, affecting the enzyme activity.
The CYP2D6*17 allele is frequently distributed among Zimbabweans [allele frequency of 34.0% (15)], Ethiopians [allele frequency of 9.0% (28)], and other African populations. Thus, more than 10% of the Zimbabwean population is homozygous for this allele, with important implications for pharmacological treatment with CYP2D6 substrates.
It seems that the C1111T mutation (yielding the T107I substitution) is always linked to the C2938T (R296C) and G4268C (S486T) mutations on the same allele (15). It cannot be excluded, however, that there are alleles that only contain the C1111T mutation or alleles where this mutation is linked to other mutations in the CYP2D6 gene. This possibility is interesting because the T107I substitution had a significant effect on the apparent Km of codeine O-demethylation, whereas no effect was seen when bufuralol was used as a substrate, a result consistent with the fact that these two substrates are structurally unrelated.
It is noteworthy that the correlation between the phenotypes obtained using different probe drugs is generally lower in black African than in other populations. The correlation coefficient between the metabolic ratio for debrisoquine and metoprolol has been shown to be only 0.16-0.67 in studies carried out on black African subjects (14, 29-31), compared with 0.81 in a Caucasian population (32). Future studies are needed to evaluate whether the distribution of the CYP2D6*17 allele, creating an enzyme with altered substrate specificity, can explain this lack of correlation.
In the absence of a crystal structure for CYP2D6 or any other mammalian
P450s, homology building models based on the bacterial P450 structures
have been used for the prediction of residue positions in the
three-dimensional structure of the enzyme (33-35). By structurally aligning these three bacterial P450s, an extended multiple sequence alignment was made, including representatives from the major mammalian P450 families (36). According to this model, the Thr107 residue is
located in the B helix, a region of the molecule known to be involved
in substrate binding. Gotoh (37) defined six SRSs for the CYP2 family,
and Thr107 is localized in SRS 1, thereby implicating an important
function for this residue in substrate binding. Modi et al.
(34) have combined homology modeling techniques and NMR studies to
build a model of the CYP2D6 active site and its interaction with
codeine. From this model, it is apparent that Thr107 is involved in the
binding of codeine and that the substitution of the hydrophilic residue
threonine to a hydrophobic isoleucine is likely to affect the substrate
binding. This is compatible with the experimental data here presented,
because the T107I CYP2D6 variant exhibited reduced affinity for codeine but not for bufuralol. This model also shows that Asp301 is involved in
the binding of the basic nitrogen found in codeine and all other CYP2D6
substrates (38). In support of this, Ellis et al. (39) have
studied the importance of this residue using site-directed mutagenesis
and shown that an acidic amino acid is required at this position,
because activities were diminished in all of the mutants except where
aspartic acid was replaced with glutamic acid.
The Arg296 residue is located in the I helix near Asp301. This helix is
the longest one found in P450s and is known to be involved not only in
substrate binding but also in delivery of catalytic protons, with the
central part of the helix coinciding with SRS 4. Because the CYP2D6 17 exhibited a more acidic pH-optimum than the wild-type enzyme, it can be
speculated that the R296C substitution affects the
pKa of some residue(s) involved in
substrate contact or electron transfer. The -pleated sheet region of
the enzyme where the Ser486 residue is located is less well conserved between bacterial and mammalian P450s and predictions are difficult to
make.
Based on this structural information, it is evident that at least both Thr107 and Arg296 are located near the CYP2D6 active site. This is in agreement with the kinetic data, which shows that these two residues were necessary for the effect observed on the enzyme's catalytic properties. It must be pointed out, however, that the substitutions introduced at these positions are much more drastic than the conserved S486T substitution, and it is difficult to predict what conformational changes in the protein structure these substitutions will cause. The combined effect of the T107I and R296C substitutions on bufuralol kinetics remains partly unexplained, because no major effect was seen when these substitutions were introduced independently. High-resolution models of CYP2D6, and especially the active site structure, are needed to further understand this combined effect.
In conclusion, we have identified a molecular explanation for the decreased in vivo activity seen in individuals with the CYP2D6*17 allele. This allele represents a unique case in which reduced CYP2D6 activity is caused by a decrease in substrate affinity; this allele is also interesting, in that a combination of substitutions is necessary to yield a P450 enzyme with altered activity. The CYP2D6 17 variant constitutes a challenging model for future structural studies and can provide important information concerning CYP2D6 active site structure-function relationships. The finding of the properties of CYP2D6 17 emphasizes the importance of the pronounced interethnic differences with respect to the CYP2D6 gene and indicates interethnic differences in the metabolism of CYP2D6 substrates also from a qualitative point of view. This might be of importance with respect to drug efficacy and drug toxicity.
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Acknowledgments |
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We are indebted to Mrs. Ann-Louise Hagbjörk for skillful technical assistance and to Mr. Roman McLellan for critical reading of the manuscript. We also extend our gratitude to Dr. Denis Pompon (Centre Nationale de Recherche Scientifique, Gif-sur-Yvette, France) for the Saccharomyces cerevisiae W(R) strain and the pYeDP60 expression vector and to Dr. Urs A. Meyer (Biozentrum, Basel, Switzerland) and Drs. Leif Bertilsson and Marja-Liisa Dahl (Division of Clinical Pharmacology, Huddinge University Hospital, Huddinge, Sweden) for generous gifts of the drugs.
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Footnotes |
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Received June 30, 1997; Accepted September 2, 1997
1 In this report we have used the new nomenclature system for CYP2D6 alleles (10).
This study was supported by grants from Stiftelsen Söderström-Königska sjukhemmet, European Commission (Biomed 2) and from the Swedish Medical Research Council.
Send reprint requests to: Dr. Magnus Ingelman-Sundberg, Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden. E-mail: magnus.ingelmansundberg{at}imm.ki.se
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Abbreviations |
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P450, cytochrome P450; PM, poor metabolizer; reductase, NADPH cytochrome P450 reductase; HPLC, high performance liquid chromatography; SRS, substrate recognition site.
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References |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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) and CYP2D6 17 (T107I+R296C+S486T; 
) variants. wt, wild-type.
) as buffer. Values are expressed as percentage of maximal activity and represent mean ± standard deviation of four independent experiments. *, p < 0.05; **, p < 0.01;
***, p < 0.001.