(Received for publication, November 20, 1996, and in revised form, April 16, 1997)
From the Departments of Surgery, ¶ Biochemistry,
and
Microbiology and Immunology, The University of Tennessee,
Memphis, Tennessee, 38163 and § The Veterans Administration
Medical Center, Memphis, Tennessee 38104
Superantigens interact with the T
cell receptor for antigen (TCR) and are, therefore, more physiological
stimulators of T lymphocytes than nonspecific polyclonal T cell
mitogens. The effects of these two classes of T cell stimulators on
methionine adenosyltransferase (MAT) and
S-adenosylmethionine (AdoMet) levels were investigated. Activation of resting human peripheral blood T lymphocytes by the
mitogen phytohemagglutinin (PHA) or the superantigen staphylococcal enterotoxin B (SEB) caused a 3- to 6-fold increase in MAT II specific activity. Although the proliferative response was higher in cultures stimulated with PHA compared with SEB, MAT II activity was comparable in both cultures. Both stimuli caused down-regulation of the MAT 68-kDa
subunit expression and induced a comparable increase in the
expression of the catalytic
2/
2
subunit mRNA and protein. However, in superantigen-stimulated cells, the expression of the noncatalytic
subunit was down-regulated and virtually disappeared by 72 h post-stimulation; whereas, no change in the expression of
this subunit was noted in PHA-stimulated cells. Thus, at 72 h
following stimulation, PHA-stimulated cells expressed MAT II
2/
2
and
subunits while SEB-stimulated cells expressed the
2/
2
subunits only; the
subunit was no longer expressed in superantigen-stimulated cells. Kinetic analysis of MAT II in extracts of PHA- and SEB-stimulated cells using reciprocal kinetic plots revealed that in the absence of the
subunit the
Km of the enzyme for L-methionine
(L-Met) was 3-fold higher than in the presence of the
subunit. Furthermore, AdoMet levels were 5-fold higher in cell extracts
lacking the
subunit (SEB-stimulated cell extracts) compared with
extracts containing MAT II
2/
2
and
subunits. We propose that
the increased levels of AdoMet in superantigen-stimulated cells may be
attributed to the absence of the
subunit, which seems to have
rendered MAT II less sensitive to product feedback inhibition by
(
)AdoMet. The data suggest that the
subunit of MAT II, which has
no catalytic activity, may be a regulatory subunit that imparts a lower
Km for L-Met but increases the
sensitivity to feedback inhibition by AdoMet. The down-regulation of
the
subunit, which occurred when T cells were stimulated via the
TCR, may be an important mechanism to regulate AdoMet levels at
different stages of T cell differentiation under physiological
conditions.
Methionine adenosyltransferase (MAT)1
(ATP:L-methionine S-adenosyltransferase, EC
2.5.1.6) is a key enzyme in cellular metabolism because it catalyzes
the formation of S-adenosylmethionine (AdoMet) from
L-methionine (L-Met) and ATP. In addition to
being the major methyl group donor and a precursor for polyamine
biosynthesis, AdoMet regulates several important intracellular
enzymatic reactions including those involved in polyamine synthesis and
one carbon metabolism (1-4). In mammals, there are at least two MAT
isozymes, designated MAT I/III and MAT II (5-7), that have distinct
tissue distribution, subunit composition, and kinetic properties (for review, see Refs. 3 and 8). MAT I/III represents a different oligomeric
state of the same 48-kDa catalytic subunit designated 1 (8). MAT I
(200 kDa), which is a tetramer, and MAT III (100 kDa), which is a dimer
of the
1 subunit, differ considerably in their physical properties
and in their Km for L-Met (4-7, 9-13),
suggesting that differences in the oligomeric state of the
1 subunit
has a profound effect on the enzyme properties (for review, see Refs. 3
and 4).
The second mammalian MAT isozyme, MAT II, appears to have a wider
tissue distribution than MAT I/III and has been detected in many
tissues including erythrocytes (14), lymphocytes (15), brain (16),
kidney (6, 17), testis (10), lens (18), and fetal liver (19, 20) and to
a lesser extent in adult liver (21). Although the catalytic
subunits of MAT I/III and MAT II are similar, they are products of
distinct genes designated MAT1A and MAT2A that
encode for the
1 and
2 catalytic subunits, respectively (8). The
1 and
2 subunits are only 84% homologous at the amino acid
level, and each consists of 395 amino acids with a predicted
Mr of 43,600; however, both subunits migrate anomalously on SDS-PAGE with an apparent size of 48-53 kDa (for review, see Ref. 3).
