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J Biol Chem, Vol. 274, Issue 46, 32613-32618, November 12, 1999
From the Division of Population Science, Fox Chase Cancer Center,
Philadelphia, Pennsylvania 19111
Human methylenetetrahydrofolate reductase
(MTHFR, EC 1.5.1.20) catalyzes the reduction of
5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate.
5-Methyltetrahydrofolate is a major methyl donor in the remethylation
of homocysteine to methionine. Impaired MTHFR can cause high levels of
homocysteine in plasma, which is an independent risk factor for
vascular disease and neural tube defects. We have functionally
characterized wild-type and several mutant alleles of human
MTHFR in yeast, Saccharomyces cerevisiae. We
have shown that yeast MET11 is a functional homologue of
human MTHFR. Expression of the human MTHFR cDNA in a
yeast strain deleted for MET11 can restore the strain's
MTHFR activity in vitro and complement its methionine
auxotrophic phenotype in vivo. To understand the domain
structure of human MTHFR, we have truncated the C terminus (50%) of
the protein and demonstrated that expressing an N-terminal human MTHFR
in met11 Elevated plasma homocysteine levels, or hyperhomocysteinemia, is
an independent risk factor for vascular disease and neural tube defects
(1, 2). In humans, homocysteine can be metabolized in one of two ways,
transsulfurated to form cysteine or remethylated to form methionine.
Disturbance in either of these pathways can lead to an accumulation of
homocysteine in plasma.
Methylenetetrahydrofolate reductase
(MTHFR)1 is a critical enzyme
in the remethylation pathway. It catalyzes the formation of a methyl
donor, 5-methyltetrahydrofolate (5-methyl-THF), for converting
homocysteine to methionine. Human MTHFR gene has been localized to 1p36.3. It encodes a protein of 656 amino acids, with a
predicted molecular mass of 74.5 kDa (3). The enzyme is a flavoprotein
that utilizes NADPH as an electron donor and FAD as a cofactor in
mammalian cells (5). Human and other eukaryotic MTHFR exist as
homodimers, whereas Escherichia coli MTHFR is a homotetramer
(6, 7). Porcine MTHFR has been shown to be allosterically inhibited by
S-adenosylmethionine (AdoMet) (5, 8). This inhibition was
proposed to play a role in preventing the depletion of
methylenetetrahydrofolate in cells (5).
Mutations in human MTHFR are known to cause clinical MTHFR
deficiency, an autosomal recessive disorder (4). The clinical symptoms
of patients with MTHFR deficiency are highly variable including
developmental delay, motor and gait abnormalities, seizures, and
premature vascular disease (9). The reductase activity from cultured
fibroblasts of patients with severe MTHFR deficiency ranges from 0 to
20% of the control (10). The severity of the disease tends to
correlate with the residual MTHFR activity (9). Several mutant alleles
of MTHFR have been identified in patients with MTHFR
deficiency (10, 11). In addition, two common polymorphisms in
MTHFR, C677T (A222V) and A1298C (E428A), have also been
described (12, 13). The C677T variant has an allele frequency of 35% in the North American population, with about 12% of the population being homozygous and 40-45% heterozygous for the mutation (14). The
homozygotes appear to have a slightly increased risk of coronary heart
disease and of giving birth to children with neural tube defects (15,
16). The A1298C mutation has an allele frequency of 33%. Neither
homozygosity nor heterozygosity of this allele is associated with high
plasma homocysteine levels or lower plasma folate concentration (13).
However, this allele is reported to interact with the C677T allele in
trans to cause reduced MTHFR activity and decreased plasma
folate level, which could be a genetic risk factor for neural tube
defects (13).
So far human MTHFR and its mutant alleles have not been
individually expressed and characterized at molecular levels. We have taken advantage of the yeast, Saccharomyces cerevisiae, as a
model organism to characterize wild-type and mutant alleles of
MTHFR. This organism serves as a good model system for
studying human biology because of the evolutionary conservation of
human and yeast genes. Over 30% of yeast open reading frames share
significant sequence similarity with known human genes, and more than
70 human cDNAs have been shown to complement yeast mutations (17).
