(Received for publication, March 22, 1995; and in revised form, June 26, 1995)
From the
Human lymphocyte methionine adenosyltransferase (HuLy MAT)
consists of heterologous subunits and
. The cDNA sequence of
the
subunit of HuLy MAT from Jurkat leukemic T cells was
identical to that of the human kidney
subunit and highly
homologous to the sequence of the extrahepatic MAT from other sources.
The 3`-untranslated sequence was found to be highly conserved,
suggesting that it may be important in regulating the expression of
MAT. The extrahepatic
subunit of MAT was found to be expressed
also in human liver, and no differences were found in the sequence of
the
subunit from normal and malignant T cells. The sequence of
two unspliced introns found in the cDNA clones from the Jurkat library
enabled us to isolate genomic clones harboring the human extrahepatic
subunit gene and to localize it to the centromere on chromosome
arm 2p, an area that corresponds to band 2p11.2.
Expression of the
subunit cDNA in Escherichia coli yielded two peptides
with the immunoreactivity and mobilities of authentic
/
`
subunits from HuLy. The K
of the
recombinant
subunit was 80 µM, which is 20-fold
higher than found for the (
`)
holoenzyme purified from leukemic lymphocytes and 4-10-fold
higher than found for the normal lymphocyte enzyme. The data suggest
that the
/
` subunits mediate the enzyme catalytic activity
and that the
subunit may be a regulatory subunit of extrahepatic
MAT.
Methionine adenosyltransferase (MAT; ()S-adenosyl-L-methionine (AdoMet)
synthetase, EC 2.5.1.6) catalyzes the formation of AdoMet from ATP and L-methionine(1, 2) . In mammals, two major
MAT isozymes have been described which differ with respect to tissue
distribution and kinetic properties (for review see (3) ).
Liver contains an isozyme unique to this organ, which has a high K
for L-methionine (S
of 0.2-0.5 mM) and is relatively insensitive to
product inhibition by AdoMet(4, 5) . This isozyme has
been studied most extensively in the rat, where it has been previously
designated the high K
form (6, 7) or the
isozyme (8) but is now
called MAT III(4) . The rat liver MAT III isozyme has been
purified to homogeneity and was found to exist as a homodimer of a
single hepatic
subunit (
) with a predicted mass
of 43.7 kDa but which migrates on polyacrylamide gel electrophoresis in
sodium dodecyl sulfate (SDS-PAGE) as a 48-kDa
protein(4, 9, 10, 41) . The
tetrameric form of this same subunit (
)
,
which is known as MAT I, has an ``intermediate'' K
for L-methionine (about
17-23 µM) (4, 5, 6, 11) and can be converted
to the dimeric form by treatment with lithium bromide(10) . Rat
MAT I and MAT III can be separated easily by hydrophobic chromatography
on phenyl-Sepharose columns(4) . Although human liver expresses
the mRNA that encodes the
subunit(12, 13) , which is highly homologous to
the rat
subunit(14) , the existence of a MAT
I and MAT III equivalent in human liver has yet to be determined. The
rat and human liver-specific
subunits are not
expressed in extrahepatic tissues.
Extrahepatic tissues contain MAT
isozymes that are very different from that of liver and are
characterized by low K values for L-methionine (3-20 µM) and by their strong
feedback inhibition by AdoMet(15, 16) . Such isozymes
have been partially purified from rat kidney(4, 8) ,
rat lens(17) , and human erythrocytes (15, 18) and completely purified from human
lymphocytes (19) and bovine brain(20) . The human
lymphocyte isozyme is strongly inhibited by
AdoMet(15, 16, 19) . The low K
isozyme is expressed in all tissues
including the liver(4, 7, 21) . However,
recent studies suggest that different tissues, or even cells from the
same tissue at different stages of development, can express different
forms of the extrahepatic
enzyme(3, 4, 7, 21) . In contrast to
the rat liver isozymes that represent different polymeric states of the
subunit (41) , the purified enzyme from human
leukemic lymphocytes and from bovine brain consists of heterologous
subunits, known as
(53 kDa) and
(38
kDa)(19, 20, 21, 22) . In human T
cells the
subunit appears to undergo some type of
post-translational modification to yield a 51-kDa protein called
`(19) . MAT from leukemic T cells has a native molecular
weight of 185,000 (19) and thus may have a possible subunit
structure of
`
, but since the molar ratio of
each subunit in the holoenzyme is not known, we will refer to this form
as (
`)
. In resting human T
cells and stage M
acute myelogenous leukemia cells, MAT
activity is primarily associated with a 68-kDa protein called
,
which is immunocross-reactive with the (
`)
form of MAT(21) . The
subunit can
exist alone or in association with
/
` and/or
(21) . The
subunit is unrelated to
/
` with
respect to peptide maps and immunoreactivity(19, 23) .