In humans, the 2 subunit of MAT II appears to undergo
post-translational modification to generate
2
subunits (15, 22). In
addition, the
2/
2
subunits have been shown to be associated with
a catalytically inactive (21, 23) 38-kDa subunit that has been
designated
(15). This hetero-oligomeric form of MAT II (185 kDa),
which consists of both
2 and
subunits, has been found in human
lymphocytes, bovine brain, Ehrlichs ascites tumor, and calf thymus (15,
16). The exact oligomeric state of MAT II is not entirely clear, and
the relative ratio of
2/
2
and
subunits in the holoenzyme is
not known. However, based on the available Mr
and amino acid composition data, we hypothesize that it may be in the
form of
2n
n,
2/
2
n, and/or
2
n
n, where n is either 2 or 3. Thus, for the time being and for simplicity, we shall refer to this form as
(
2/
2
)x
y.
Studies of MAT II from human leukemia cells revealed that the
(2/
2
)x
y form has a low Km
for L-Met (3.5-20 µM) and is strongly and
synergistically inhibited by all three of its end products,
Pi, PPi, and AdoMet (15, 24). However, recent
data showed that recombinant
2/
2
subunits expressed in
Escherichia coli had a Km for
L-Met of 80 µM (23), suggesting that the
association of the
2/
2
subunits with the
subunit lowers the
Km for L-Met.
Despite the availability of detailed information on the kinetic
behavior of pure MAT II, the regulation of MAT II in mammalian tissues
remains poorly understood, and the relative contribution of the
different MAT subunits to enzyme activity and/or regulation is at
present not clear. In an effort to address this problem, we have
focused our studies on MAT II from human T lymphocytes. We recently
reported that in addition to the 2/
2
and
subunits, normal
resting human lymphocytes have another 68-kDa protein, which has MAT
activity, that we have designated
(21). The
protein is also
found in human liver and is the only form of MAT in certain types of
myelogenous leukemia cells (21). When T lymphocytes are stimulated to
divide,
disappears while the
2/
2
subunits, which have higher
MAT activity than
, concomitantly increase (21). Activation of T
cells and the switch in the expression of MAT subunits is accompanied
by a significant increase in enzyme activity as well as AdoMet levels
and turnover (21, 25, 26). Inasmuch as AdoMet is a strong inhibitor of
MAT, the paradoxical simultaneous increase in both AdoMet turnover and
AdoMet levels suggests the existence of a cellular regulatory mechanism
that allows MAT to remain active in the presence of high concentrations of its products (24). To further investigate this phenomenon, it was
necessary to conduct our investigation under more physiological conditions.
Under physiological conditions, T cells are activated via the T cell receptor (TCR) for antigen. The engagement of TCR by antigen transmits important biochemical signals that, together with other essential costimulatory signals derived from antigen-presenting cells (APC), program T cells to divide and differentiate (for review, see Refs. 27-29). Nonspecific polyclonal mitogens (e.g. PHA) are nonphysiological stimulators of T cells, and studies have documented differences in the biochemical signals triggered as a result of TCR occupancy versus those elicited by the interaction of polyclonal mitogens with their respective receptors on T cells (30-33). Differences in the signals used, and hence the biochemical pathways triggered, can have major effects on T cell differentiation and function.
Superantigens are a newly characterized class of molecules that
activate T cells by engaging their TCR (for review, see Refs. 34 and
35). Whereas the binding of nominal antigen to the TCR is highly
specific and is dependent on the five variable elements of the TCR,
namely V, D
, J
, V
, and J
, the binding of superantigen to
the TCR is somewhat less specific because superantigens interact with T
cells based on their V
type without regard for the fine specificity
of the TCR (for review, see Refs. 34 and 35). Each superantigen has a
characteristic affinity to a set of V
elements and can stimulate
virtually all T cells that express them (34). This number can reach
5-40% of resting T cells, compared with
0.001% of cells that can
be stimulated by a typical antigen. Consequently, biochemical signals
triggered by superantigen in resting T cells can be readily measured.
The ability of superantigens to stimulate large numbers of T cells,
together with the fact that stimulation occurs via the TCR, provides an
ideal system for studying biochemical events triggered in activated T
cells under physiological conditions that mimic antigenic stimulation (32).