This structural and functional conservation has allowed the functional characterization of human genes in yeast and identification of mutations in human genes. Some examples of characterizing mutations and
polymorphisms of human genes in yeast include p53 (18), cystathionine
In this paper we describe the development of a yeast system for
functionally and structurally characterizing human MTHFR, and we show the utility of the yeast system in both mutation analysis and structure-function analysis.
Strains
E. coli strain XL-1 Blue was used for all DNA
manipulations. Yeast strains used were as follows: W303-1A, Mat
a, ade2-1, can1-100, ura3-1, leu2-3, 112, trp1-1,
his3-11,15 and XSY3-1A, Mat a, ade2-1, can1-100,
ura3-1, leu2-3,112, trp1-1, his3-11,15, met11 Plasmids
phMV2.1--
Human MTHFR cDNA was obtained from a
human cDNA library (27) by PCR amplifications using Klentaq
(CLONTECH). The final PCR product containing the
entire coding region of MTHFR was ligated to PCR 2.1 (Invitrogen). The amplified cDNA carries the common C677T mutation.
pC677T--
An XhoI/KpnI (about 1.8 kb)
fragment containing the entire MTHFR coding region from
phMV2.1 was isolated and ligated to
SalI/KpnI-digested pBBP-GAL (28). This generates
a plasmid carrying the 5' end HA (hemagglutinin A) epitope-tagged MTHFR
open reading frame under the control of the inducible yeast
GAL1 promoter and the yeast URA3 gene as a
selectable marker.
phMTHFR--
The C677T mutation in phMV2.1 was reverted to
wild-type (C-677) by site-directed mutagenesis using the Transformer
Mutagenesis kit (CLONTECH). A 20-bp oligonucleotide
with the altered base in the middle was used as the mutagenic primer.
The resulting plasmid, phMA2.1, was digested with KpnI and
XhoI, and a 1.8-kb DNA fragment containing wild-type MTHFR
cDNA (C677) was gel-purified. To incorporate the wild-type cDNA
into the expression plasmid, the yeast gap repair technique was used. A
1.8-kb DNA fragment from phMA2.1 was co-transformed into XSY3-1A
strain with an SmaI (partial)/EcoNI-linearized
pC677T with a region containing T677 being removed. The transformants
carry a gap repaired phMTHFR identical to pC677T except C-677
(wild-type).
pG164C, pT980C, pC1141T--
These plasmids were generated using
the same strategy as above. Essentially, phMV2.1 was mutated at the
desired positions by site-directed mutagenesis. The mutagenic primers
are all 20-mers with the altered nucleotide in the middle. The final
products were sequenced to confirm the incorporation of the mutations. These plasmids were then digested with XhoI and
KpnI. The 1.8-kb fragments containing various mutations were
isolated and ligated to SalI/KpnI-digested
pBBP-GAL (28). These plasmids express HA-tagged MTHFR
variants from the yeast GAL1 promoter.
pG458T and pA1298C--
These two plasmids were generated
through PCR mutagenesis. Briefly, for pA1298C, primer sets
5'-GCCATGGTGAACGAAGCCAGAGGAA (forward)/5'-AAAGACACTTGCTTCACTGG
(reverse) and 5'-CCAGTGAAGCAAGTGTCTTT (forward)/5'-TCATGGAGCCTCCGTTTCTCTCGC (reverse) were used to
generate two PCR fragments containing A1298C mutation. These two
fragments were than co-transformed with a linearized gap repair vector
into XSY3-1A. The gap repair vector is essentially identical with
phMTHFR except most of the MTHFR coding region has been
removed. The yeast transformants carry the pA1298C plasmid. This
plasmid put the HA-tagged A1298C allele under the control of the yeast
GAL1 promoter. pG458T was constructed the same way. Only two
PCR primers containing the G458T mutation were used.
pNhMTHFR--
This plasmid was constructed to express the
N-terminal MTHFR. A DNA fragment encoding the N terminus was amplified
by PCR using primers 5'-GCCATGGTGAACGAAGCCAGAGGAA (forward) and
5'-GAAGTCATTGTCCACCAGGTTCAGGGGTAGGGGACGCCTGA (reverse).
