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J Biol Chem, Vol. 273, Issue 46, 30599-30607, November 13, 1998
From the Flavin-containing monooxygenases
(FMOs) are NADPH-dependent flavoenzymes that catalyze
the oxidation of heteroatom centers in numerous drugs and xenobiotics.
FMO2, or "pulmonary" FMO, one of five forms of the enzyme
identified in mammals, is expressed predominantly in lung and differs
from other FMOs in that it can catalyze the N-oxidation of
certain primary alkylamines. We describe here the isolation and
characterization of cDNAs for human FMO2. Analysis of the sequence
of the cDNAs and of a section of the corresponding gene revealed
that the major FMO2 allele of humans encodes a polypeptide
that, compared with the orthologous protein of other mammals, lacks 64 amino acid residues from its C terminus. Heterologous expression of the
cDNA revealed that the truncated polypeptide was catalytically
inactive. The nonsense mutation that gave rise to the truncated
polypeptide, a C The flavin-containing monooxygenases
(FMOs)1 (EC 1.14.13.8) are
NADPH-dependent flavoenzymes that catalyze oxidation of
soft nucleophilic heteroatom centers in a range of structurally diverse compounds, including drugs, pesticides, and other xenobiotics (1, 2).
Functionally, FMOs differ from other monooxygenases in that the active
oxygenating species, the C(4a)hydroperoxide derivative of the flavin
cofactor, exists stably within the protein in the absence of substrate,
thereby enabling the enzyme to oxidize any soft nucleophile able to
gain access to the active site. FMO was originally characterized by
using a homogeneous enzyme preparation isolated from pig liver (3).
Proteins with equivalent catalytic properties and substrate profiles
were later isolated from the livers of several other species (4-6).
However, contemporaneous studies indicated that rabbit lung contained a
form of FMO which, although clearly related to the liver enzyme,
possessed distinct physicochemical properties (7) and substrate
preferences (8, 9). The purification of this "lung" or
"pulmonary" FMO (10-12) confirmed the existence of two distinct
FMOs, distinguishable both immunochemically and by substrate preference
(10-12), and the subsequent isolation and characterization of the
corresponding cDNA clones (13) demonstrated that each was the
product of a separate gene.
Following the identification of "liver" and "lung" FMO as
discrete enzymes, evidence accumulated indicating the presence in these
tissues of additional forms of the enzyme. The existence of multiple
mammalian FMOs was subsequently confirmed when new forms were
identified, either by direct sequencing of purified proteins (14, 15),
or via the isolation and characterization of cDNA clones
(16-18). To date, five distinct forms of FMO, designated FMOs 1-5,
each of which is encoded by its own gene and which exhibits approximately 50-60% pairwise amino acid sequence similarity, have
been identified in mammals (19). According to the present nomenclature
(19) "liver" and "lung" FMO are designated FMO1 and FMO2,
respectively, whereas the forms identified subsequently have been
designated FMOs 3, 4, and 5.
Although detected at other sites, such as the kidney (13, 20, 21), FMO2
is expressed predominantly in pulmonary tissue (10, 11, 13, 20-23)
where, in the rabbit, it has been localized to the nonciliated
bronchiolar epithelial (clara) cells and endothelial type I and II
cells (24). FMO2 gene expression has been demonstrated to be
regulated by sex hormones in experimental animals (25, 26) and putative
glucocorticoid responsive elements have been identified in the
5'-flanking region of the rabbit FMO2 gene (27). FMO2
displays catalytic characteristics that distinguish it from other forms
of FMO. For instance, although able to oxidize many typical FMO
substrates, it is inactive toward certain tertiary amines, such as
imipramine and chlorpromazine (9, 10), that are good substrates for
other forms of the enzyme. Furthermore, in contrast to other
FMOs, FMO2 is capable of mediating the N-oxidation of some
primary alkylamines to their oximes, via an
N-hydroxylamine intermediate (28, 29).
We have previously reported the isolation of cDNAs encoding human
FMOs 1 (30), 4 (16) (previously designated FMO2), and 3 (31) and have
determined that the corresponding genes, plus the genes encoding human
FMOs 2 and 5, are all located on the long arm of chromosome 1 (16, 30,
32, 33). In this report we describe the isolation and characterization
of cDNA clones encoding human FMO2 and demonstrate that, in common
with human FMOs 1 and 3 (31), expression of the corresponding gene is
subject to both ontogenic and tissue-specific regulation. Furthermore, we report that, in contrast to apparently all other mammalian species,
including closely related primates, in humans the major FMO2
allele encodes a truncated polypeptide which is catalytically inactive.
Oligonucleotide Synthesis--
Oligonucleotides were
synthesized on a PCR-MATE DNA synthesizer (model 391, Applied
Biosystems, Warrington, UK).
Isolation of RNA and Genomic DNA--
Human adult and fetal
tissue samples were obtained as described previously (31) except for
single adult lung and kidney samples, which were obtained from the
International Institute for the Advancement of Medicine (Exton, PA).
Total RNA was prepared by the guanidinium thiocyanate/LiCl method (34),
resuspended in diethylpyrocarbonate-treated water and stored in
aliquots at Amplification of a Partial-length cDNA Encoding Rabbit
FMO2--
Reverse-transcription of rabbit (New Zealand White) lung
total RNA (20 µg) was primed with 200 pg of random hexamer
oligonucleotides (Pharmacia Biotech, St. Albans, UK) and catalyzed by
200 units of Moloney murine leukemia virus reverse-transcriptase (Life
Technologies, Paisley, Scotland) in a total volume of 20 µl according
to the supplier's recommendations. The reaction mix was then incubated for 5 min at 75 °C and the volume increased to 100 µl with water. Oligonucleotides 102 (5'-GACGCAGTCATGGTCTGCAGTGGC-3') and 180 (5'-GATGTAATTGGTCTGCAGTTTCTG-3'), designed from the rabbit FMO2 cDNA sequence (13) and which incorporated PstI
restriction endonuclease sites present within the rabbit sequence, were
used to prime the amplification, by PCR, of 879 bp of coding region.