In this study, the effects of a nonspecific mitogen and
superantigen activation of T cells on MAT activity and the temporal expression of its subunits were compared. We show that, although both
mitogen and superantigen induced an increase in MAT activity, they had
different effects on MAT II subunit expression and consequently on the
kinetic properties of the enzyme. Specifically, we show that the
expression of the subunit is down-regulated in
superantigen-stimulated T cells and that this phenomenon is associated
with a marked increase in intracellular AdoMet concentration and with a
form of MAT II that has a reduced affinity for L-Met but is
less susceptible to feedback inhibition by AdoMet. These results
suggest that the role of the
subunit may be regulatory, and the
findings underscore the importance of conducting studies of MAT in T
cells under physiological conditions.
Aprotinin, antipain, chymotrypsin, leupeptin, pepstatin A, phenylmethanesulfonyl fluoride, benzamidine, o-phenanthroline, bicinchoninic acid, SEB, and PHA were from Sigma. All tissue culture reagents were purchased from Cellgro. The ECL chemiluminescence detection system was from Amersham Life Science, Inc. Nitrocellulose membranes were from Fisher Scientific, and the RX medical x-ray film was from Fuji.
Cell CultureAll cells were cultured in RPMI medium
supplemented with 10% heat-inactivated fetal bovine serum, 50 µg/ml
streptomycin, and 50 units/ml penicillin (referred to as RPMI
complete). PBMC were isolated from peripheral blood by Ficoll-Hypaque
density centrifugation, washed twice, and then resuspended in RPMI
complete. The cells were adjusted to 1-2 × 106
cells/ml and stimulated with either PHA (Boehringer Mannheim) or the
staphylococcal superantigen SEB (Sigma). At specific times after the
initiation of culture, the cells were harvested and used for
determination of MAT II activity and kinetic properties, MAT 2
subunit mRNA expression, MAT
,
2/
2
, and
subunits protein expression, and AdoMet levels as detailed below. Parallel cultures were set up in 96-well plates and harvested at 48 or 72 h
for assessment of the proliferative response.
PBMC were cultured at 105 cells/200 µl RPMI complete in 96-well round bottom plates. The cells were stimulated with either PHA or SEB and incubated at 37 °C in 5% CO2 and 95% humidity. PHA- and SEB-induced blastogenic responses were assessed by measuring [3H]thymidine uptake at 24, 48, and 72 h post-stimulation. Five h prior to harvesting, cells were pulsed with 1 µCi/well of [3H]thymidine (specific activity = 6.7 Ci/mmol, DuPont Co., Wilmington, DE), harvested onto glass fiber filters using an automated cell harvester, and counted in a Packard liquid scintillation counter. The precipitable radioactivity are presented as mean cpm [3H]thymidine uptake of triplicate cultures ± S.E.
DNA content was measured by the method of Burton (37) in acid-insoluble precipitate of cells extracted with 2 N perchloric acid (PCA).
Preparation of Cell ExtractsMAT activity was assayed in extracts prepared from cell pellets by three cycles of freeze-thawing. The enzyme extraction buffer consisted of 50 mM Tris, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 4 mM DTT, and a mixture of the following proteolytic inhibitors: 50 µg/ml aprotinin, 25 µg/ml leupeptin, 10 µg/ml phenylmethanesulfonyl fluoride (15, 21). The protein concentration in the cellular extracts was determined by the Bradford method (36) with the dye-binding kit from Bio-Rad and/or by the bicinchoninic acid method (37) from Sigma.
MAT AssayMAT activity was assayed as described previously (15, 21). The standard assay contained 20 µM L-Met, 5 mM ATP in 50 mM TES buffer, pH 7.4, 50 mM KCl, 15 mM MgCl2, 0.3 mM EDTA, and 4 mM DTT. The Met concentration was varied as indicated in the kinetic analysis. One unit of MAT activity is defined as the amount of enzyme that catalyzes the formation of 1 nmol of AdoMet in 1 h.
Analysis of kinetic data was performed both manually by the Lineweaver-Burk and the S/v versus S plots and confirmed by PSI-Plot software (Poly Software International) and the Marquardt algorithm. The kinetic data were fit to nonlinear least squares analysis to the Adair-Scatchard equation (45) for one and two catalytic sites.
SDS-PAGE and Western blottingThe same cell extracts
assayed for MAT activity as described above were diluted in loading
buffer (60 mM Tris-Cl, pH 6.8, 2% SDS, 5%
2-mercaptoethanol, and 5% glycerol), heated in a boiling water bath
for 4 min, and analyzed by SDS-PAGE (10% total acrylamide, 2.7%
bisacrylamide) as described previously (21). After electroblotting the
proteins onto the nitrocellulose for 1 h at 25 to 30 V/cm, the
blots were blocked overnight with 6% nonfat dry milk in TBS (50 mM Tris, pH 7.5, and 150 mM NaCl), washed in
TBS, and incubated with primary polyclonal anti-holoenzyme antisera, or
polyclonal antisera generated against synthetic peptides of the or
the
subunits (22). The blots were developed with secondary
anti-rabbit antibodies conjugated to horseradish peroxidase and the
luminol-chemiluminescence reagents (Amersham ECL) (21). The processed
blots were exposed to x-ray film, and the autoradiograms were analyzed.