The reverse primer contains a point mutation, G1059A, that
changes a Trp codon to an OPA. This DNA fragment was used to gap repair
as above. The resulting yeast transformants carry plasmids that express
HA-tagged N-terminal MTHFR under the control of the yeast GAL1
promoter.
Disruption of Yeast Met11
The chromosomal copy of MET11 in strain W303-1A was
disrupted using the one-step gene disruption method (29). The DNA
fragment used for disruption was generated by the fusion PCR (30). In this fragment, 200 bp of 3' and 5' MET11 flanking regions
were fused to the yeast TRP1 gene at each end. Briefly, a
PCR fragment was generated that contained the 5'-flanking region of
MET11 plus 20 bp at the 3' end homologous to the 5' end of
the yeast TRP1 gene. Another PCR fragment containing the
3'-flanking region and 20 bp at the 5' end complementary to the 3' end
of the TRP1 was also generated. These two DNA fragments and
the TRP1 gene were connected by PCR. PCR primers for the
5'-flanking region were 5'-CACCGTACGTGAGCACATG (forward) and
5'-GTCGTGACTGGGAAAACCCTGGCGCTGTGGTGGTTCTGTTTGC T (reverse).
For the 3'-flanking region, the primers were
5'-TCCTGTGTGAAATTGTTA TCCGCTCACAG (forward) and 5'-TTTCTGAATTGGATGAGTAC
(reverse). The fusion PCR product was gel-purified and transformed into
W303-1A. Ten Trp+ transformants were obtained, and their
chromosomal DNA was isolated for PCR analysis to verify that
TRP1 gene was integrated into the MET11 locus.
One of the ten transformants was confirmed to be
met11 Complementation Assay and Growth Curve Measurement
To test for growth, yeast cells were inoculated on SD-Met-Ura
plates and SG-Met-Ura plates. The plates were then incubated at
30 °C for 3-4 days.
For growth curve measurement, yeast cells were inoculated into
SG-Met-Ura liquid media and incubated at 30 °C overnight. The cultures were diluted to about 0.1 × 107 cells/ml the
next day. The number of cells in 1 ml of media was counted using a
hemocytometer every 2 h.
Yeast Extract Preparation and Immunoblot Analysis
Yeast extracts and immunoblot analysis were done essentially as
described (31). Monoclonal antibody 12CA5 (against HA) was purchased
from Roche Molecular Biochemicals, and alkaline phosphatase-conjugated goat anti-mouse secondary antibody was from Bio-Rad. Color developing reagents, nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, were from Sigma.
MTHFR Enzyme Activity Assay
The assay is described by Rosenblatt and Erbe (32). The assay
measures the reverse reaction that MTHFR catalyzes under physiological conditions. Briefly, 250 µg of extract was used for each reaction. The reaction mix also contained 0.18 M potassium phosphate,
pH 6.3, 3.6 mM menadione bisulfite, 1.4 mM
EDTA, 7.2 mM ascorbic acid, 178 µM flavin
adenine dinucleotide, 420 µM 5-[14C]MeTHF.
The reaction temperature was 37 °C, and the reaction time was 1 h. For heat treatment, the reaction mixes without
5-[14C]MeTHF were heated 50 °C for 20 min.
5-[14C]MeTHF was then added back to the mix and proceeded
as the samples without heat treatment. The 5-[14C]MeTHF
(barium salt) was from Amersham Pharmacia Biotech. All other chemicals
were from Sigma.