The cDNA of MAT subunit from human kidney has been cloned and
sequenced and was found to be 99% homologous to the rat kidney MAT
subunit protein(24) . The kidney
subunit and the
liver
subunit are clearly different; however, at the
protein level they are 85% identical. Inasmuch as studies of the MAT
protein expression in the kidney showed no expression of the
subunit that is expressed in lymphocytes and brain tissues, we
hypothesized that differences may exist in the sequence of the
subunit of MAT from different tissues and that these differences may
affect the association between the
and the
subunits of
extrahepatic MAT. The present study was undertaken to determine whether
differences exist between: (i) MAT
subunit from different human
extrahepatic tissues, namely kidney versus lymphocytes; and
(ii) MAT
subunit from normal and malignant tissues. We report
that the sequence of the MAT
subunit from human kidney and normal
or malignant lymphocytes is identical and that the gene for the
subunit of MAT is located on chromosome 2p11.2. We report also the
expression of the
subunit in Escherichia coli and
demonstrate that, although it is catalytically active in the absence of
subunit, its kinetics are different from the native
(
`)
form of MAT found in normal
and malignant lymphocytes.
For the random-primed and specific-primed
libraries, filters were prehybridized for 3-5 h at 42 °C in 6
NET, 4
Denhardt's solution, 0.05% pyrophosphate,
0.1% SDS, and 200 µg/ml nicked and denatured salmon sperm DNA.
Filters were hybridized overnight at 42 °C in the same solution
minus salmon sperm DNA with a probe consisting of a
P-labeled 36-mer oligodeoxynucleotide (oligo
see below), which was complementary to a previously determined
cDNA sequence. Following hybridization, filters were washed three times
for 20 min each with 6
NET, 0.1% SDS at 23 °C and then
twice for 5-10 min each at 45 °C in 2
NET, 0.1% SDS.
Plaques giving positive signals were purified by replating.
phage
DNA was prepared by the method of Chisholm(35) , and EcoRI inserts were subcloned in pT7T3 19U for DNA sequencing.
pBluescript SK
phagemids were obtained from
ZAP
derivatives by in vivo excision with helper phage as
recommended by Stratagene.
Figure 1:
Nucleotide sequences of cloned DNA. Panel A shows the cDNA for the /
` subunit of human
lymphocyte MAT. The sequence was determined from clones isolated from
three different Jurkat T cell libraries. The deduced amino acid
sequence is shown for the ORF. Nucleotides are numbered beginning at
the first position of the ORF. Amino acid positions are shown in parentheses. The positions of putative introns A and B are
shown as well as the HindIII site and a polyadenylation
signal. Shaded amino acid residues correspond to tryptic
peptide sequences that were chemically determined from purified
/
` subunit. Panel B shows the sequence of partial
intron A, which consists of 245 bp upstream of position 45 in isolate
HLy29.1, and the sequence of the 191-bp intron B, which is complete and
was found between positions 768 and 769 in isolate
HLy45.1.