The PCR was performed in a volume of 40 µl containing 15 pmol of each
primer, 66.7 mM Tris-HCl (pH 8.4), 1.25 mM
MgCl2, 16.7 mM
(NH4)2SO4, 0.1% (v/v) Tween 20, 50 µM of each dNTP, and a 5-µl aliquot of the reverse-transcription product. After an initial denaturation at 94 °C for 1 min, 0.5 µl (2.5 units) of Taq DNA
polymerase (BioLine, London, UK) was added, and the reaction mix was
incubated (TR1, Hybaid, Ashford, UK) for 32 cycles at 94 °C for
45 s, 58 °C for 45 s, and 72 °C for 1 min, followed by
an additional 5 min at 72 °C. The PCR product was incubated with
PstI, gel-purified, and ligated into
PstI-digested pBS (Stratagene, Cambridge, UK) to give the
plasmid pRABLUNG. The identity of the cDNA insert was confirmed by
restriction mapping and partial DNA sequencing.
cDNA Library Screening--
The insert from pRABLUNG was
excised by incubation with PstI, gel-purified, radiolabeled
by the oligonucleotide random primer method (37) to a specific
radioactivity of approximately 109 cpm/µg with
[ 5'-RACE-PCR of Human and Macaque FMO2 cDNAs--
Reverse-transcription of human or cynomolgus macaque (Macaca
fascicularis) lung total RNA (10 µg), and first-round and
seminested 5'-RACE-PCRs, using the human FMO2 cDNA-specific
primers 498 (5'-CAGGAAGTTTGGAAAATCTTCAGGC-3') and 423 (5'-CTCAAGTCCCTCATCCACACAGC-3'), respectively, and the common
adapter primer 365 (31), were performed as described previously (31).
The products of the seminested 5'-RACE-PCRs were blunt-ended with T4
DNA polymerase, phosphorylated with T4 polynucleotide kinase (New
England Biolabs, Hitchin, UK), then purifed (SpinBind, FMC Bioproducts,
Rockland, ME) and inserted into EcoRV-digested pBS.
3'-RACE-PCR of Human FMO2 cDNA--
3'-RACE-PCR was
performed essentially as described by Frohman et al. (38).
Reverse-transcription of human lung total RNA (10 µg) was performed
as described previously (31), except that reactions were primed with
100 ng each of the anchored oligo-d(T) primers 366, 367, and 368 (31).
First-round and seminested 3'-RACE-PCRs were performed as described
above for 5'-RACE-PCR, except that they were primed with 15 pmol each
of the universal primer 365 (31) and the FMO2 cDNA-specific primers
359 (5'-ATGATGTCCCAAGTCGTCTACT-3') and 356 (5'-TACTCTGTGGAGCCATCAAG-3'), respectively. Seminested 3'-RACE-PCR
products were cloned as descibed above for 5'-RACE-PCR products.
Amplification of Full-length cDNAs Encoding Human and Macaque
FMO2--
Reverse-transcription of human or macaque lung total RNA (10 µg) and the subsequent removal of RNA with RNases A and H were performed as described previously (31), except that
reverse-transcription was catalyzed by 200 units of SuperScript II
reverse-transcriptase (Life Technologies) in a volume of 20 µl
according to the supplier's recommendations.
Oligonucleotides 392 (5'-CTAGAATTCTAGAATGGCAAAGAAGGTAGCTGTGATTG-3') and 394 (5'-TACTGGATCCTGACAAGATAATAAAGCCCAAAG-3'), containing unique
XbaI and BamHI restriction enzyme sites,
respectively, were designed to prime the amplification of the entire
protein-coding region of the human FMO2 cDNA plus a small stretch
of the 3'-untranslated region. PCR was performed in a volume of 40 µl
containing 15 pmol of each primer, 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.8), 2 mM MgSO4,
0.1% (v/v) Triton X-100, bovine serum albumin (100 µg/ml), 200 mM of each dNTP, and a 2-µl aliquot of the
reverse-transcription product. After an initial denaturation step at
94 °C for 1 min, 1 µl (2.5 units) of Pfu DNA polymerase
(Stratagene) was added and the reaction incubated for 36 cycles at
94 °C for 75 s, 52 °C for 75 s, and 72 °C for 4 min,
and then for an additional 10 min at 72 °C. The resulting PCR
product was incubated with XbaI and BamHI,
gel-purified, and ligated into pBS, previously digested with
XbaI and BamHI, to give the plasmid pBSFMO2/2.
Amplification of the entire protein-coding region of the cynomolgus
macaque FMO2 cDNA, plus small stretches of both the 5'- and
3'-untranslated regions, was achieved with the forward primer 618 (5'-CGTTGAATTCCAAGGGAGAAAACTATTCTGTC-3'), designed from the sequence of
the 5'-RACE-PCR products (data not shown), and the human FMO2
cDNA-specific reverse primer 394. PCR conditions were as described
above for the amplification of the partial-length rabbit FMO2 cDNA,
except that a 5-µl aliquot of the reverse-transcription product was
used as template and the reaction mix was incubated for 35 cycles at
94 °C for 30 s, 56 °C for 1 min, and 72 °C for 2 min,
followed by an additional 5 min at 72 °C. The PCR product was
purified from the reaction mix (SpinBind, FMC) and sequenced directly.
Amplification of a Region of the FMO2 Gene--
An equivalent
235-bp region of the human, gorilla (Gorilla gorilla), and
chimpanzee (Pan troglodytes) FMO2 gene was
amplified by PCR using the primers 422 (5'-TTCAAAGATCCTAAACTGGCTGTGAG-3') and 394. Reaction mixtures
containing approximately 200 ng of genomic DNA as template were
incubated for 38 cycles at 94 °C for 45 s, 56 °C for 45 s, and 72 °C for 45 s, followed by an additional 5 min at
72 °C. Reaction conditions were as described above for the
amplification of the partial-length rabbit FMO2 cDNA. PCR products
were purified from the reaction mix (SpinBind, FMC) and sequenced directly.