Yeast MET11 Encodes MTHFR--
To characterize human
MTHFR in yeast, a strain lacking endogenous MTHFR
was constructed. We searched for yeast proteins with sequence
similarities to human MTHFR in the Saccharomyces Genome Data
Base. Protein sequence alignments showed that a polypeptide encoded by
yeast MET11 shared the highest degree of identity with the
human MTHFR (38.3%). To determine if MET11 encodes the
yeast MTHFR enzyme, we constructed a deletion allele of
MET11 in yeast strain W303-1A (see "Materials and
Methods"). We anticipated that yeast lacking MTHFR activity would
require methionine for growth since 5-MeTHF is required for the
synthesis of methionine from homocysteine. As expected, the resulting
met11 Human MTHFR Can Functionally Substitute for the Yeast MET11
Gene--
We next determined whether expression of human
MTHFR in the met11
Yeast met11 Complementation Analysis of MTHFR Alleles Found in Patients with
MTHFR Deficiency--
To detect biological consequences caused by
mutations in human MTHFR gene, we constructed four
expression plasmids carrying mutant alleles of MTHFR. Each
of the alleles contains a single MTHFR mutation (G164C,
G458T, T980C, and C1141T), previously found in patients with clinical
MTHFR deficiency (see Table I) (11). These mutations were engineered into MTHFR cDNA by either
site-directed or PCR mutagenesis (see "Materials and Methods"). The
cDNA fragments were then cloned into a yeast expression vector
individually (see "Materials and Methods"). The resulting plasmids
are identical to phMTHFR, except each carries a mutant allele. The
plamids were then transformed into the met11
We ascertained that MTHFR proteins were expressed from the mutant
MTHFR alleles by immunoblot analysis of yeast whole cell extracts using the anti-HA antibody. The extracts were made from yeast
cells grown in galactose medium to induce the expression of the alleles
(see "Materials and Methods"). Immunoblot analysis of the extracts
indicates that all the alleles were expressed at a similar level to the
wild type (see Fig. 3).
We next examined the ability of the mutants to complement the
auxotrophic phenotype of the met11
However, one of the alleles tested, C1141T, was able to
complement the methionine auxotrophic phenotype of the
met11 Complementation Analysis of Two Common Variants of MTHFR--
We
next used this system to characterize two common polymorphisms found in
the human MTHFR, C677T and A1298C. We constructed plasmids
pC677T and pA1298C that express C677T and A1298C from the
GAL1 promoter, similar to phMTHFR (see "Materials and
Methods"). The two plasmids were transformed into
met11 Enzyme Activity in Extracts Made from Yeast Cells Expressing
Various MTHFR Alleles--
We then determined whether the ability of a
human MTHFR allele to complement the yeast growth phenotype
correlated with its enzymatic activity in vitro. We measured
the MTHFR activities using yeast whole cell extracts containing various
MTFHR mutants (see "Materials and Methods"). As a
negative control, MTHFR activity was also measured using an extract
made from cells grown in glucose media where the expression of an
allele was repressed. Since the gene product of the C677T allele is
thermolabile (12), we have also examined thermal stability of MTHFR
variants that display detectable reductase activity. As shown in Fig.
2, the mutant alleles that failed to complement the yeast growth
phenotype showed less than 7% of wild-type activity, whereas extracts
from cells carrying the complementing alleles, C677T, A1298C, and
C1141T, all exhibited high levels of enzyme activity. After heat
treatment (55 °C for 20 min), we found wild-type human MTHFR
retained about 40% of its original activity, whereas C677T lost almost
all its activity (Fig. 2). Alleles A1298C and C1141T retained about
40% of their enzyme activity similar to that of wild-type
MTHFR. Thus A1298C and C1141T are indistinguishable from the
wild type in their thermal stability and MTHFR activity in
vitro.
Catalytic Domain of Human MTHFR--
Our results show that A1298C
and C1141T mutations do not cause any dysfunction of the enzyme under
our assay conditions. One common feature of these two mutations is that
they are located in the C-terminal domain of the protein. Comparison of
human MTHFR and E. coli MetFp (encoding MTHFR in E. coli) revealed that the C terminus of human MTHFR was absent in
E. coli MTHFR (Fig. 4). This
suggested that the catalytic domain of human MTHFR was located in the N
terminus of the protein. To verify this, we constructed a plasmid
(pNhMTHFR) that expresses a truncated human MTHFR (residues 1-349).