Figure 2:
Orientation of cloned cDNA segments from
the human lymphocyte and human liver phage libraries. The nine
clones from the lymphocyte libraries are shown at the top, and
the two clones from the liver library, XHM4.1 and XHM4.2, are shown at
the bottom of the figure. A 245-bp putative partial intron
(intron A) is shown at the upstream boundary of isolate HLy29.1. A
191-bp complete putative intron (intron B) is shown within the sequence
of isolate HLy45.1. The scale begins at position 1 of the ORF
&cjs2110;. The cDNA spans a total of 2,693 bp, not including the
poly(A) region. The ends of all phage inserts contain EcoRI
sites (not shown), which were introduced during library construction.
The only internal EcoRI site was found in intron
B.
Ten clones were obtained from the
random-primed library, and one of these, designated HLy45.1, was
characterized in detail. It contained the sequence between positions
-94 and +847 but was also found to have an additional 191 bp
between positions +768 and +769. The sequences at the 5` and
3` junctions of this segment, CAG/GT and AG/GG, are characteristic for
an unspliced intron, which we have designated intron B ( Fig. 1and Fig. 2). The single EcoRI site in this
putative intron is unlikely to be a cloning artifact because it does
contain the adjacent NotI site present in the adapter used for
cloning. The final cDNA sequence of 2,787 bp contains a 395-codon ORF
encoding a 43.6-kDa polypeptide in which all five of our chemically
determined tryptic peptide sequences are present (Fig. 1). The
putative start codon at position +1 is found within the sequence
CAACATGA, which is identical to the -4 to +4 consensus
initiator sequence C(A/G)CCATGG (37) at 6 of 8 positions. The
Met-Asn sequence that begins this ORF would be expected to be
acetylated in eukaryotes to give N-acetyl-Met-Asn(38) , which would explain our finding
of a blocked amino terminus for the purified /
` peptide. The
sequence of the HuLy MAT was identical to that of human kidney
MAT(24) . However, the Jurkat library provided additional
3`-untranslated sequence totaling 1,505 bp, not including the poly(A)
tail and including the polyadenylation signal sequence AATAAA (39, 40) beginning 23 bp upstream of the poly(A)
region.
No sequence differences were noted in the various isolates
obtained from the three Jurkat T cell libraries. The two clones
obtained from the adult human liver library corresponded to positions
830-2068 (XHM4.1) and positions 854-2236 (XHM4.2) and were sequenced
for about 500 bp from both ends. The sequence of the XHM4.1 and the
XHM4.2 fragments was identical to the subunit cDNA sequence from
Jurkat cells. An A and a T located at positions 837 and 846 of human
lymphocyte and kidney cDNA were replaced by a G and a C in the XHM4.1
and XHM4.2 fragments from human liver. This is most likely a cloning
artifact related to a 5-methyl cytosine spontaneous mutation in E.
coli strains that are able to perform the methylation reaction. In
these strains the mutation creates a GC to AT transition. However,
these base substitutions, which occurred in the third positions of
codons 278 and 281, gave synonymous codons for glycine and
phenylalanine, respectively. XHM4.1 contained a C at position 2222 in
the 3`-untranslated region in place of the T found in human lymphocyte
cDNA. These data show that the extrahepatic
subunit of MAT is
also expressed in human liver.
Figure 3:
Chromosomal localization of the human
subunit of MAT. Hybridization was done using P1 clone 2007 that
harbors the
subunit gene. The gene was localized to chromosome
arm 2p, an area that corresponds to band 2p11.2. Chromosome 2
centromere-specific probe (D2Z1) was labeled with biotin dUTP and
cohybridized with the P1 clone labeled with digoxigenin dUTP. Specific
hybridization signals were detected using fluorescein-conjugated
antidigoxigenin and Texas red avidin followed by counterstaining with
4,6-diamidino2-phenylindole.
Figure 4:
Construction of expression vector pMAT355.