DNA Sequencing--
Plasmid DNA and purified PCR products were
sequenced by the dideoxy chain-termination method (39) using DNA
sequencing kits (Sequenase II, Amersham International; Cyclist,
Stratagene) and either vector- or insert-specific primers. The insert
of pBSFMO2/2 and the subcloned products of the human FMO2 3'-RACE-PCRs
and of the human and cynomolgus macaque FMO2 5'-RACE-PCRs were
sequenced completely on both strands (Fig. 1), whereas the full-length
cDNA encoding cynomolgus macaque FMO2 was only partially sequenced. Sequence data were analyzed with MacVector (Oxford Molecular, Oxford,
UK) or Genetics Computer Group (Madison, WI) sequence analysis software.
RNase Protection Assay--
The human FMO2 RNase protection
plasmid, pBSFMO2/2/15, was constructed by excising a 985-bp section
from within the insert of pBSFMO2/2 (by digestion with
HindIII and StyI), followed by blunt-ending of
the DNA with T4 DNA polymerase and self-ligation of the larger vector
fragment. pBSFMO2/2/15 (10 µg) was linearized by digestion with
MscI, after which the reaction mix was treated with
proteinase K and SDS (40), and extracted with phenol-chloroform (1:1,
v/v). The linearized plasmid was ethanol-precipitated and resuspended
in diethylpyrocarbonate-treated water. In vitro synthesis of
radiolabeled antisense RNA, probe purification, and RNase protection assays were performed as described previously (31, 40, 41). Comparison
of the autoradiographic signal derived from the protected species with
a standard curve of undigested probe permitted quantification of FMO2
mRNA in terms of molecules/µg of total RNA. This was converted to
molecules/cell by using the average RNA content of a mammalian cell (5 pg) (42).
Southern Blot Hybridization--
Human genomic DNA (20 µg) was
incubated overnight at 37 °C with 300 units of EcoRI or
HindIII (New England Biolabs), extracted with
phenol-chloroform (1:1, v/v), ethanol-precipitated, electrophoresed through a 0.8% agarose gel, and transferred to a nylon membrane (Hybond, Amersham International). The membrane was prehybridized at
42 °C for 3 h in 50% deionized formamide, 5× SSPE (1× SSPE, 0.18 M NaCl, 10 mM sodium phosphate (pH 7.4), 1 mM EDTA), 5× Denhardt's reagent, 2% (w/v) SDS, and
denatured salmon sperm DNA (100 µg/ml), then hybridized overnight at
42 °C in 50% deionized formamide, 5× SSPE, 2% (w/v) SDS and 10%
(w/v) dextran sulfate, containing radiolabeled probe (synthesized as
described above for cDNA library screening) at a final
concentration of 2-5 ng/ml. After hybridization the membrane was
washed once in 2× SSPE/1% (w/v) SDS, (15 min at room temperature),
twice in 1× SSPE/1% SDS (15 min each at room temperature), and twice
in 0.1× SSPE/1% SDS (15 min each, once at room temperature then at
55 °C), then autoradiographed for 72 h at Northern Blot Hybridization--
RNA samples (15 µg) were
denatured in formaldehyde and electrophoresed through a 1% agarose gel
(43). After transfer to a nylon membrane (BDH, Poole, UK) RNA was
immobilized by baking the membrane at 80 °C, followed by UV
cross-linking (Stratalinker 1800, Stratagene). The membrane was
prehybridized, hybridized, washed, and subjected to autoradiography as
described above.
Construction of Recombinant Baculovirus--
To construct
FMO2X472 recombinant baculovirus, pBSFMO2/2 was incubated with
XbaI and PstI, and the smaller of the resulting restriction fragments, comprising the 1413-bp ORF, 192 bp of associated 3'-untranslated region, and approximately 50 bp of pBS, was ligated to
XbaI/PstI-digested pFastBac (Life Technologies)
to give pFastFMO2/2/8. To construct recombinant virus containing
FMO2Q472, two oligonucleotides, 576 (5'-CGAGCTCGCCTTAGAGATAGGTGCG-3')
and 394, were used to prime the amplification of a 330-bp
contiguous region of the FMO2 gene that encompassed codon
472 and the second in-frame stop signal located 192 bp downstream (see
Fig. 2). PCR conditions were as described above for FMO2
gene amplification, except that genomic DNA (100 ng) isolated from an
individual previously identified as being heterozygous for TAG and CAG
triplets at codon 472 was used as a template. The PCR product was
ligated directly into EcoRV-linearized pBS, and, after
propagation in Escherichia coli, a plasmid clone
containing CAG at codon 472 in the amplified region of the
FMO2 gene was identified by DNA sequencing. The insert of
this plasmid was excised by incubation with SacI and
BamHI, gel-purified, and ligated, in a three-fragment
reaction, with a SacI/XbaI restriction fragment
of pBSFMO2/2 that contained the remainder of the FMO2 coding region,
and XbaI/BamHI-digested pBS, to give
pBSFMO2/2/16. The extended FMO2 ORF was excised from pBSFMO2/2/16, by
incubation with XbaI and PstI, then gel-purified
and ligated into pFastBac to give pFastFMO2/2/16.
In order to bring the translational initiation codon of each of the
inserted cDNAs closer to the position of that of the original polyhedrin gene, approximately 50 bp were excised from the multiple cloning sites of both pFastFMO2/2/8 and pFastFMO2/2/16 by digestion with EcoRI and XbaI. After religation of the
blunt-ended vector portions to give pFastFMO2X472 and pFastFMO2Q472,
the respective recombinant baculoviruses, AcFMO2X472 and AcFMO2Q472,
were generated by transfection of Spodoptera frugiperda
(Sf) 9 cells with the corresponding recombinant bacmid DNA,
obtained via site-specific transposition using the
Bac-to-Bac system (Life Technologies) according to the supplier's recommendations.