Yeast met11 We have expressed and characterized wild-type and mutant human
MTHFR in S. cerevisiae. The work is based on the fact that human MTHFR can functionally substitute for the yeast
MET11 gene. Deletion of the chromosomal copy of
MET11 results in a haploid yeast strain becoming a
methionine auxotroph, due to the lack of MTHFR to produce 5-MeTHF for
methionine biosynthesis. Expression of wild-type human MTHFR in
met11 yeast cells restores MTHFR activity to the cells and
complements the methionine auxotrophic phenotype. However, mutant
alleles of human MTHFR with severely reduced enzyme activities cannot complement the growth phenotype of
met11 To characterize the effect of mutations in human MTHFR, six
previously described mutations were engineered individually into wild-type MTHFR cDNA. These included four rare mutations
G164C, G458T, T980C, and C1141T found in patients with severe MTHFR
deficiency and two common polymorphisms, C677T and A1298C (12, 13).
Mutation G164C was originally described in a patient who is a compound heterozygote for G164C and 249-1G/T, a 3' splice-site mutation. Enzyme
activity from crude fibroblast extracts shows the patient has about
1.6% MTHFR activity of the control (11). The G458T was found in a
homozygous patient with 4% MTHFR activity of the control (11). The
T980C and C1141T were found in an individual who is a compound
heterozygote of these two mutations alone with heterozygosity for
677C/C677T (11). The patient has about 2% MTHFR activity of the
control (11). Among the four suspected pathogenic alleles we tested,
G164C, G458T, and T980C did not complement the methionine auxotrophy
phenotype and show less than 7% MTHFR activity of the wild type
expressed in yeast. These results are in agreement with their
pathogenic effect on humans.
However, the C1141T allele did complement and displayed high levels of
MTHFR activity. This variant also had comparable thermal stability as
that of wild type, indicating that this mutation alone may not lead to
MTHFR deficiency in the patient. The cause of the MTHFR deficiency in
the patient is complicated by the presence of two other mutations
(T980C and C677T) in the patient's MTHFR. We have tested the
possibility that T980C is a dominant-negative allele. When yeast
extracts containing the gene products of T980C and wild-type
MTHFR were mixed together at various ratios, no reduction of
MTHFR activity was observed (data not shown). In addition,
co-expression of wild-type and T980C in met11 The common polymorphism C677T allele has been reported to be
thermolabile and retains 40-50% enzyme activity at normal temperature (14). The point mutation causes an alanine to valine substitution. Both
the heterozygous and the homozygous state of this mutation are
associated with reduced specific activity (12, 34, 35). The
corresponding mutation in E. coli MTHFR was shown to have reduced affinity for the cofactor FAD (33). We found that this allele
was able to complement the methionine auxotrophic phenotype of the
met11 The A1298C is another commonly occurring mutation with an allele
frequency of 33% (13). This mutation causes an amino acid substitution
of a glutamate into an alanine. However, this mutation is not known to
associate with either higher plasma homocysteine or a lower plasma
folate concentration (13). Our data show that A1298C can functionally
rescue the yeast growth phenotype, and the yeast extract containing
A1298C allele gene product retains high levels of MTHFR activity.
Unlike the C677T mutant, the A1298C has comparable thermal stability to
that of the wild-type enzyme. Similar to C1141T, A1298C, by itself,
does not cause obvious phenotype alterations in human and yeast,
suggesting that A1298C is a benign polymorphism.
It is interesting that the two mutant alleles (C1141T and A1298C) are
both located in the C-terminal domain. Based on the sequence similarity
between human MTHFR and E. coli MTHFR (MetFp), a hypothesis
stating that the N terminus contains the catalytic domain has been
proposed by Guenther et al. (33). To verify this we have
shown that expression of a C-terminal truncated human MTHFR in
met11 It has been reported previously that porcine MTHFR can be easily
cleaved with trypsin to produce two fragments of 36 and 39 kDa (6).