A regulatory region containing the tac promoter with
downstream Shine-Dalgarno sequences (SD) (42) was
derived from pDR540 as a HindIII/BamHI fragment and
inserted into pT7T3 19U. Isolate HLy31.1 provided a 1.3-kb fragment
containing the coding region for the /
` subunit of human
lymphocyte MAT extending from position 145 (relative to the translation
start site) to a position 282 bp downstream of the translation
terminator. The first 144 bp of the ORF were obtained by PCR with
template from isolate HLy45.1 and contained an upstream BamHI
site suitable for ligation immediately downstream of the regulatory
region. The lower portion of the figure shows the sequence of the
regulatory region containing the tac promoter (-35 and
-10 regions) with Shine-Dalgarno sequences followed by the first
three codons of the ORF for the
subunit.
The lacI strain
NM522, containing either pMAT355 or pMAT356, was grown on 2
YT
medium to a density of about 4
10
cells/ml, and
IPTG was added at 0.5 mM. Control cultures were treated in the
same way but did not receive IPTG. After an additional 2.5 h of growth,
cells were harvested, and the expression of the
subunit was
examined. Immunoblots of an SDS-PAGE gel probed with a polyclonal
antibody to highly purified human lymphocyte MAT holoenzyme (23) showed two bands in extracts from IPTG-induced NM522
(pMAT355), which migrated almost identically with the 53-kDa
band
and the 51-kDa
` band of the human lymphocyte MAT holoenzyme (Fig. 5). These two bands were not present in the extract from
uninduced NM522 (pMAT355). By comparison with standards and by assuming
equivalent immunoreactivity, we estimated that the
subunit
protein accounted for approximately 6% of the total protein in induced
NM522 (pMAT355). No band corresponding to the 38-kDa
subunit was
noted in bacterial extracts.
Figure 5:
An
immunoblot of the human lymphocyte MAT /
` subunit expressed
in E. coli. Portions of crude extracts from NM522 (pMAT355)
that had been grown with and without the inducer IPTG were analyzed by
SDS-PAGE together with a human lymphocyte MAT
/
` standard,
and immunoblots were prepared with polyclonal rabbit antiserum against
the highly purified lymphocyte enzyme(23) . The quantities
shown represent the amounts of total protein applied to the gel from
either the crude extracts or a preparation of human lymphocyte MAT that
was approximately 90% pure. The positions of the
,
`, and
subunits of the human lymphocyte enzyme are indicated and migrate
with apparent molecular masses of 53, 51, and 38 kDa. No bands were
noted with the extract from uninduced NM522 (pMAT355). The
subunit band was not detected in the E. coli extracts.
The existence of multiple isozymes of MAT in mammalian
tissues is well established (for review see (3) and (43) ). Whereas the liver-specific enzyme appears to be a
homodimer or tetramer of a single subunit(41) , the extrahepatic enzyme that is expressed
in all tissues appears to consist of nonidentical
and
subunits. Several complementary pieces of evidence indicate that the
cDNA we have characterized from Jurkat T cell libraries is that of the
subunit of human lymphocyte MAT. First, the deduced amino acid
sequence is identical to that of the
subunit from human kidney (24) and highly homologous to that of other MATs (see below).
Second, the sequence contains the five tryptic peptide sequences that
were chemically determined from purified, native
/
` subunit.
Third, expression of this cDNA in E. coli gave two peptide
bands that comigrated with authentic
and
` subunits in
SDS-PAGE and reacted with antiserum to human lymphocyte MAT. The fact
that the
subunits of MAT in human lymphocytes, kidney, and liver
are identical suggests that the sequence of this subunit is the same in
all tissues.
Several studies have reported that MAT activity is
elevated in malignant
cells(6, 22, 44, 45, 46) .
Furthermore, Liau et al.(6, 7, 47, 48) demonstrated the
presence of an altered form of MAT in tumors. In this study we show
that the sequence of the subunit is the same in normal and
malignant lymphocytes. These findings do not rule out the possibility
that the expressed protein may be altered post-translationally in
malignant lymphocytes. However, immunoblot analysis of cell extracts
shows a very similar pattern for leukemic and normal activated T
lymphocytes(21, 22) . Additional studies are needed to
determine whether differences exist in the regulation and expression of
MAT in normal and malignant tissues. These studies may facilitate the
development of specific inhibitors that specifically block MAT in
malignant tissues(44, 49, 50, 51) .