Baculovirus-mediated Expression of FMO2X472 and
FMO2Q472--
Sf9 and Tricoplusia ni insect cells
were grown and passaged in shaker cultures using Sf-900II (Life
Technologies) and Excell 405 (JRH Biosciences-Europe, Marlow, UK)
serum-free media, respectively, containing amphotericin B (5 µg/ml),
penicillin G (100 units/ml), and streptomycin sulfate (50 µg/ml). For
amplification of virus, Sf9 cells were infected at a
multiplicity of infecton of Western Blotting--
Western blotting was performed as
described previously (44). Blots were incubated with goat anti-(rabbit
FMO2) serum (1 in 3000 dilution; gift of R. Philpot) and a rabbit
anti-(goat immunoglobulin G)-alkaline phosphatase conjugate (Sigma
Chemical Company (UK), Poole, UK), and antigen visualized (Color
Development Kit, Bio-Rad Laboratories Ltd., Hemel Hempstead, UK). The
concentration of each expressed FMO2 was determined by scanning
densitometry (Bio-Rad, model GS-670) using a standard curve of
authentic rabbit FMO2 (gift of R. Philpot).
FMO Assays--
FMO activity was determined using the substrate
methimazole according to the method of Dixit and Roche (45). The assay
was performed in a 1-ml cuvette, held at 37 °C, containing 0.1 M Tris-HCl (pH 8.4), 1 mM EDTA, 0.02 mM dithiothreitol, 0.06 mM
5,5-dithiobis-(2-nitrobenzoate), 0.1 mM NADPH, 2 mM methimazole, and microsomal membranes. When used,
MgCl2 was added to both sample and reference cuvettes. The reaction was started by addition of methimazole (in 10 µl of buffer) to the sample cuvette and 10 µl of buffer to the reference cuvette. Reaction buffer was aerated for 30 min before use, and reaction mixtures were allowed to equilibrate for 2 min after addition of
substrate. The progress of the reaction was monitored by measuring the
decrease in the concentration of nitro-5-thiobenzoate, as revealed by
decreased absorbance at 412 nm.
The Human FMO2 Gene Contains a Premature In-frame Termination
Codon--
A screen of 6 × 105 plaques of a human
lung cDNA library with the insert of pRABLUNG, which comprised an
879-bp fragment of rabbit FMO2 cDNA, yielded four positive clones,
L1, L2, L3, and L8, containing inserts of 0.6, 2.0, 1.6, and 0.9 kb,
respectively. Sequence analysis confirmed that the inserts of all four
clones contained sequences that encoded the human ortholog of FMO2.
However, two of the clones, L2 and L3, also contained sequences that
were not related to FMO2. A search of the GenBankTM data
base revealed that the latter sequences encode pulmonary surfactant-associated protein and mitochondrial 12 S ribosomal RNA,
respectively. In each case, the FMO2 cDNA sequence was found to be
joined to the unrelated sequence by an EcoRI restriction site, indicating that both clones were chimeric artifacts formed during
library construction.
A consensus sequence for the human FMO2 cDNA derived from these
four clones contained a short stretch of 5'- untranslated region and
almost 1100 bp of protein-coding region (Fig.
1). The latter region was considerably
shorter than the corresponding regions of FMO2 cDNAs of rabbit and
guinea pig and did not contain an in-frame stop codon. To obtain
sequence encoding the carboxyl-terminal portion of human FMO2 we
carried out 3'-RACE-PCR. Two independent 3'-RACE-PCR products (Fig. 1)
were found to have identical sequences, which contained the remainder
of the protein-coding region and 45 bp of an apparent 3'- untranslated
region. To confirm and extend the sequence of the 5'-untranslated
region we performed 5'-RACE-PCR. The products of two independent
5'-RACE-PCRs (Fig. 1) were each found to contain 60 bp of
5'-untranslated region, the sequences of which were identical with each
other and with the overlapping regions of the shorter stretches of 5'-
untranslated region contained within the clones L3 and L8. A cDNA,
FMO2/2 (Fig. 1), containing the entire protein-coding region of FMO2,
plus a short stretch of 3'-untranslated region, was obtained by
reverse-transcription PCR.
The sequence of human FMO2 cDNA (Fig.
2) was assembled from the partial
sequences of the phage clones L1, L2, L3, and L8, the complete
sequences of the 5'- and 3'-RACE-PCR products and the complete sequence
of the full-length cDNA obtained by reverse-transcription PCR. The
sequence contains 60 bp of 5'-untranslated region, followed by an ORF
of 1413 bp, a TAG translational termination codon and 237 bp of an
apparent 3'-untranslated region that lacks a consensus polyadenylation
signal. The high degree of sequence identity with FMO2 cDNAs of
rabbit (87%) (13) and guinea pig (86%) (46) confirmed the identity of
the human clone. However, in contrast to FMO2 proteins of rabbit (13),
guinea pig (46) and rhesus macaque (23), each of which contains 535 amino acid residues, the ORF of the human cDNA that we have
isolated encodes a polypeptide of only 471 residues, with a calculated
molecular mass of 53,639 daltons. Comparison of the human FMO2 cDNA
sequence with the corresponding sequences of rabbit, guinea pig, and
macaque (data not shown) revealed that the TAG codon terminating the
human ORF aligns with a CAG codon, encoding Gln-472, in the nucleotide
sequences of FMO2s of the other three species. The human cDNA
contains another in-frame TAG triplet, 192 bp downstream of that at
codon 472. This second in-frame stop codon aligns exactly with the
termination codons of FMO2s of rabbit, guinea pig, and rhesus macaque.
As the upstream TAG triplet was present in both of the 3'-RACE-PCR products and in the full-length FMO2 cDNA obtained by
reverse-transcription PCR it is unlikely to be the result of a cloning
artifact.
To confirm the presence of the apparent premature stop codon in the
human FMO2 gene, a region of the gene encompassing both stop
codons and part of the 3'-untranslated region was amplified from each
of 12 unrelated individuals and the resulting PCR products directly
sequenced. All 12 were found to be homozygous for a TAG triplet at
codon 472 (Fig. 3A and data
not shown). Therefore, in humans, the FMO2 gene contains a
premature stop codon and thus encodes a polypeptide that is 64 amino
acid residues shorter than FMO2s of other mammalian species such as
rabbit, guinea pig, and macaque monkey. Southern blot hybridization of
human genomic DNA revealed that the full-length cDNA hybridized to
fragments of 7.0, 6.4, 4.7, 3.7, and 3.1 kb in
EcoRI-digested DNA (Fig. 4, lane 1), and to fragments of 9.2, 7.2, and 6.9 kb in
HindIII-digested DNA (Fig. 4, lane 2). This gives
an estimated minimum size for the human FMO2 gene of
approximately 23 to 25 kb, similar to that of the human FMO3
gene (47) and suggests that humans contain only one FMO2
gene.