This limited proteolysis abolishes the inhibitory effect of AdoMet, but
the enzyme activity remains unchanged suggesting that one of the
domains functions as a regulatory region (6). Considering our data
indicating that the N terminus is the catalytic domain, it is likely
that the C terminus is the regulatory domain that allows AdoMet to
carry out the inhibitory effect. The C terminus may also be involved in
stabilizing the enzyme. We observed a major reduction of the amount of
this protein, when the C-terminally truncated MTHFR was expressed in
yeast. When E. coli MTHFR that naturally lacks the C
terminus was expressed under the same conditions, the protein level was
also low (data not shown). These results imply that the C-terminal
domain could be important for the protein stabilization in eukaryotes.
Interestingly, met 11 In conclusion, we have shown that yeast MET11 is a
functional homologue of human MTHFR. The
met11 We thank Drs. Elizabeth Henske, Cynthia
Keleher, and Kent Hunter for critical review of the
manuscript and Alex Shipman for helping to clone the human MTHFR
cDNA. We are grateful to Dr. David Rosenblatt for providing the
MTHFR assay protocol.
*
This work was supported by a grant from the W. W. Smith
Charitable Foundation and by National Institutes of Health Grant
HL57299.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 abbreviations used are:
MTHFR, methylenetetrahydrofolate reductase;
5-MeTHF, 5-methyltetrahydrofolate;
PCR, polymerase chain reaction;
kb, kilobase pairs;
bp, base pairs;
HA, hemagglutinin;
AdoMet, S-adenosylmethionine.
Functional Characterization of Human Methylenetetrahydrofolate
Reductase in Saccharomyces cerevisiae*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
yeast cells rescues the growth
phenotype, indicating that this region contains the catalytic domain of
the enzyme. However, the truncation leads to the reduced protein
levels, suggesting that the C terminus may be important for protein
stabilization. We have also functionally characterized four missense
mutations identified from patients with severe MTHFR deficiency and two
common missense polymorphisms found at high frequency in the general
population. Three of the four missense mutations are unable to
complement the auxotrophic phenotype of met11
yeast cells and show less than 7% enzyme activity of the wild type
in vitro. Both of the two common polymorphisms are able to complement the growth phenotype, although one exhibited thermolabile enzyme activity in vitro. These results shall be useful for
the functional characterization of MTHFR mutations and analysis
structure/function relationship of the enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-synthase (19), galactose-1-phosphate uridylyltransferase (20), and
the Wilson disease gene (21). In addition, yeast systems, due to their
genetic tractability, have proven useful in structure-function analysis
of human proteins. For example, yeast systems have been used to
identify functional domains of the human melatonin receptor (22), p53
(18), and cystathionine -synthase (23).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
::TRP1. Media preparation and
bacterial and yeast transformation were done according to standard
procedures (24-26).
::TRP1 (TRP1
integrated at MET11 locus). This strain, XSY3-1A, was used
for complementation studies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
::TRP1 strain, XSY3-1A, fails to grow in
media lacking methionine (see Fig. 1). To
confirm further that MTHFR activity was depleted in the
met11 (XSY3-1A) yeast cells, we compared the
enzyme activity of whole cell extracts made from the
met11 strain and the parental strain W303-1A
(see "Materials and Methods"). As shown in Fig.
2, extracts made from the
met11 (XSY3-1A) cells contain no detectable
MTHFR activity. The genetic and enzyme activity data both indicate that
MET11 encodes yeast MTHFR and that the MET11 gene
is essential for cell growth on media lacking methionine.
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Fig. 1.
Growth of yeast strains on SD-Met-Ura
(glucose) and SG-Met-Ura (galactose) plates. MET11
strain is W303-1A and met11 strain is XSY3-1A, which is
isogenic W303-1A except for the deletion of MET11. The
expressing plasmid indicated on the sectored circle is carried by the
XSY3-1A. The locations of these strains on the plate are indicated
correspondingly on the circle and are described in Table I.
The plates were incubated at 30 °C for 3 days.
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Fig. 2.