Comparisons of the deduced amino acid sequence for the human
lymphocyte or kidney subunit with those of non-mammalian MAT (not
shown) revealed identities of 49% with the E. coli enzyme(52) , 68% with the Aradiposis thaliana enzyme(53) , and 69% with the two yeast
enzymes(54, 55) . Identities were found at 84% of
positions with the liver
subunit from human, rat, and
mouse(13, 14, 56) , and at 98.7% of positions
with the rat kidney enzyme(57) . This homology at the molecular
level confirms previous reports showing that the
subunit of human
lymphocytes is immunocross-reactive with MAT from E. coli,
yeast, and rat liver(23) . Therefore, earlier reports by Abe et al.(9) , which failed to detect cross-reactivity
between the hepatic and the kidney enzymes in rat, may represent
differences in the amount of proteins or the antibodies used in the
immunologic studies.
Earlier studies by Horikawa et al.(57) suggested that the rat kidney subunit is not
expressed in the liver, but later studies showed that the human kidney
subunit is expressed at very low levels in adult
liver(12) . However, the data presented here clearly show that
the
subunit is expressed in human liver because the sequence of
the XHM4.1 and XHM4.2 cDNA fragments, which were isolated from an adult
human liver library, was identical to that of the extrahepatic
subunit. The finding that the extrahepatic
subunit is expressed
in liver confirms earlier findings by Liau et al. (7) of a human liver isozyme with a low K
for L-Met, and a more recent report by De La Rosa et
al.(21) in which immunoblots of human liver extracts
revealed that adult human liver also expresses the extrahepatic form of
MAT.
The deduced amino acid sequences of the human and rat
extrahepatic subunit differ at only 5 of 395 positions (Fig. 6). The cDNA sequences for the human lymphocyte
subunit and rat kidney enzyme were 93.5% identical. This high degree of
identity was even more striking at the 3`-untranslated region, where
only a single difference exists (position 39) in the 73 bp downstream
of the TGA terminator codons (to position 1260). The 3`-untranslated
region was also conserved for the extrahepatic
subunit cDNA
expressed in adult liver because fragment XHM4.1 contained an identical
sequence in this region differing by only 1 base where a C at position
2222 in the 3`-untranslated region was found in place of the T found in
human lymphocyte cDNA. Although the sequences of the 3`-untranslated
regions of the hepatic
subunit and the extrahepatic
subunit are quite different, the 3`-untranslated region is highly
conserved across different species, suggesting that this region may
exert an important regulatory function.
Figure 6:
Comparison
of the deduced amino acid sequence of the subunit of human
lymphocyte MAT with those of the rat liver and rat kidney (57) enzymes. The amino acid sequence shown for the rat liver
enzyme is from cDNA that was cloned and sequenced in this laboratory
and is identical to that reported by Horikawa et al.(14) except at amino acid position 345, where the Horikawa
sequence contains an extra Asp. Residues in the rat sequences that are
identical to the human lymphocyte
subunit sequence are shaded (&cjs2110;).
The finding of the two
introns in the clones derived from the Jurkat library was useful in
that it allowed us to select genomic clones that harbor the
extrahepatic subunit gene. DNA from these clones enabled us to
determine that the gene for the human extrahepatic
subunit of MAT
is located on chromosome arm 2p, an area that corresponds to band
2p11.2. Recently, the gene for the mouse liver enzyme was localized to
chromosome 7(56) . To our knowledge there are no reports of the
chromosomal localization of the human liver
subunit.
Studies that investigate the genomic organization and regulation of MAT
isozymes will undoubtedly reveal molecular mechanisms controlling the
expression of these enzymes in different mammalian tissues as well as
in normal and malignant tissues.