For some experiments, the autoradiograms were scanned using a Howtek
Scanmaster-3 scanner (Protein Data Base, Inc., Huntington Station, NY),
and the intensity of the desired band was integrated and expressed in
arbitrary units.
Normal, resting PBMC (2 × 106) were stimulated with PHA or SEB for specific time intervals. At the desired times, the cells were harvested and counted, and total RNA was extracted using RNAzol-B (Tel-Test Inc., Friendswood, TX) as described previously (38). The RNA was treated with RNase-free DNase to remove contaminating DNA that could interfere with the PCR analysis. First-strand cDNA was prepared from 2 µg of RNA using superscript reverse transcriptase (Promega, Madison, WI), and random hexanucleotides (Boehringer Mannheim). The reaction contained in 20 µl: 2 µl of 10 × PCR buffer (0.2 M Tris-HCl, pH 8.4, 0.5 M KCl, 15-20 mM MgCl2, and 100 µg/ml nuclease-free bovine serum albumin); 2 µl of 10 mM dNTPs mix; 2 µg of random hexanucleotides; 2.5 mM DTT; 1 unit of RNase inhibitor; and 200 units of RT/µg of RNA. The reaction was incubated for 45 min at 37 °C and terminated by heating at 95 °C for 10 min followed by rapid chilling in ice-water for 2 min.
The cDNA was amplified in the presence of Taq polymerase
(0.25 units) using primers that are specific for MAT II 2 subunit (forward primer, 5
-GCC-GCT-GCT-CCT-TCG-TAA-G-3
; reverse primer, 5
-ACC-CCA-ACC-GCC-ATA-AGT-GT-3
) and that amplified an 870-bp product.
Amplification of a 590-bp glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA was included in each reaction as an internal control.
All primers were used at 0.3 µM, and the concentration of
Mg2+ was optimized for each primer set. Quantification of
the amplified products was achieved by the inclusion of
32P-labeled reverse primers (106 cpm of
each/reaction) that were labeled using T4 polynucleotide kinase (Promega) and [
-32P]ATP (Dupont) (38). After
20-25 PCR cycles consisting of 95 °C denaturation (1 min), 55 °C
annealing (1 min), and 72 °C extension (1 min), the radiolabeled PCR
products were separated on 2% agarose gels in TBE buffer, pH 8.0 (0.09 M Tris-base, 0.09 M boric acid, and 2 mM EDTA). The gels were dried, subjected to
autoradiography, and scanned using Howtek Scanmaster-3 scanner (Protein
Data Base, Inc.). For each experiment, the cDNA from each culture
was titrated in pilot PCR reactions to determine the optimal amount
that can be used for linear amplification. Results of the
semi-quantitative PCR are expressed in arbitrary scanning units.
Integrated values for the MAT II
2 subunit bands are normalized to
the integrated values of the GAPDH internal control to allow
comparisons among samples. To rule out reagent contamination, controls
include reactions with no added template, no added primers, or
reactions with untranscribed RNA.
At specific times poststimulation, total RNA was
extracted (108 cells yield ~300 µg of RNA), and in some
experiments, mRNA was isolated using oligo(dT)-cellulose. Total RNA
(20 µg) or the eluted mRNA (5 µg of mRNA) was precipitated
and resuspended in MOPS buffer (40 mM MOPS, 10 mM Na-acetate, 1 mM EDTA final concentration) at pH 7.1, 3.3 µl of 37% formaldehyde, and 10 µl of formamide to a
final 20 µl in diethyl pyrocarbonate-treated water. The samples were
incubated for 15 min at 65 °C and then chilled on ice prior to
loading. Samples were centrifuged briefly, mixed with 2 ml of loading
buffer (1 mM EDTA, pH 8.0, 0.25% w/v bromphenol blue, 0.25% w/v xylene cyanol, 50% v/v glycerol), immediately loaded onto a
1.2% agarose gel in MOPS buffer with 0.01% formaldehyde, and
subjected to electrophoresis at 125 V constant for 3.5 h. In one
lane, total RNA was loaded to allow staining and photographing of the
28 and 18 S ribosomal subunits. The gel was briefly washed in
DEPC-treated water and then soaked in 20 × SSC buffer (3 M NaCl, 300 mM Na-citrate, pH 7.0) for 30 min.