Analysis of Human FMO2 mRNA--
The sequence flanking the
proposed translational initiation codon of human FMO2 mRNA,
GAGCUGAUGG (initiation codon underlined) (Fig. 2), conforms
poorly to the consensus sequence, GCC(AG)CCAUGG, for
efficient initiation of translation of vertebrate mRNAs (48). Of
particular note is the lack of a purine at the
Northern blot hybridization of total RNA isolated from the lungs of
three human individuals revealed the presence of a single species of
FMO2 mRNA of 5.7 kb (Fig. 5,
lanes 1-3), which is similar in size to the mRNAs
encoding FMO2 of macaque (5.0 kb) (Fig. 5, lane 4) (23),
rabbit (a single major transcript of 4.8 kb and minor transcripts of
6.0, 2.6, and 2.4 kb) (13), guinea pig (6.0 kb) (46), and pig (6.0 kb)
(46). The human FMO2 mRNA thus contains a 3'-untranslated region of
approximately 4 kb. The much shorter length of 3'-untranslated sequence
in the 3'-RACE-PCR products that were obtained (Figs. 1 and 2),
together with the absence of a consensus polyadenylation signal,
suggests that the reverse-transcription step of the 3'-RACE procedure
was primed from an internal adenosine-rich sequence rather than from
the poly(A) tail of the mRNA. In support of this, both rabbit and guinea pig FMO2 cDNAs contain adenosine-rich sequences at the corresponding positions within their respective 3'-untranslated regions
(13, 46).
The Human FMO2 Gene Is Subject to Developmental and Tissue-specific
Regulation--
The tissue distribution and developmental expression
of the mRNA encoding human FMO2 was investigated by quantitative
RNase protection. A representative autoradiogram is shown in Fig.
6A and demonstrates that a
discrete protected RNA, of the expected size, was obtained from the
FMO2 antisense RNA probe. FMO2 mRNA was expressed relatively
abundantly (13-36 molecules/cell) in adult lung samples from four
different individuals (Fig. 6B). It was also expressed, but
in lower abundance (3 molecules/cell), in one of two adult kidney
samples analyzed (Fig. 6A and data not shown), but was not
detected in the other kidney sample or in either of two adult liver
samples, even after prolonged exposure. In fetal tissues, FMO2 mRNA
was not detected in samples of liver, kidney or brain (data not shown)
but was present in moderate abundance (5-12 molecules/cell) in each of
four lung samples (Fig. 6B).
The Nonsense Mutation in the Human FMO2 Gene Occurred after the Pan
and Homo Lines Diverged--
The FMO2 gene of rhesus monkey
encodes a polypeptide of 535 amino acid residues (23), the same length
as the orthologous protein of rabbit (13) and guinea pig (46). Analysis
of the sequence of cDNA for FMO2 of the cynomolgus monkey, a close
relative of the rhesus monkey, revealed that a stop codon was not
present at position 472 (data not shown). The mutation that gave rise to the premature stop codon present in the human FMO2 gene
must therefore have occurred within the Hominoidea, sometime after this
primate superfamily, which includes apes and humans, diverged from the
Cercopithecoidea superfamily, which includes the Old World monkeys. To
determine at what stage during the evolution of the Hominoidea the
mutation occurred, a 235-bp region of the FMO2 gene
encompassing codon 472 was amplified by PCR from genomic DNA of gorilla
and chimpanzee. Within this region the sequence of the FMO2
gene of these species is 98% identical to that of the corresponding
region of the FMO2 gene of humans (data not shown). Both of
these non-human hominoids were homozygous for a CAG triplet, encoding
Gln, at codon 472 of FMO2 (Fig. 3B and data not
shown) and contained a translational stop codon at position 536 (data
not shown), corresponding to that present in FMO2 of rabbit,
guinea pig, and rhesus and cynomolgus monkeys. The mutation that gave
rise to the truncated FMO2 of present day humans must therefore have
occurred in the human lineage sometime after the divergence, some 4-5
million years ago, of humans from their closest relative, the chimpanzee.
Subsequent analysis, with a PCR-restriction enzyme assay, of
individuals of various racial and ethnic backgrounds, including 27 European Caucasians, 18 Orientals (10 Japanese, 8 Chinese), 41 of
African descent (20 Africans, 16 African-Americans, 5 UK Afro-Caribbeans), 6 New Guinea Aboriginals, 2 Indians and 2 Maoris, revealed that the allele encoding the truncated FMO2,
FMO2X472, occurs at a frequency of essentially 100% in all
groups investigated, with the exception of populations of African
descent, in which an allele containing a CAG triplet at codon 472 (Q472) is present at a frequency of approximately
4%.2
The Truncated Protein Encoded by the Human Gene Is Catalytically
Inactive--
FMO2X472, the truncated form encoded by the major
FMO2 allele of humans, and FMO2Q472, a "full-length"
form containing 535 amino acid residues (see Fig. 2), were produced
via heterologous expression of the corresponding cDNAs
in insect cells via the baculovirus expression system.
Western blotting with antibody against rabbit FMO2 detected proteins of
53 and 57 kDa, respectively, in microsomal membranes isolated from
cells infected with virus containing cDNA encoding FMO2X472
(AcFMO2X472) (Fig. 7A,
lane 5), or FMO2Q472 (AcFMO2Q472) (Fig. 7A,
lane 6). Authentic rabbit FMO2 (Fig. 7A,
lane 7) had an estimated molecular mass of 56 kDa. The
concentrations of heterologously expressed FMO2X472 and FMO2Q472 were
14 and 19 pmol/mg microsomal membrane protein, respectively. Immunoreactive protein was undetectable in microsomal membranes isolated from noninfected insect cells (Fig. 7A, lane
1) or from cells infected with wild-type virus, AcNPV (Fig.
7A, lane 2), or in the cytosolic fractions of
cells infected with AcFMO2X472 (Fig. 7A, lane 3)
or AcFMO2Q472 (Fig. 7A, lane 4).