Expression of the subunit in E. coli produced two immunoreactive peptides that are probably
equivalent to the
and
` peptides of HuLy
MAT(19, 23) . Total identity would not be expected
because of differences in amino-terminal processing, which would give
NH
-Met-Asn or perhaps NH
-Asn in E. coli(58, 59) and N-acetyl-Met-Asn in
lymphocytes(38) . Such differences may account for the slightly
faster migration of the E. coli-expressed products in SDS-PAGE (Fig. 5). It seems likely, however, that the E. coli products with apparent masses of 53 and 51 kDa are related to each
other in the same way that the lymphocyte
and
` subunits are
related. Since mRNA processing and specific post-translational
modifications are generally very different between prokaryotes and
eukaryotes, we favor a relatively nonspecific process such as
proteolysis or deamidation to account for differences between the
and
` subunits of the lymphocyte enzyme and between their
counterparts produced by the E. coli expression strain. Amino
acid sequences of the lymphocyte
/
` subunit that are not
present in the rat liver enzyme are possible targets for such a process
since the latter gives only a single band in SDS-PAGE. It is of
interest in this regard that the deduced
/
` sequence contains
two Asn-Gly dipeptides (at positions 6-7 and 170-171) that
are not present in the rat liver enzyme (Fig. 6). The
asparaginyl residue in this sequence is known to be particularly
susceptible to nonenzymatic succinimide formation with subsequent
hydrolysis to an isoaspartyl residue(60, 61) . If the
difference between the
and
` subunits is due to such a
modification, one would expect the rat kidney enzyme also to show two
bands in SDS-PAGE because its deduced sequence contains the same two
Asn-Gly dipeptides. We are unaware of any report on the subunit
structure of the rat kidney enzyme.
The 43.6-kDa polypeptide
predicted from the ORF of our cDNA is considerably smaller than the
values of 53 and 51 kDa estimated by SDS-PAGE for the and
`
subunits of human lymphocyte MAT(19, 23) . This
discrepancy also exists for the HuLy
subunit expressed in E.
coli and for the rat liver
subunit, which has a
predicted mass of 43.7 kDa (14) but an estimated mass of about
48 kDa by SDS-PAGE
analyses(4, 9, 10, 62) . Anomalously
slow migration in SDS-PAGE has been reported for other proteins and in
some instances has been attributed to excess charge density (63) or more specifically, to an unusually high content of
acidic amino acids(64, 65) . Although the entire
deduced
/
` peptide has a net charge of only -7, many of
the basic residues are located in the carboxyl-terminal portion, and
the first 240 amino acids have a net charge of -19. A similar
asymmetric charge distribution is found in the rat liver enzyme.
The
extrahepatic form of MAT has been studied extensively in human
lymphocytes(16, 19, 21, 22, 23, 66, 67) ,
human erythrocytes(15, 18) , rat
lens(17, 68) , rat kidney(4) , and bovine
brain(20) . The presence of the and
subunits has
been demonstrated in human
lymphocytes(19, 21, 22, 23) , human
mature erythrocytes(18) , and bovine brain (20) and has
been reported to exist in Erlich ascites tumor and calf
thymus(20) ; there have been no reports of the existence of the
subunit in kidney tissues. There is strong evidence to suggest
that the
subunit is the catalytic subunit, whereas
may have
a regulatory function. Extracts from the E. coli expression
strain contained levels of MAT activity which were 20-fold higher than
those from E. coli itself. However, a preliminary kinetic
analysis of unpurified enzyme indicates that it has a K
of 80 µM, which is 20-fold higher than the 4
µM value of MAT from leukemic T cells and 5-fold higher
than the 16 µM value for enzyme in crude resting human
lymphocytes. Perhaps these findings only reflect differences between
lymphocytes and E. coli in amino-terminal processing or some
other post-translational modification, but it is intriguing to
speculate that the function of the
subunit may be to impart the
kinetic properties that distinguish the lymphocyte enzyme from the
hepatic enzyme, i.e. a low K
for L-methionine and sensitivity to product inhibition by
AdoMet(16, 19) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L43509[GenBank].