The RNA was transferred to positively charged nylon membranes
(Boehringer Mannheim) for 1.5 h using the Posiblot apparatus
(Stratagene). The membranes were washed for 5 min in 6 × SSC at
25 °C, and the RNA was cross-linked to the membrane using a UV
cross-linker. The membranes were washed briefly in 6 × SSC at
25 °C and incubated for 5 h at 42 °C in prehybridization
buffer (25 mM KH2PO4, pH 7.4, 5%
Denhardt's solution, 50% formamide, 5 × SSC, and 50 µg/ml of
salmon sperm DNA). Blots were incubated for at least 5 h at
42 °C with the appropriate probes in hybridization buffer
(prehybridization buffer containing 100 mg/ml dextran sulfate). The
probe for the 2 subunit was a purified 314-bp PCR cDNA product
that was amplified with specific primers (forward primer,
5
-GTT-AAA-GGA-GGT-CTG-TGC-AG-3
; reverse primer,
5
-TAA-CCT-ACG-CCA-ACA-AGT-CT-3
) that had been internally labeled
using Taq DNA polymerase and [
-32P]ATP
(Dupont). After hybridization, the blots were subjected to various
stringency washes, dried, and exposed to x-ray films, and the
autoradiograms were scanned as described above for the PCR method. The
integrated values for MAT II
2 subunit bands were normalized to the
corresponding GAPDH bands to allow comparisons of samples. The size of
the hybridized bands was determined by comparing the relative migration
to that of the 28 and 18 S ribosomal subunits.
PBMC were cultured with medium alone or stimulated with either PHA or SEB as described above and then harvested at the desired time and counted. The cells were resuspended in 500 µl of ice-cold PBS and centrifuged in microcentrifuge tubes at 1800 × g, washed with ice-cold PBS, and frozen in liquid nitrogen. Cell extracts were prepared for analysis of AdoMet by lysing cells and precipitating proteins with 2 N perchloric acid (25). The acid-soluble extract was neutralized with 2 N KOH and 1 N KHCO3, chilled on ice for 10 min, and centrifuged to remove KClO4. The acid-insoluble precipitate was extracted again with 2 N PCA and used for the determination of DNA by the method of Burton (37). The neutralized acid-soluble extract was used for the determination of intracellular AdoMet levels by HPLC as detailed elsewhere (25) using a Waters Model 840 HPLC system with model 510 solvent delivery system, model 490 dual variable UV detector, and Model 710B intelligent sample process (WISP) with refrigeration.
Neutralized, cleared PCA extracts were injected onto a Whatman Partisil-10 SCX cation exchange column (0.46 × 25 cm) and eluted isocratically at 1 ml/min with 0.2 M ammonium formate, pH 4.0, and 10% acetonitrile (25). The absorbance was monitored at 260 and 280 nm, and the peaks were identified and integrated using an on-line digital computer and Waters HPLC software. Biologically active (-)AdoMet, used as the HPLC standard, was prepared using E. coli MAT (10 µg/ml) incubated with 1 mM L-Met in 100 mM Tris, pH 8.3, 100 mM KCl, 20 mM MgCl2, 2.5 mM ATP, 2 mM DTT, and 150 µg/ml of bovine serum albumin, for 1 h at 37 °C and was purified chromatographically on an HPLC C18 column (Waters microBondapak) as described previously (15).
Statistical AnalysesExperiments were conducted a minimum of three times, and statistical differences were evaluated by Student's t tests.
Polyclonal mitogens and superantigens activate T cells
by distinct mechanisms. Both types of T cell activators stimulate large numbers of T cells to proliferate; however, PHA usually stimulates at
least twice as many cells as SEB (34). The difference in the mode of
interaction of mitogen and superantigens with T cells can be reflected
in the biochemical pathways used to program the cells to divide and/or
differentiate (32). To investigate whether mitogen and superantigen may
have different effects on the induction of MAT activity, PBMC were
stimulated with optimal concentrations of either PHA or SEB, and we
then measured the proliferative response and MAT activity. As expected,
the proliferative response to PHA was significantly higher than that to
SEB (Fig. 1). However, despite this difference in
proliferative responses, there was no significant difference in MAT
activity in PBMC cultures stimulated with either SEB or PHA
(p > 0.8, Fig. 2); both stimuli induced
a similar and significant increase in MAT activity compared with
unstimulated cells. After 48 h in culture with PHA or SEB, MAT
activity in PBMC increased by 2.4- and 3.2-fold, respectively, compared
with unstimulated PBMC (p < 0.05).