The function of FMO2X472 and FMO2Q472 was investigated by determining
their ability to catalyze the S-oxidation of methimazole, an
excellent substrate for FMO2s of other mammalian species (Fig. 7B). Microsomal membranes isolated from cells infected with
AcFMO2Q472 catalyzed the S-oxidation of methimazole with
essentially maximal specific activity of 0.50 nmol of methimazole oxide
formed/min/mg microsomal protein ([S] = 2 mM;
Km = 411 µM) (51)
(kcatapp Our results demonstrate that the major FMO2 allele of
humans encodes a polypeptide that, in comparison with the orthologous protein of rabbit (13), guinea pig (46), and rhesus macaque (23), lacks
64 residues from its carboxyl terminus. This is due to the presence in
the human gene of an in-frame TAG translational termination triplet at
codon 472, 64 codons upstream of a second in-frame termination signal
the position of which corresponds exactly with that present in
FMO2 of rabbit, guinea pig, and macaque. As the latter
species all contain a CAG triplet, encoding glutamine, at codon 472, it
appears that the premature stop codon present in the human
FMO2 gene arose as the result of a C to T transition at the
first position of codon 472.
The presence of nonsense mutations within prokaryotic and eukaryotic
genes is frequently associated with decreased abundance of the
corresponding mRNA, due to an increase in the turnover rate of the
mutant transcript (52). This process, termed nonsense-mediated mRNA
decay (53), is dependent upon the relative position of the mutation
within the protein-coding region; mutations located within the region
encoding the amino-terminal two-thirds of a polypeptide accelerate
degradation, whereas those within regions encoding sequences closer to
the carboxyl terminus may have little or no effect upon mRNA
stability (54). Consequently, as the nonsense mutation present in the
FMO2 mRNA is located in the 3'-most one-third of the protein-coding
region, it would be expected to exert little effect on the stability of
the mRNA. In support of this, we found no evidence of degraded FMO2
mRNA in any of the human lung samples analyzed by Northern blot
hybridization, and, furthermore, RNase protection analyses demonstrated
that FMO2 mRNA was moderately abundant in all samples of human lung
examined. Thus, the primary transcript of the human FMO2
gene is apparently correctly processed in pulmonary tissue to produce a
stable and abundant mRNA that should be available for potential
translation. The conservation throughout mammalian evolution of a long
3'-untranslated region in FMO2 mRNA suggests that it may be
important for some aspect of the metabolism or function of the
mRNAs, such as stability.
The results of RNase protection assays demonstrate that in humans
expression of the FMO2 gene in both the adult and fetus is
essentially restricted to the lungs, with the gene being inactive in
the liver. The complete absence of hepatic expression of
FMO2 has been demonstrated for other species (13, 21, 23)
and confirms that FMO2 is essentially a pulmonary-specific FMO. The higher concentration of the FMO2 mRNA present in adult, compared with fetal, human lung indicates that the gene is also regulated developmentally. Thus, in humans, FMO2, in common with
FMO1 and FMO3 (31), is subject to both ontogenic
and tissue-specific regulation. The similarities in the size and
pattern of expression of FMO2 mRNAs of humans and other species of
mammals indicate that the human FMO2 gene has suffered no
mutations that affect either the expression of the gene or the
processing or stability of the corresponding mRNA.
Heterologous expression studies showed that the loss of 64 amino acid
residues from the carboxyl terminus of human FMO2 had no effect on
targeting of the protein to the membranes of the endoplasmic reticulum,
but abolished its catalytic activity. This is in accord with
site-directed mutagenesis studies of rabbit FMO2 (55, 56), which showed
that, although the removal of up to 26 amino acid residues from the
carboxyl terminus of the enzyme had no effect on catalytic activity, a
deletion of 40 or more residues inactivates the enzyme. It is possible
that the truncated human FMO2 has some other, unidentified, catalytic
activity. However, even if this were so, Williams et al.
(57) found that FMO2, although abundant in the lungs of rhesus macaque,
was undetectable, by Western blotting, in all but one of 29 human lung
samples. The results of our Northern blot hybridization and RNase
protection experiments demonstrate that FMO2 mRNA is moderately
abundant in human lung. Consequently, the absence of FMO2 protein in
this tissue cannot be due to deficient gene transcription or RNA
processing, or to instability of the mRNA. The relatively poor
conformity of the Kozak region of the human FMO2 mRNA to the
consensus sequence may compromise the translational efficiency of the
mRNA. However, this seems unlikely as the FMO2 of rhesus macaque,
which is abundant in the lungs (23, 57), is encoded by a mRNA that
contains a Kozak region that, apart from a single base difference at a noncritical position, is identical to that of the human mRNA. A
more likely explanation for the lack of pulmonary expression of FMO2 in
man is that, owing to the lack of 64 residues from its carboxyl
terminus, the truncated polypeptide is unable to fold correctly and is
thus detected by cellular surveillance systems, such as the ubiquitin
pathway (58), and rapidly degraded.
The absence of FMO2 in human lung is in marked contrast to the
situation in all other species of mammals investigated, in which it
represents the major, if not only, form of FMO in adult lung (10, 11,
20-23). In humans, the only member of the FMO family present in adult
lung is FMO5 (31, 59). However, as FMO5 has a very restricted substrate
range that is quite distinct from that of any other FMO (18, 60), it
would be unable to substitute for FMO2. The lung plays a significant
role in the metabolism of certain foreign compounds (61, 62). Although the pharmacological or toxicological significance of the absence of
FMO2 in human lung remains to be established, it is clear that caution
should be exercised when extrapolating pulmonary drug metabolism data
from experimental animals to man if an FMO-mediated metabolic pathway
is suspected.
Recent reports (63, 64) have indicated the presence in human brain of
an FMO that is immunoreactive with, and catalytically inhibited by,
antibodies raised against rabbit FMO2. However, as we have demonstrated
here, almost all individuals have two FMO2X472 alleles and
would thus express a truncated form of the enzyme, which would be
nonfunctional and, quite likely, rapidly degraded. The FMO detected by
these workers is therefore unlikely to be FMO2. This is supported by
the observation that it is able to effectively catalyze the
N-oxidation of the non-FMO2 substrate imipramine (63, 64).