Enzyme activity of different alleles of human
MTHFR. About 250 µg of crude yeast extract was used
in each assay. For heat treatment, samples were incubated at 55 °C
for 20 min before the substrate was added to the reaction mixture. Heat
treatment was only performed on alleles with detectable reductase
activity. The reaction time was 1 h at 37 °C, and measurements
were done in triplicates.
yeast cells
could complement the methionine auxotrophic phenotype. We constructed a
yeast centromere plasmid, phMTHFR, carrying human MTHFR cDNA
controlled by the galactose-inducible yeast GAL1 promoter (see "Materials and Methods"). In the presence of galactose, this plasmid expresses wild-type human MTHFR with a hemagglutinin A (HA)
epitope at its N terminus. The HA epitope allowed us to detect the
protein by immunoblot analysis of whole cell extract with a monoclonal
anti-HA antibody, 12CA5. This plasmid was transformed into the
met11 yeast cells. As shown in Fig.
3, a doublet band was observed in the
extract made from cells grown in galactose media but not in the extract
from glucose-grown cells. The apparent molecular mass of the upper band
is about 78 kDa, which is very close to the predicted size (74.5 kDa)
for human MTHFR. The absence of the lower band in the extract of cells
grown in glucose media suggests that it is related to MTHFR protein.
This band could be either a proteolytic product or the result of
post-translational modification.
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Fig. 3.
Immunoblot of yeast whole cell extracts.
The extracts were made from yeast cells carrying different
MTHFR alleles. G indicates galactose media, and
D indicates glucose media. About 200 µg of extract was
loaded in each lane. Anti-HA antibody (12CA5) was used as primary
antibody and goat anti-mouse IgG was used as secondary antibody.
cells carrying phMTHFR were then
tested for growth on galactose and glucose media lacking methionine. As
shown in Fig. 1, expression of human MTHFR, induced by galactose,
supported the growth of met11 cells on media
lacking methionine. No growth was observed when the expression of human
MTHFR was repressed in a glucose media lacking methionine. In fact,
yeast met11 expressing human MTHFR grow as
well as the same strain expressing yeast MTHFR
(MET11). The ability of human MTHFR to complement the auxotrophic phenotype indicates that human MTHFR and
yeast MET11 encode functional homologues.
cells.
Plasmids carrying mutant MTHFR alleles
yeast
cells. As shown in Fig. 1, met11 cells
expressing three of the mutant alleles, G164C, G458T, and T980C, were unable to grow in either glucose (repressed) or galactose (induced) media lacking methionine, indicating that these cells lacked
MTHFR activity. Since immunoblot analysis (Fig. 3) showed that the
mutant proteins were present in the cells, we conclude that these
mutant proteins are non-functional. Our data show that the inability of
these mutants to complement met11 yeast cells
correlates with their pathogenic effects in humans.
cells and was phenotypically
indistinguishable from the wild-type MTHFR in yeast. It
should be noted that this mutation was found in a patient heterozygous
for C1141T, C677T, and T980C. Thus it is possible that
C1141T might be deleterious only in the context of these other
mutations (see "Discussion").
cells, and expression of the two alleles
was confirmed by immunoblotting with anti-HA antibody (see Fig. 3). As
shown in Fig. 1, these alleles were able to rescue the auxotrophic
phenotype of the met11 cells. This is not
unexpected for two reasons. First, human lymphocyte extracts from
individuals homozygous for C677T have 40-50% MTHFR activity of the
control (14), and those A1298C homozygotes have 62% MTHFR activity of
wild-type (13). Second, in our yeast cells these alleles were expressed
from the strong yeast GAL1 promoter, resulting in high
levels of MTHFR protein. This would elevate the reductase activity in
the cell and compensate for the low MTHFR activity of the mutant alleles.
cells carrying this plasmid were
able to grow on galactose medium lacking methionine but not on glucose
medium lacking methionine (see Fig. 5).
Further analysis (Fig. 6) showed those
yeast cells expressing the truncated MTHFR grew slower than those
expressing the full-length protein, suggesting that the truncated
protein was less active or the protein level was low. We then
determined MTHFR activity using extracts made from cells expressing
truncated MTHFR. The result in Fig. 2 shows that the C-terminal
truncated protein does have MTHFR activity but at a lower level
compared with the full-length protein. Immunoblot analysis, shown in
Fig. 3, indicated that cells expressing the C-terminal truncated MTHFR contain a significantly lower amount of protein, compared with cells
expressing wild-type MTHFR. This result suggests that the truncated
protein has lower stability, which might reflect a role of the C
terminus in stabilizing the protein.