Effect of Mitogen and Superantigen Activation of T Cells on MAT mRNA Expression
Inasmuch as PHA stimulates 80-90% of
resting T cells, and whereas SEB is only capable of stimulating T cells
that bear the appropriate V elements (usually 20-40% of resting T
cells) (34), the finding that MAT activity was similar in both cultures
suggests that, on a per cell basis, SEB may be inducing higher levels
of MAT activity than that induced by PHA. Superantigen-stimulated cells
may have higher MAT activity than PHA-stimulated cells due to a higher
induction of MAT subunit expression or differences in MAT regulation
elicited by SEB stimulation. To distinguish between these
possibilities, the expression of MAT mRNA and protein was examined
in PHA- and SEB-stimulated cells. PBMC were incubated with specific
stimulators for 0, 8, 12, 24, 36, 48, and 72 h. At each time
point, replicate cultures were harvested, and MAT
2 subunit mRNA
expression was determined by RT-PCR and Northern blots. As shown in
Fig. 3, the kinetics of
2 subunit mRNA induction by PHA and SEB was similar, peaking at 4-8 h after addition of the
stimulus and declining to near base-line levels by 12 h (Fig. 3).
Both PHA and SEB induced a 5-8-fold increase in the amount of
2
subunit mRNA compared with unstimulated cells (Fig. 3).
Effect of Mitogen and Superantigen Activation of T Cells on the Expression of MAT Subunit Proteins
PBMC were incubated with PHA
or SEB for 0, 8, 12, 24, 36, 48, and 72 h. At each time point,
cell extracts were analyzed by immunoblots to determine the expression
of MAT subunit proteins. As shown in Fig. 4, stimulation
with either mitogen or superantigen caused a time-dependent
decrease in protein, which nearly disappeared by 48 h after
stimulation, while the amount of both
2 and
2
subunits increased
concomitantly, relative to unstimulated cells cultured in medium alone.
The relative increase in the
2
compared with the
2 subunit
correlated with a higher MAT activity in SEB-stimulated cultures.
However, the most striking difference between extracts of PHA- and
SEB-stimulated cells was the finding that stimulation with superantigen
induced a dramatic down-regulation of the
subunit expression,
which was no longer detectable by 72 h (Fig. 4A). These
findings were reproducible when PBMC from different individuals were
stimulated with this superantigen (Fig. 4B).
Kinetic Properties of MAT from Mitogen- and Superantigen-activated Cells
We reported recently that PHA activated PBMC that expressed
high levels of 2/
2
, and
subunits appeared to have two
kinetically different forms of MAT with a Km for
L-Met of 16 and 80 µM (21). In addition, we
reported that recombinant
2/
2
expressed in E. coli
without the
subunit has a Km for L-Met of 80 µM (23). It was of interest,
therefore, to determine the consequence of SEB-induced down-regulation
of the
subunit on MAT kinetic properties. The Km
for L-Met of MAT in extracts from 72-h cultures stimulated
with either PHA or SEB was compared by the Lineweaver-Burk and the
S/v versus S plots, which are commonly used to
determine the presence of multiple enzymes catalyzing the same reaction
(46). As shown in Fig. 5, MAT in the 72-h PHA-stimulated
cell extracts, which consisted of
2/
2
and
subunits (Fig. 4),
appeared to exist in two kinetic forms. These observations were also
confirmed by computer curve fitting by nonlinear least squares. For,
example, when MAT data from PHA-stimulated cell extracts, which has the
2/
2
and
subunits, were fit to both single- and two-site
Adair-Scatchard equations, the data clearly fit the two-site model
better as indicated by improvement in correlation coefficient,
coefficient of determination and
2. By analyzing the
values for low (2.5-20 µM) and high (20-80 µM) L-Met concentrations separately, the data
were consistent with the co-existence of two enzyme species with
calculated Km values for L-Met of 53-74
µM (Vmax 9.7-12, correlation
coefficient = 0.998, sum of squares = 0.071) and 23 µM (Vmax 5.3, correlation coefficient = 0.999, sum of squares = 0.013), respectively.
Conversely, the kinetic data of MAT from SEB-stimulated cell extracts
that lacked the
subunit clearly fit the single-site model based on the same criteria, with a Km value of 55-67
µM and a Vmax of 12-13.5,
respectively (correlation coefficient = 0.997). Thus, in the
absence of the
subunit, the Km for
L-Met is higher, suggesting that
may be a regulatory
subunit of MAT.