The precise identity of this FMO is thus unclear.
Our results indicate that the FMO2X472 allele, which encodes
a truncated, nonfunctional protein, arose as the result of a point
mutation that occurred in the human lineage sometime after the
divergence of the Homo and Pan clades took place some 4-5 million
years ago and has subsequently spread to attain a frequency of close to
100% in the present day human population. FMO2 thus represents a very unusual case of a gene that has become non-functional in humans but not in other primates. The average time (in generations) for fixation of a neutral mutation is approximately 4N,
where N is the effective population size (65). For humans,
N has been estimated to have been approximately 10,000 for
most of the last 1 million years (66). Assuming a generation time of
about 15 years, the average time required for fixation of a neutral
mutation in humans is thus 4 × 10,000 × 15 = 6 × 105 years. The FMO2X472 allele could therefore
have become virtually fixed in the human population merely due to the
effects of random genetic drift on a neutral mutation, i.e.
one that conferred no, or very little, selective advantage or
disadvantage. An alternative, more intriguing, possibility is that the
nonsense mutation may have conferred an evolutionary advantage,
resulting in a rapid spread of the mutant allele due to a directional
selective sweep.
We thank R. Philpot for authentic rabbit FMO2
and anti-(rabbit FMO2) serum, K. Reid for the human lung cDNA
library, and M. Yacoub for human lung samples.
*
This work was supported by grants from the Wellcome Trust
(036025 and 045229).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y09267.
§
Present address: Dept. of Pharmacy, King's College London, Manresa
Road, London SW3 6LX, UK.
The abbreviations used are:
FMO, flavin-containing monooxygenase; ORF, open reading frame; pBS, pBluescript; PCR, polymerase chain reaction; RACE, rapid amplification
of cDNA ends; bp, base pair(s); kb, kilobase pair(s).
2
C. T. Dolphin, R. L. Smith, E. A. Shephard, and I. R. Phillips, manuscript in preparation.
The Flavin-containing Monooxygenase 2 Gene (FMO2)
of Humans, but Not of Other Primates, Encodes a Truncated,
Nonfunctional Protein*
§,,
,,
Laboratory of Molecular Biology, Department
of Biochemistry, Queen Mary and Westfield College, University of
London, London E1 4NS, United Kingdom, the ¶ Department of
Biochemistry and Molecular Biology, University College London,
London WC1E 6BT, United Kingdom, and ** Molecular Toxicology, Imperial
College School of Medicine, London W2 1PG, United Kingdom
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
T transition in codon 472, is not present in the
FMO2 gene of closely related primates, including gorilla
and chimpanzee, and must therefore have arisen in the human lineage
after the divergence of the Homo and Pan clades. Possible mechanisms
for the fixation of the mutation in the human population and the
potential significance of the loss of functional FMO2 in humans are discussed.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
80 °C. RNA concentration was determined from
absorbance at 260 nm. Human genomic DNA was isolated from whole blood
by the method of Lahiri and Nurnberger (35) and from solid tissue by
use of a commercial DNA isolation kit (Nucleon Biosciences, Coatbridge, Scotland). Gorilla and chimpanzee genomic DNA was isolated by the
method of Blin and Stafford (36) from post-mortem tissue samples.
-32P]dCTP (800 Ci/mmol, Amersham International,
Amersham, UK), and used to screen an adult human lung cDNA library
constructed in 
gt11 (gift of Dr. K. Reid) plated at a density of
1.5 × 105 plaque-forming units per 20 × 20-cm
plate (Nalge Nunc, Hereford, UK). Duplicate filters were prehybridized,
hybridized, washed, and subjected to autoradiography as described
previously (30). Because of loss of the EcoRI cloning sites
during library construction, insert DNAs could not be excised
from positive phage clones by restriction digestion and were
therefore recovered by PCR as described previously (30).
80 °C with an
intensifying screen.
0.1. For expression, 100 ml of
T. ni cells, grown to a density of 1 × 106 cells/ml in a 500-ml Erlenmeyer flask, were infected
with virus at a multiplicity of infecton of 7 and incubated by shaking
at 130 rpm in an orbital shaker (New Brunswick Scientific (UK) Ltd., Hatfield, Herts) at 28 °C for 60 h. Cells were pelleted,
resuspended in 30 ml 0.154 M KCl, 50 mM
Tris-HCl (pH 7.4), 0.2 mM phenylmethylsulfonyl fluoride,
and subjected to two 30-s bursts of sonication (Dynatech, model 150) on
ice. The cell lysate was centrifuged at 10,000 × g for
15 min at 4 °C. The microsomal fraction was obtained by centrifugation of the resulting supernatant at 100,000 × g for 1 h at 4 °C. Microsomal pellets were
resuspended in 5 ml of 0.154 M KCl, 10 mM HEPES
(pH 7.5), 1 mM EDTA, 20% (v/v) glycerol, and stored in
aliquots (150 µl) at 80 °C until use. Protein concentration was
determined by the BCA method (Pierce & Warriner (UK) Ltd., Chester, UK)
using bovine serum albumin as standard.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (18K):
[in a new window]
Fig. 1.
Strategy for sequencing cDNAs
encoding human FMO2. A representation of human FMO2 cDNA is
shown (top) relative to the locations of four partial-length
cDNA inserts recovered from the clones, L3, L8, L2, and L1, the
5'- and 3'-RACE-PCR products and the full-length cDNA, FMO2/2,
obtained by reverse-transcription PCR. FMO2/2 and the 5'-RACE-PCR
products were fully sequenced on both strands, whereas the
partial-length clones and the 3'-RACE-PCR products were only partially
sequenced. The locations of the 5'- and 3'-untranslated regions
(solid lines), the truncated ORF terminated by
the premature translational stop signal at codon 472 (shaded
box), and the extended ORF of 535 residues generated by
mutagenesis (open box) are indicated.
Arrows represent the extent and direction of sequencing
runs. Reactions were primed with either universal M13 primers
(vertical line) or gene-specific oligonucleotides
(closed circle). Restriction sites used to obtain
fragments for subcloning of FMO2/2 are indicated.