View larger version (21K):
[in a new window]
Fig. 4.
Comparison of domain structures of human
MTHFR (hMTHFR), yeast MTHFR (yMTHFR), and E. coli MTHFR. The
mutations studied in this work are indicated in the diagram of hMTHFR
with amino acid changes in the parentheses. Homologous domains are
shaded with the same pattern.
View larger version (50K):
[in a new window]
Fig. 5.
Growth of yeast strains. Yeast strain
XSY3-1A carrying different plasmids was tested for growth on SD-Met-Ura
(glucose) and SG-Met-Ura (galactose) plates. The plates were incubated
at 30 °C for 3 days.
View larger version (11K):
[in a new window]
Fig. 6.
Growth rate of yeast strains. The growth
rate of yeast strain XSY3-1A expressing full-length or N-terminal MTHFR
were measured. The cultures were incubated in an orbital shaker at
30 °C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
yeast cells. Thus, the
met11 strain, XSY3-1A, provides an
experimental tool for functionally investigating the effect of
mutations in human MTHFR and revealing the domain structure of the
enzyme. For example, the catalytic domain of human MTHFR has been
proposed to reside in the N terminus, based on structural comparison
with the E. coli enzyme (33). By using the yeast system, we
show that the N terminus, 50% of the protein, is capable of
complementing the auxotrophic phenotype, confirming that the N-terminal
domain alone contains the catalytic site.
cells did not result in any impaired growth of the strain, even when
T980C was expressed at significantly higher levels than the wild type
(data not shown). These results do not support the dominant negative
hypothesis. However, it is possible that the T980C allele could
interact specifically with C677T and/or C1141T to cause the detrimental
effect on the MTHFR activity. This can be tested by our yeast system
should the exact genotype (cis/trans arrangement of the mutations) of
the patient become available. It is also conceivable that the C1141T
alteration is only a rare benign polymorphism but is genetically linked
to an undiscovered mutation. The yeast system offers a powerful tool in
analyzing the contribution made by each single mutation toward the
detrimental effect caused by a combination of mutations.
strain. The enzyme activity of crude
yeast extracts from cells expressing the C677T allele was about 50% of
the wild-type level and was extremely thermolabile, as seen in human
cells. Thus, our results are consistent with the clinical data that the
C677T alteration has a modest effect on enzyme activity.
yeast cells can complement the growth
phenotype. In fact, the two alleles (C1141T and A1298C) with mutations
located in the C terminus retain the ability to complement the growth
phenotype of met11 cells as well. In addition,
these two mutations do not result in thermolability of the enzyme,
unlike the C677T mutation that is located in the N terminus of the
protein. It is likely that the mutations in the C terminus do not
produce as severe a phenotype as the N-terminal mutations because they
do not affect the catalytic domain. Since E. coli MTHFR also
requires FAD as a cofactor, the lack of the C-terminal domain in
E. coli MTHFR argues that the C terminus of human MTHFR is
not required for FAD binding.
yeast cells expressing
N-terminal MTHFR grew significantly slower than the parental
MET11 strain (W303-1A) although the enzyme activity of N
terminus human MTHFR was only moderately less (78%) than that of
Met11p according to our in vitro assay using crude yeast
extracts. Unless this level of enzyme activity happens to be just below
a threshold, the growth rate of cells seems to depend on more than just
the reductase activity. This observation cannot be easily explained by
the AdoMet regulation or protein stability. The growth rate discrepancy
of the two strains suggests that the C terminus may be involved in
other regulatory functions in vivo.
yeast strain serves as model system for
characterizing the effects of sequence alterations in the human MTHFR
cDNA and understanding the domain structure of the enzyme. Our
findings should be helpful in understanding the molecular basis of
MTHFR deficiency and the domain structure of the enzyme; this in turn
could improve the strategy for treating the disorder.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Division of Population
Science, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA
19111. Tel.: 215-728-3030; Fax: 215-728-3574; E-mail:
wd_kruger@fccc.edu.
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