Effect of Mitogen and Superantigen Activation of T Cells on Intracellular AdoMet Levels
Intracellular AdoMet levels were
determined by HPLC at different times following stimulation of PBMC
with either mitogen or superantigen. Levels of AdoMet were higher in
PHA- and SEB-stimulated cells compared with unstimulated cells (Fig.
6); however, the levels of AdoMet were markedly higher
in SEB-stimulated compared with PHA-stimulated cells (Fig. 6). At
72 h, steady-state levels of AdoMet were 10-fold higher in
SEB-stimulated cells compared with control cells (p < 0.01) and 4-6-fold higher than in PHA-stimulated cells
(p < 0.02). The difference between AdoMet levels in
cells stimulated with SEB or PHA was highest and more significant at the time point of total disappearance of the MAT II subunit in
SEB-stimulated cells. Based on an estimated volume of 0.69 µl/106 stimulated PBMC (26, 25), these data indicate that
AdoMet concentrations can exceed 100 µM in cells
stimulated via a physiological route.
Effect of AdoMet on MAT Activity in Extracts of Mitogen- and Superantigen-activated Cells
According to the kinetic analysis of
2/
2
MAT II from human T cells, AdoMet is a potent inhibitor
of its own synthesis, particularly in the presence of Pi
and PPi (24). The finding that both MAT activity and AdoMet
levels were higher in superantigen-activated compared with
mitogen-activated cells suggested that the enzyme may be differentially
regulated in cells stimulated by these distinct stimuli. Guided by the
knowledge that AdoMet activates hepatic MAT III (
1)2 and
moderately inhibits MAT I (
1)4 (6), we hypothesized that
in the absence of
, the
2/
2
polymeric forms of MAT II may be
less susceptible to feedback inhibition by AdoMet. We, therefore,
investigated whether the SEB-induced down-regulation of the
subunit
has an effect on the feedback inhibition of MAT by AdoMet. As shown in
Table I, MAT from 72-h activated SEB extracts (
2/
2
but no
subunits) was almost 2-fold less inhibited by AdoMet compared with MAT from PHA-stimulated cells (
2/
2
and
subunits).
|
Activation of resting T cells triggers biochemical signals that initiate a cascade of events leading to cellular differentiation and proliferation. AdoMet plays a key role in these events, and as the major methyl group donor, it regulates protein activity, RNA stability, and gene expression in addition to acting as a cofactor for several enzymatic reactions and as a precursor for the polyamines (39-41). It is reasonable, therefore, to assume that the levels of AdoMet need to be tightly regulated to meet specific cellular demands at the different stages of T cell activation and differentiation.
The regulation of cellular AdoMet concentrations can be achieved at the
level of its synthesis by MAT and/or its utilization via
transmethylation and polyamine synthesis pathways. Our studies have
focused on the regulation of MAT activity in human lymphocytes as a
means for regulating AdoMet levels. In previous studies, we reported
that activation of T cells is accompanied by an increase in MAT
activity that was associated with a decline in the subunit and a
concomitant increase in the
2 and
2
catalytic subunits (21). In
this study, the use of a superantigen as a T cell stimulator revealed
that stimulation of T cells via the physiological route induces
specific changes in MAT subunit expression and kinetic and regulatory
properties that were not observed in nonphysiologically stimulated,
mitogen-activated T cells.
Superantigens are potent T cell stimulators that stimulate T cells in a
manner similar to that of conventional antigen because they interact
with the T cell via the TCR (for review, see Refs. 34 and 35).
However, superantigens interact with specific V
elements found on
the outside of the variable region of the TCR
chain (V
elements)
and, thus, are less specific than antigen. Each superantigen has a
characteristic affinity for 1-5 of the 25 human TCR V
elements and
can stimulate all T cells expressing them. Consequently, a given
superantigen can stimulate from 5 to 40% of resting T cells compared
with
0.001% stimulated by an antigen (34). The ability of
superantigen to stimulate large numbers of T cells, plus the fact that
stimulation is via the TCR, provides an ideal system for studying
biochemical events triggered in T cells under conditions that mimic
antigenic stimulation (32).
In previous studies, we have shown that the signaling and molecular
requirements for T cell stimulation by nonspecific mitogens are
different from those for superantigen (31-33). Here we showed that
like PHA, superantigen induces down-regulation of and up-regulation of
and
expression; however, unlike PHA, superantigen seems to
down-regulate the expression of the
subunit. We have reported that
and
2/
2
are capable of catalyzing the synthesis of AdoMet; whereas,
subunit has no known catalytic function (21). The data
from this study suggest that the
subunit may exert a regulatory effect on the enzyme activity.