View larger version (96K):
[in a new window]
Fig. 2.
Nucleotide sequence of a cDNA encoding
human FMO2 and the deduced amino acid sequence. The translational
initiation codon, the premature stop signal at codon 472 and the second
in-frame stop signal located further downstream are boxed.
Annealing sites for oligonucleotides used to prime the amplification of
the full-length cDNA (solid arrows) and the
pairs of nested gene-specific oligonucleotide primers used in the 5'-
and 3'-RACE-PCRs (dashed arrows) are indicated.
The deduced amino acid sequences for both the truncated and extended
open reading frames are shown underneath the nucleotide sequence.
View larger version (24K):
[in a new window]
Fig. 3.
Sequence of a region of the FMO2
gene of human (A) and chimpanzee
(B). A section of the gene was amplified by PCR from
genomic DNA and sequenced directly. Nucleotide and deduced amino acid
sequences are shown to the right of each sequencing gel.
Nucleotides that differ between the two species are boxed.
The positions on the gel of bands corresponding to the variant
nucleotides are indicated by arrowheads.
View larger version (32K):
[in a new window]
Fig. 4.
Southern blot analysis of human genomic
DNA. Genomic DNA was digested with EcoRI (lane
1) or HindIII (lane 2) and the filter
hybridized with the cDNA insert of FMO2/2. Sizes of hybridizing
fragments, indicated by arrows, are given in kilobase
pairs.
3 position (the A of
the AUG codon is designated +1), as pyrimidines at this position are
rarely associated with functional initiation codons (48, 49). However,
the sequence contains a G at the +4 position, which has been shown
experimentally (50) to compensate for the otherwise unfavorable
presence of a pyrimidine at 3. The remaining 5'-untranslated sequence
of the mRNA contained no additional AUG triplets which might serve
as alternative translational initiation codons (Fig. 2). The region
flanking the initiation codon of the FMO2 mRNA of cynomolgus
macaque, determined from the sequence of a 5'-RACE-PCR product (data
not shown), also has a poor match to the consensus Kozak sequence, as
it differs from the human sequence at only the 4 position, at which
an A replaces a G. In contrast, the Kozak regions of mRNAs encoding
FMO2s of rabbit, GAGACGAUGG, and guinea pig,
GGGGCAAUGG, conform better to the consensus and contain a
purine at the important 3 position.
View larger version (25K):
[in a new window]
Fig. 5.
Northern blot analysis of lung RNA.
Samples of total RNA isolated from the lungs of three humans
(lanes 1-3) and a single cynomolgus macaque (lane
4) were hybridized to the cDNA insert of FMO2/2. The position
of RNA markers (28, 23, 18, and 16 S) (Bio-Rad) are indicated by
arrows and their sizes are given in kilobase pairs.
View larger version (20K):
[in a new window]
Fig. 6.
RNase protection assays of FMO2 mRNA in
human tissues. A, representative autoradiogram. An
antisense probe for FMO2 mRNA was hybridized with 15 µg of tRNA
(t) or of total RNA isolated from adult kidney (lane
1), adult lung (lane 2), adult liver (lane
3), fetal kidney (lane 4), fetal lung (lane
5), or fetal liver (lane 6). The position of the
protected RNA species is indicated by an arrow and sizes of
selected standards (1-kb ladder, Life Technologies) are indicated in
nucleotides. P, undigested probe. B,
quantification of FMO2 mRNA in adult and fetal human lung.
Concentrations of FMO2 mRNA in total RNA isolated from each of four
adult (shaded bars) and four fetal
(hatched bars) lung samples were determined by
RNase protection.
View larger version (11K):
[in a new window]
Fig. 7.
Analysis of heterologously expressed human
FMO2. A, immunoblot of subcellular fractions of
T. ni cells. The microsomal fraction of noninfected cells
(200 µg, lane 1), and of cells infected with AcNPV (200 µg, lane 2), AcFMO2X472 (100 µg 
1.4 pmol,
lane 5) or AcFMO2Q472 (100 µg 1.9 pmol,
lane 6), and the cytosolic fraction of cells infected with
AcFMO2X472 (200 µg, lane 3) or AcFMO2Q472 (200 µg,
lane 4), together with 1.0 pmol of a recombinant rabbit FMO2
(lane 7), were analyzed by Western blotting. FMO2s were
detected through the use of goat anti-(rabbit FMO2) serum and a rabbit
anti-goat IgG-alkaline phosphatase conjugate. The positions and sizes
(in kDa) of molecular mass markers are indicated. B, time
course of methimazole oxidation-dependent
nitro-5-thiobenzoate oxidation. Methimazole oxidation, monitored
indirectly as the time-dependent difference in absorbance
at 412 nm, was catalyzed by microsomal membrane proteins isolated from
noninfected T. ni insect cells (×------×, 420 µg), or from cells infected with AcNPV (
------, 300 µg),
AcFMO2X472 (
------, 555 µg 8.0 pmol) or AcFMO2Q472, in
the absence (
------, 540 µg 10.3 pmol) or presence
(
------, 540 µg 10.3 pmol) of 100 mM
Mg2+.
27 min
1). In the presence of 100 mM
Mg2+, a known effector of FMO2 (7), this was increased
approximately 6-fold to 2.9 nmol/min/mg microsomal protein
(kcatapp 155 min1). In contrast, microsomal membranes isolated from
noninfected insect cells, or from cells infected with either AcNPV or
AcFMO2X472, failed to catalyze methimazole oxidation. Thus, although
the full-length human FMO2 that we have produced can catalyze the
S-oxidation of methimazole and respond to Mg2+
in similar ways to FMO2s of other mammals, the truncated form of the
protein encoded by the major FMO2 allele of humans appears to be catalytically inactive.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of an Overseas Research Studentship from the
Committee of Vice-Chancellors and Principals of the United Kingdom.
To whom correspondence should be addressed: Dept. of
Biochemistry, Queen Mary and Westfield College, University of London, Mile End Road, London E1 4NS, UK. Tel.: 44 171 982 6338; Fax: 44 181 983 0531; E-mail: I.R.Phillips{at}qmw.ac.uk.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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