Regulation of MAT activity by differential subunit oligomerization has
been reported for the hepatic isozymes MAT I/III (4, 6). The tetrameric
form, MAT I (1)4 has a Km for L-Met of 3-14 µM, whereas, the
1 dimer,
MAT III, has a Km of 200 µM (4, 6,
13). The physiologic relevance for the existence of different
oligomeric states of the hepatic form of MAT is not entirely clear;
however, these isozymes are starkly different in their kinetic and
physical properties as well as in their regulation by AdoMet (4, 6). At
physiological concentrations of L-Met, MAT I was reported
to be 10-fold more active than MAT III (7). In addition, MAT I is
feedback-inhibited by AdoMet, whereas, MAT III shows positive
cooperativity and is markedly activated by concentrations of AdoMet
between 50-500 µM (6). Whether a balance between MAT I
and MAT III is important for normal liver function and metabolism is
not entirely clear; however, studies by Mato and coworkers (4, 42-44)
revealed that the ratio of MAT I to MAT III can be regulated, in part,
by oxido/reduction of the enzyme sulfhydryl group and that the ratio of
reduced form (GSH) of glutathione/oxidized form (GSSG) of glutathione
can affect the enzyme oligomeric state.
The data presented here, together with several previous observations
(21, 23), suggest that MAT II may also be regulated by differential
oligomerization of its subunit proteins 2,
2
, and
. In our
earlier studies, we reported that MAT II purified from leukemic cells,
which consist of the
2,
2
, and
subunits, had a
Km for L-Met of 3.8 µM
(24). Detailed kinetic analysis of this pure enzyme allowed us to
develop a steady-state kinetic model that correctly predicted the
in situ MAT activity in human leukemia cells expressing the
2/
2
and
subunits (25). However, when the model was applied
to resting, freshly isolated T cells from healthy individuals, the
predicted value for MAT activity was higher than the measured value
(25). This difference was explained by the finding that in resting T
cells MAT exists as a less active 68-kDa
form (21, 25). Activation
of T cells resulted in the replacement of
with the
2/
2
subunits (21). The increase in the
2/
2
subunits was accompanied
by an increase in MAT activity, suggesting that the
2/
2
subunits
are more catalytically active than the
subunit. Attempts to
separate the
subunit from the holoenzyme revealed that this subunit
has no MAT catalytic activity (21, 23). The possibility that the
subunit may have a regulatory role in MAT II activity was first suggested from experiments showing that purified recombinant
2/
2
subunits expressed in E. coli had a Km
for L-Met of 80 µM, which was considerably
higher than that of the pure
2/
2
holoenzyme form (23).
The data presented in this study support the role of the subunit in
regulating MAT activity. In SEB-stimulated cells,
was no longer
detected after 72 h of culture, and kinetic analysis revealed that
MAT in extracts of these cells has a Km for
L-Met of 55-67 µM and a
Vmax of 12-13.5. By contrast, PHA-stimulated cells, which expressed the
2,
2
, and
subunits, appeared to have two kinetic forms of MAT with Km for
L-Met of 53-74 µM
(Vmax 9.7-12) and 23 µM
(Vmax 5.3), respectively. We propose that at
72 h, the relative amount of
2/
2
subunits is higher than
the
subunit in PHA-stimulated cells, allowing both homomeric and
heteromeric association of MAT subunits, where the
2/
2
homomer
has a Km of 53-74 µM and the
2/
2
heteromer has a Km of 23 µM. Clearly, these possibilities need to be investigated
directly with more detailed structural analyses. Nonetheless, the data
presented here suggest that the
subunit lowers the
Km of the enzyme for L-Met while
rendering it more susceptible to feedback inhibition by AdoMet. The
negative regulatory role of
may explain the finding that AdoMet
levels were 3-5-fold higher in SEB compared with PHA-stimulated cells. Down-regulation of the
subunit may be required in physiologically stimulated cells to relieve MAT from being feedback inhibited by its
product, thereby allowing a concomitant increase in both synthesis and
steady-state levels of this important molecule. Indeed, AdoMet levels
reached 100 µM in SEB-stimulated cells. It is tempting to
speculate that this mode of regulation of MAT via differential subunit
association may be necessary to achieve appropriate levels of AdoMet
that are required for physiological activation and functional
differentiation of T cells at specific stages of the cell cycle.
The authors are grateful to Dr. Harry Jarrett for helpful discussion regarding computer-assisted analysis of kinetic data and to Mr. Hesham Basma for help with photographic scanning.